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 2, 4-5, 7, 9-10, 12-13, 15-18, 21, 23-25 are amended. Claims 3, 8, 11, 14, 22 are cancelled. Claims 1-2, 4-7, 9-10, 12-13, 15-21, 23-25 are examined on the merits.
Priority
The applicant’s application is a U.S. National Stage application of PCT International Patent Application Serial No. PCT/SG2022/050125, filed 03/11/2022, which itself claims the benefit of Singapore Patent Application Serial No. SG10202102572X, filed 03/12/2021 is acknowledged.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Claim Objections
Claim 1 is objected to because of the following informalities: reciting an abbreviation without spelling out what the abbreviation is in the first instance (E. coli).
Claim 1 is objected to because of the following informalities: recites “for the in vivo”, it should be changed to “for in vivo”.
Claim 4 is objected to because of the following informalities: recites “E. coli strains genome” isn’t grammatically correct. It should be changed to “the genome of the E. coli strain.” Alternatively, it could be “E. coli strain’s genome.”
Claim 25 is objected to because of the following informalities: recites “such the expression”, it should be changed to “such that the expression”.
Appropriate correction 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.
Claims 1-2, 4-7, 9-10, 12-13, 15-21, 23-25 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.
Claim 1: “Stringently controlled” is a term of degree that lacks an objective standard for determining the scope of the claim. The claim does not define what level of repression or induction constitutes “stringent”. The specification does not provide a specific objective for what constitutes “stringently”.
Claim 5: The claim refers to “the E. coli arabinose operon”, which lacks of antecedent basis, because its specific identity has not been previously introduced in claim 1. The claim does not limit the E. coli arabinose operon to one that would be inherently present in the E. coli and reads on other E. coli arabinose operons that may be located in a plasmid, for example. Thus, recitation of E. coli in claim 1 does not provide sufficient antecedent basis for this claim limitation.
Claim 7: The term “preferably” makes the recitation of SEQ ID NO 9 optional; the examiner is unable to determine the metes and bounds of the claim. It is unclear whether the claim is limited to the specific SEQ ID NO 9, or if any sequence performing the function of an “scIHF2” is sufficient.
Claim 9: The term “preferably” makes the recitation the expression of both, IntC3 and scIHF2, is stringently controlled by the same inducible expression control sequence, “optional”. The claim fails to provide a clear indication of whether a method using a different control sequence would infringe this claim.
The term “Stringently controlled” is a term of degree that lacks an objective standard for determining the scope of the claim. The claim does not define what level of repression or induction constitutes “stringent”. The specification does not provide a specific objective for what constitutes “stringently”.
Claim 10: The term “preferably” makes the recitation of SEQ ID NO 1 optional; the examiner is unable to determine the metes and bounds of the claim. It is unclear whether the claim is limited to the specific SEQ ID NO 1, or if any expression cassette containing the selection maker is sufficient.
Claim 13: The term “preferably” makes the requirement for an expression control optional. It is unclear if the claim is limited to genes that are actively regulated or if it encompasses any gene regardless of its expression status.
Claim 17: The term “Stringently controlled” is a term of degree that lacks an objective standard for determining the scope of the claim. The claim does not define what level of repression or induction constitutes “stringent”. The specification does not provide a specific objective for what constitutes “stringently”.
Claim 18: The term “Stringently controlled” is a term of degree that lacks an objective standard for determining the scope of the claim. The claim does not define what level of repression or induction constitutes “stringent”. The specification does not provide a specific objective for what constitutes “stringently”.
Claim 21: The term “preferably” makes the recitation of SEQ ID NO 9 optional; the examiner is unable to determine the metes and bounds of the claim. It is unclear whether the claim is limited to the specific SEQ ID NO 9, or if any sequence performing the function of an “scIHF2” is sufficient.
Claim 23: The term “preferably” makes the recitation the expression of both, IntC3 and scIHF2, is stringently controlled by the same inducible expression control sequence, “optional”. The claim fails to provide a clear indication of whether a method using a different control sequence would infringe this claim.
Claim 25: The term “preferably” makes the use of the strain MG1655 optional. This creates two different options of the claim within one sentence either any E. coli or E. coli MG1655.
The term “Stringently controlled” is a term of degree that lacks an objective standard for determining the scope of the claim. The claim does not define what level of repression or induction constitutes “stringent”. The specification does not provide a specific objective for what constitutes “stringently”.
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, 4-7, 9-10, 12-13, 15-21, 23-25 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 1 requires the provision of a genus of functional variants or fragments thereof of SEQ ID NO 2. The claims encompass any number of mutations of any type, such as an insertion, deletion or substitution, relative to the sequence of SEQ ID NO: 2, as long as the variant has an integrase activity. Thus, the claim encompasses the provision of a large genus of amino acid sequences that must function as an integrase.
Claims 7 and 21 requires the provision of a genus of functional variants or fragments thereof of SEQ ID NO 9. The claims encompass any number of mutations of any type, such as an insertion, deletion or substitution, relative to the sequence of SEQ ID NO: 9, as long as the variant has an integrase factor activity. Thus, the claim encompasses the provision of a large genus of amino acid sequences that must function as an integrase.
Claim 16 requires the provision of a genus of functional variants thereof of repeated lambda integrase recombination sequences consisting of attP and attB, attL and attB, attL and attL. The claim encompass any number of combinations, relative to attP, attB, attL, as long as the variant has a recombination sequence. Thus, the claim encompasses the provision of a large genus of combination of recombination sequences.
Claim 18 requires the provision of a genus of functional variants or fragments thereof of SEQ ID NO 2. The claims encompass any number of mutations of any type, such as an insertion, deletion or substitution, relative to the sequence of SEQ ID NO: 2, as long as the variant has an integrase activity. Thus, the claim encompasses the provision of a large genus of amino acid sequences that must function as an integrase.
Claim 25 requires the provision of a genus of functional variants or fragments thereof of SEQ ID NO 2. The claims encompass any number of mutations of any type, such as an insertion, deletion or substitution, relative to the sequence of SEQ ID NO: 2, as long as the variant has an integrase activity. Thus, the claim encompasses the provision of a large genus of amino acid sequences that must function as an integrase.
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 envisions a method for the in vivo production of seamless DNA vectors in E.coli, said seamless DNA vectors comprising a DNA sequence of interest and a phage lambda integrase recombination sequence, the method comprising: (i) providing an E. coli strain comprising a nucleotide sequence encoding a mutant phage lambda integrase (lntC3) having the amino acid sequence set forth in SEQ ID NO:2 or a functional variant or fragment thereof, wherein the expression of said nucleotide sequence is stringently controlled by an inducible expression control sequence; (ii) transforming a bacterial plasmid comprising a DNA sequence of interest and a bacterial backbone sequence flanked by two directly repeated lambda integrase recombination sequences that are recombination substrates for the mutant phage lambda integrase into the E.coli strain of (i), wherein the bacterial backbone sequence comprises a selection marker; (iii) cultivating the transformed E. coli cells under conditions selective for the selection marker comprised in the bacterial plasmid; (iv) inducing the expression of the mutant phage lambda integrase to facilitate recombination of the two directly repeated lambda integrase recombination sequences in the bacterial plasmid to obtain a dimeric DNA catenane consisting of a first circular DNA molecule that carries the bacterial backbone and a second circular DNA molecule that carries the DNA sequence of interest and a phage lambda integrase recombination sequence that is a hybrid of the two directly repeated lambda integrase
recombination sequences; and (v) isolating the second circular DNA molecule that carries the DNA sequence of interest and a phage lambda integrase recombination sequence (e.g., lines 21- , pages 2-3). The specification envisions the nucleotide sequence encoding a mutant phage lambda integrase having the amino acid sequence set forth in SEQ ID NO:2 or a functional variant or fragment thereof is stably integrated into the E. coli strains genome. The inducible expression control sequence may be the E. coli arabinose operon. In such embodiments, the induction in step (iv) may be triggered by the addition of arabinose. If the inducible expression control sequence is the E. coli arabinose operon, the nucleotide sequence encoding a mutant phage lambda integrase (lntC3) may be inserted into the genomic arabinose operon of E. coli. The insertion may occur immediately downstream of the arabinose promoter, for example by using the start codon of the endogenous araB gene as the start codon for the nucleotide sequence encoding a mutant phage lambda integrase. In various embodiments, the E. coli strain of (i) further comprises a nucleotide sequence encoding for single chain integration host factor 2 (sclHF2). sclHF2 may have the amino acid sequence set forth in SEQ ID NO:9 or a functional variant or fragment thereof (e.g., line12, page 3). The specification envisions lntC3 and sclHF2 are comprised in an expression cassette that is stably integrated into the genome of the E. coli strain. In such embodiments, the expression of both, lntC3 and sclHF2, may be stringently controlled by the same inducible expression control sequence, for example the endogenous arabinose operon. The expression cassette may comprise further elements, for example a selection marker, optionally flanked by recombination sites for later excision (e.g., line 28, page 3). The specification envisions the second circular DNA construct comprising the DNA sequence of interest, i.e. the seamless DNA vector, does not contain bacterial sequences, with the exception of the phage lambda integrase recombination sequence. Said phage lambda integrase recombination sequence is an individual recombination sequence that is generated as a hybrid from the two recombination sites present in the bacterial plasmid as a result of the recombination (e.g., line 1, page 4). The specification envisions the two directly repeated lambda integrase recombination sequences that are recombination substrates for the mutant phage lambda integrase are selected from the group consisting of attP (SEQ ID NO:11) and attB (SEQ ID NO:12), attl (SEQ ID NO:13) and attB (SEQ ID NO:12), attl (SEQ ID NO:13) and attl (SEQ ID NO:13), as well as functional variants thereof. These functional variants are recombination competent. Functional variants of att sites may consist, without limitation, of pairs of attH4x (SEQ ID NO:14) and attP4x (SEQ ID NO:15), attl4x (SEQ ID NO:16) and attH4x (SEQID NO:14), attR4x (SEQ ID NO:17) and attH4x (SEQ ID NO:14), and attl4x (SEQ ID NO:16) and attR4x (SEQ ID NO:17) (e.g., line8, page 4). The specification envisions that the methods described herein comprise the step of providing an E coli strain comprising a nucleotide sequence encoding a mutant phage lambda integrase (lntC3) having the amino acid sequence set forth in SEQ ID NO:2 or a functional variant or fragment thereof, wherein the expression of said nucleotide sequence is stringently controlled by an inducible expression control sequence. The term "phage lambda integrase" as used herein refers to any phage lambda-derived integrases that possess endonuclease and ligase activities. As known in the art, the phage lambda integrase like Cre and Flp belongs to the integrase family of the sequence-specific conservative DNA recombinases and catalyses the integrative recombination between two different recombination att sites. The term "functional variant", as used herein in relation to the integrase, relates to integrases that differ from the amino acid sequence set forth in SEQ ID NO:2 by one or more amino acid substitutions, additions or deletions but retain the functionality of the reference sequence. In such variants the amino acid positions that define the reference integrase C3, namely the positions 43F, 319G, and 336V may be invariable. The term also encompasses variants that comprise the sequence set forth in SEQ ID 20 NO:2 but comprise N- and/or C-terminal extensions of 1 or more amino acids. Generally, the term "variant" covers such integrases that have at least 90% sequence identity with the sequence set forth in SEQ ID NO:2 over their entire length, preferably at least 91, 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity. In these variants, the positions 43F, 319G, and 336V may still be invariable. The identity of nucleic acid sequences or amino acid sequences is generally determined by means of a sequence comparison (e.g., lines 4-25, page 8). The specification envisions the term "functional fragment" or "fragment", as used herein in relation to the integrase, relates to integrases that differ from the amino acid sequence set forth in SEQ ID NO:2 by a deletion of one or more amino acids from its C- and/or N-terminus. Said fragments preferably retain full functionality. In various embodiments, such fragment differs from the reference sequence and that they lack 1-20 amino acids from their N- and/or C-terminus, for example 1-15 amino acids or 1-10 amino acids or 1-5 amino acids (e.g., line 34, page 8). The specification envisions that the mutant integrases disclosed herein are, in contrast to the wild-type integrase, able to perform the recombination reaction without a co-factor, such as IHF. However, as described below, the addition of the co-factor gene, in particular of sclHF2 (SEQ ID NO:9), can have beneficial effects in that it seems to substantially reduces the lag phase in cultivation and thus can shorten incubation times needed to reach the desired cell density (e.g., line 1, page 9).
The working examples discloses the engineering of Escherichia coli (E. coli) strain MG1 655 carrying an inducible lambda integrase C3 with or without sclHF2 (e.g., lines 5-33, page 14). The working examples disclose the seamless vector production using engineered E. coli strain MG1655-ISC, using recombination substrate variants attL /attB and attB /attP (e.g., Examples 3-4).
The examples described in the specification does not meet the limitation of the rejected claims containing a functional variant or fragment thereof of SEQ ID NO 2, SEQ ID NO 9 and functional variants of attP, attB, attL. The specification only provides data for SEQ ID NO 2, SEQ ID NO 9 and variants attL /attB and attB /attP. This is not representative of the very large variations allowed by the claims, there is insufficient guidance provided indicating any of the elements that are critical to the functioning of the IntC3 variants or the scIHF2 variants, thus it cannot be determined which amino acid can be changed without disrupting the function of the IntC3 variant or fragment thereof; thus, further experimentation would be required to determine which variants of e.g., SEQ ID NO: 2 and SEQ ID NO 9 are functional and which are not. There is insufficient guidance provided indicating any of the elements that are critical to the functioning of the functional variants of attP, attB, attL.
The state of the art with respect to using IntC3 integrase functional variants is under developed and unpredictable. Lee et al (PNAS, 2004) teaches that a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates (e.g., abstract; Figs 2-3). Kazmierczak et al. (Nucleic Acid Res., 2002) teaches that Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100‐fold reduced recombination activity. Alanine scanning mutagenesis of the C‐terminus indicates that two mutants, W350A and I353A, cannot perform site‐specific recombination although their DNA binding, cleavage and ligation activities are at wild‐type levels. Two other mutants, R346A and R348A, are deficient solely in the ability to cleave DNA (e.g., abstract; Table 2). Lorbach et al. ( J. Mol. Biol., 2000) teaches that while wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination; two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are proficient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells (e.g., abstract; Fig. 4). Bao et al (Gene, 2004) teaches that shortened either one or both linkers by one amino acid in scIHF2, thereby generating three new variants termed scIHF1, 3, and 4. These variants exhibit distinct DNA-binding properties, different phenotypes in site-specific integrative and excisive recombination by phage λ integrase in vitro, as well as in pSC101 replication assays in a DIHF E. coli host. K45E substitution within α domain of scIHF3 and based on electrophoretic mobility shift assays (EMSAs), argue that it significantly changes the DNA trajectory within the protein–DNA complex (e.g., abstract). Furthermore, while all four newly described scIHFs, like scIHF2, promote excisive recombination in vitro, both scIHF1 and scIHF3-K45aE appear unable to trigger integrative recombination to a significant extent in vitro. A critical factor for this restricted functionality may be the significantly increased Kd for all three E cognate sites at 100 mM salt (Table 1). A recombinogenic integrative intasome with attP may not be stably formed under these conditions to allow synaptic complex formation with the protein-free attB (e.g., 2nd, paragraph, left column, page 7; Fig. 5). Bao et al (J. Mol. Biol, 2007) teaches that scIHF2-K45αE in in vitro recombination assays using protein-to-DNA stoichiometries that, with each variant of IHF tested, lead to at least 80% complex formation on individual IHF target sites. Finding that scIHF2-K45αE is marginally active as a co-factor in intramolecular integrative recombination on supercoiled pλIR (Figure 2(a), lane 3), but it triggers excisive recombination on supercoiled pλER that contains attL and attR instead of attB and attP, respectively, to an extent comparable with either scIHF2 or wild-type IHF (e.g., paragraph 2nd, right column, page 2; Fig. 2a).
As such, the prior art teaches about the unpredictability of the use of Int integrase, scHIHF2, and recombination sequences attP, attB, attL.
The 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 1-2, 4-7, 9-10, 12-13, 15-21, 23-25.
The claims listed in the statement of rejection but not otherwise discussed are rejected because they are similarly not limited to particular amino acids that are considered to be adequately described by the specification.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-2, 4-5, 12-13, 15-16, 18, 25 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kay et al. (“Kay”, Nature Biotechnology, 2010, cited as reference 13 on IDS filed 12/07/2023).
Under the broadest reasonable interpretation, the scope of the claim is interpreted as not to be limited to the SEQ ID NO 2. The claim requires ”a functional variant or fragment thereof” that is selected from SEQ ID NO 2, “which reads on selection of “a sequence from the recited sequence, where “a functional variant or a fragment” reasonably reads on variants that differ by one or more amino acid substitutions, additions or deletions but retain the functionality or a protein of two or more consecutive amino acids. See page 8, lines 15-17, of the specification.
Regarding claims 1, 4-5, Kay teaches a method for the in vivo production of minicircle DNA vectors in E. coli, comprising a human factor IX with ApoE promoter/enhancer) (It reads on DNA sequence of interest) and a phage integrase recombination ΦC31 integrase (e.g., abstract; Fig. 1b). Kay teaches (i) relocation of genes encoding ΦC31 integrase and I-SceI from the small amount of parental plasmids used to generate minicircle DNAs, from the minicircle producer plasmid to the bacterial genome, both genes under the control of L-arabinose-inducible araC.BAD (e.g., paragraph 4th, right column, page 1287; paragraph bridging pages 1287-1288; Fig. 1b; Supplementary Fig. 1); (ii) transforming into the E. coli BW27783 strain a bacterial plasmid comprising the ApoE,hFIX sequence flanked by two directly repeated integrase recombination sequences attP and attB, and wherein the bacterial backbone sequence comprises a Kanamycin resistance selection marker (e.g., Fig. 1b); (iii) cultivating the transformed E. coli cells under conditions selective for the selection marker (e.g., Fig. 1b [iii]); (iv) inducing the expression of the ΦC31 integrase in presence of L-arabinose to facilitate recombination to obtain a minicircle DNA carrying hFIX gene (e.g. Fig. 1b [iii]); and (v) isolating the second minicircular DNA molecule by plasmid purification column (e.g., Fig. 1b [iii]).
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Regarding claim 2, Kay teaches that upon L-arabinose induction, ΦC31 integrase is expressed to mediate the formation of minicircle and plasmid backbone circle and the homing endonuclease I-SceI to induce the destruction of plasmid backbone circle (e.g., Fig. 1b).
Regarding claim 12, Kay teaches upon L-arabinose induction, ΦC31 is expressed to mediate the formation of minicircle and plasmid backbone circle and I-SceI (a homing endonuclease) to induce the destruction of plasmid backbone circle. Kay teaches the isolation of the minicircle comprising the hFIX gene (e.g., Fig. 1b).
Regarding claim 13, Kay teaches pMC.ApoE.hFIX, minicircle producer plasmid; hFIX, human factor IX; sApoE, promoter/enhancer (e.g., Fig. 1b).
Regarding claim 15, Kay teaches that the minicircle DNA comprising ApoE.hFIX and the phage lambda integrase recombination site attR (e.g., Fig. 1b).
Regarding claim 16, Kay teaches the attB and attP, the bacterial and phage attachment sites of the ΦC31 integrase (e.g., Fig. 1b).
Regarding claims 18, 25, Kay teaches relocation of genes encoding ΦC31 integrase and I-SceI from the small amount of parental plasmids used to generate minicircle DNAs, from the minicircle producer plasmid to the bacterial genome, both genes under the control of L-arabinose-inducible araC.BAD (e.g., paragraph 4th, right column, page 1287; Fig. 1b; Supplementary Fig. 1).
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 7, 9-10, 19, 21, 23 are rejected under 35 U.S.C. 103 as being unpatentable over Kay et al. (“Kay”, Nature Biotechnology, 2010, cited as reference 13 on IDS filed 12/07/2023) as applied to claims 1-2, 4-5, 12-13, 15-16, 18, 25 above, and further in view of Makhija et al. (“Makhija”, US 2017/0362606 A1, cited as reference 1 on IDS filed 12/07/2023).
The teachings of Kay et al. are described above and applied as before.
Kay does not teach the phage lambda integrase IntC3 and the single chain integration host factor 2, as required by the instant claims. However, this is cured by Makhija.
Makhija teaches a method of generating a circular DNA construct essentially consisting of a DNA sequence of interest (e.g., paragraph 0024). Makhija teaches a kit for use in the presently disclosed method of stably integrating a DNA sequence of interest into a target genomic DNA sequence of a host cell, wherein the kit comprises any one or more components selected from the group consisting of (a) a bacterial plasmid comprising the DNA sequence of interest of step (i), (b) a phage lambda integrase mediating the intramolecular recombination of step (ii), (c) an endonuclease linearizing the second circular DNA construct of step (iii), ( d) a means for isolating the first circular DNA construct of step (iv), (e) a means for introducing the first circular DNA construct into the host cell of step (v), (f) a phage lambda integrase mediating the genomic integration of step (vi) or an expression plasmid encoding the phage lambda integrase, and (g) a means for determining the genomic integration events of step (vi) (e.g., paragraph 0036). Makhija teaches the method may be performed in all types of cells in vitro, ex vivo, or in vivo. The host cell may be a eukaryotic cell, preferably a mammalian cell, more preferably a human cell. For example, the host cell may be a bacterial cell, a yeast cell, a plant cell, or a human cell (e.g., paragraph 0060). Makhija teaches the phage lambda integrase Int-C3 SEQ ID NO 8 with 99.7% identity to SEQ ID NO 2 of the instant claims (e.g., paragraph 0014).
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Makhija teaches that the intramolecular recombination of step (ii) is carried out in the presence of an IHF variant such as scIHF2 of SEQ ID NO 10 with 100% identity to SEQ ID NO 9 of the instant claims (e.g., paragraph 0017).
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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 substitute the ΦC31 integrase taught by Kay with the phage lambda integrase Int-C3 and scIHF2 taught by Makhija; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of obtaining a method for the in vivo production of minicircle DNA vectors carrying a DNA of interest in E. coli, using the phage lambda integrase IntC3 and the single chain integration factor under the control of the arabinose inducible system.
Claims 17, 24 are rejected under 35 U.S.C. 103 as being unpatentable over Kay et al. (“Kay”, Nature Biotechnology, 2010, cited as reference 13 on IDS filed 12/07/2023) in view of Freddolino et al. (“Freddolino”, WO 2019/213374 A1).
Kay does not teach E. coli MG1655, as required by the instant claims. However, this is cured by Freddolino.
Freddolino teaches compositions and methods for production of proteins in microbial systems. In particular, provided herein are compositions and methods for expressing exogenous genes from specific chromosomal locations of E. coli designed to yield very high expression levels by virtue of the chromosomal context of the integration (e.g., line 6, page 2). Freddolino teaches particular site specific recombination systems, including FLP/FLP recombination target (FRT), CRE/LOX, lambda-integrase (using attP/B recognition sites), lambda red mediated recombination (from phage lambda), CRISPR/CAS9, transposases and cpC3 l integrase (e.g., line 26, page 7). Freddolino teaches the E. coli strain MG1655 (e.g., line 26, page 11).
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 substitute the E. coli BW27783 strain taught by Kay with the E. coli strain MG1655 designed to yield very high expression levels by virtue of the chromosomal context of the integration taught by Freddolino; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of obtaining a method for the in vivo production of minicircle DNA vectors carrying a DNA of interest in E. coli strain MG1655, using the phage lambda integrase IntC3 and the single chain integration factor under the control of the arabinose inducible system.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to use E. coli MG1655 that allow genetic recombination and yield high levels of expression of genes of interest.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JULIO GOMEZ RODRIGUEZ whose telephone number is (571)270-0991. The examiner can normally be reached Monday - Friday 8:00 am - 5:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Jennifer Dunston can be reached at 5712722916. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/JULIO WASHINGTON GOMEZ RODRIGUEZ/Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
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