DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I, encompassing claims 1-36, and the species of integration into the 18S coding region in the reply filed on 02 January 2026 is acknowledged. The search and examination has been extended to the species of non-integrated fragments resulting in a circular plasmid. Claims 5 and 37-39 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-4 and 6-36 are pending and are being examined on the merits.
Priority
The application is a 371 PCT/ of EP2021/050173 filed 01/07/2021 that claims priority to application 62/958,088 filed 01/07/2020.
Information Disclosure Statement
The information disclosure statement filed 01/17/2023.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code in 00150 and 00151. 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.
The use of the term New England Biolabs, Biorad, Thermo Fisher Scientific, Surmodics, Lonza, FastDigest, to name a few, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term. A cursory review of the specification has revealed these trademarks or names. It would be remedial to check the specification for additional trademarks or names and amend all upon amendment.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 7 is objected to because of the following informalities: Claim 7 appears to be missing the work ‘wherein’ after the comma. Appropriate correction is required.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 33 and 35 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Niimi (Niimi et al. Methods in Molecular Biology, vol. 824 2011).
Niimi teaches that one of the main advantages of a L. tarentolae expression system is the mammalian-type posttranslational modification of target proteins [abstract]. Nimii teaches that in a L. tarentolae expression system, integration of the expression cassette into the chromosomal small subunit rRNA locus (ssu) enables the generation of large numbers of transcripts for constitutive expression. The expression cassette of pLEXSY-2 expression vectors for L. tarentolae is flanked by two fragments of the small subunit rRNA locus (5 ¢ and 3 ¢ ssu) for homologous recombination [pg. 309, para 2].
Claims 33 and 35 are product by process claims. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) MPEP 2113.
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.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 7-18, 23, 32-33 and 35 are rejected under 35 U.S.C. 103 as being unpatentable over Li (Li et al. 2007 Nature Medicine Vol. 4, No. 3, pg. 251-256) in view of Basile (Basile et al. Mol Biotechnol (2009) 43:273–278).
Regarding claims 1-2, 7-18 and 23, Li teaches a new cloning method, sequence and ligation–independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing [abstract]. Li teaches a ten-fragment assembly (regarding claim 23) in E.coli cells with nine PCR-generated fragments of sizes ranging between 275 and 980 bp with 40 bp overlaps (regarding claims 9-10 and 13-18) inserted into a 3.1 kb vector (regarding claims 7-8, 11-12) and demonstrated that the assembly of complex recombinant DNA assemblies can be achieved using in vitro homologous recombination. [pg. 254, col. 1 para 2 – col. 2, para 1; Fig. 4d]. Li teaches that this method allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology[abstract]. The PCR fragments as shown in Fig. 4d comprises a 5' homologous region and/or a 3' homologous region; wherein the 3' homologous region of the each of the two or more DNA fragments is homologous to a 5' homologous region of another one of the two or more DNA fragments. The nucleotide sequences outside of the homologous regions of the PCR fragments as shown in Fig. 4d are not homologous to each other; are not homologous to a sequence in the cell's genome; and/or have no homologies within the respective DNA fragment.
Li do not teach where the fragment assembly occurs in Leishmania tarentolae.
Regarding claims 1, 32-33 and 35, Basile teach that Leishmania tarentolae, has been suggested as candidate for heterologous genes expression because of its ability to produce mammalian like complex type N-glycosylation, easy genetic manipulation, straightforward adaptation to large-scale production, and minimal nutrition requirement [pg. 277, col. 1, para 2]. Basile teach that, therefore, Leishmania tarentolae is a promising host for large-scale recombinant protein production for both pharmaceutical purpose and structural studies [pg. 277, col. 1, para 2].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to substitute the cell of Li with the Leishmania tarentolae cell of Basile for the advantage of generating recombinant DNA that can be used to produce large-scale recombinant proteins.
Claims 1-4, 7-18, 21-35 are rejected under 35 U.S.C. 103 as being unpatentable over Shao (Shao et al. Nucleic Acids Research, 2009, Vol. 37, No. 2) in view of Basile (Basile et al. Mol Biotechnol (2009) 43:273–278).
Regarding claims 1-4, 12, and 23, Shao teaches a new method, DNA assembler, which allows the assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae [abstract]. Shao teaches that DNA assembler can rapidly assemble up to eight gene fragments (regarding claim 23), and three distinct functional biochemical pathways with sizes ranging from 9 to 19 kb (regarding claim 12), with high efficiencies (70–100%) either on a plasmid (i.e., not integrated and circular, regarding claim 7-8) or on a yeast chromosome (i.e., integrated, regarding claims 3-4) [abstract; Scheme 1; pg. 2, col. 1, para 2; Figs 1-3]. Shao teaches that the 5’-end of the first gene expression cassette is designed to overlap with a vector (or a helper fragment carrying a selection marker and a sequence sharing homology with a targeted locus of a chromosome for integration), while the 3’-end is designed to overlap with the second cassette [pg. 3, col. 2, para 3]. Shao teach that each successive cassette is designed to overlap with the two flanking ones, and the 3’-end of the last cassette overlaps with the vector (or the targeted locus of the chromosome for integration) [pg. 3, col. 2, para 3 – pg. 4, col. 1, para 1]. The fragments as shown in Scheme 1 and Figs. 1-3 comprises a 5' homologous region and/or a 3' homologous region; wherein the 5' homologous region of the each of the two or more DNA fragments is homologous to a 3' homologous region of another one of the two or more DNA fragments or the 3' homologous region of the each of the two or more DNA fragments is homologous to a 5' homologous region of another one of the two or more DNA fragments. The nucleotide sequences outside of the homologous regions of the PCR fragments as shown in Scheme 1 and Figs. 1-3 are not homologous to each other; are not homologous to a sequence in the cell's genome; and/or have no homologies within the respective DNA fragment.
Regarding claims 9-11, Shao teachings of the nucleic acid overlaps (i.e., homology regions) being 50, 125, and 270-430bp where the three-gene assembly comprises fragments of 3263 bp, 2477 bp, and 3879 bp is a teaching where the nucleotide sequence of the first or second fragment outside the homologous region is at least 1000-3000 or and all DNA fragments is at 5000 nucleotides to 10000 nucleotides in length [Table 1; Fig. 1].
Regarding claims 13-18, Shao teach that with both plasmid-based and chromosome based three-gene DNA assembly, high efficiencies (80–100%) were obtained for all three different lengths of overlaps (~50, ~125, and ~270–430 bp) [pr. 4, last paragraph; Table 1]
Regarding claim 21, Shao teach that the resulting multiple expression cassettes are co-transformed into S. cerevisiae with the linearized vector (or the helper fragment) through electroporation (i.e., transfection), which allows the entire pathway to be either assembled into a vector or integrated onto the chromosome [pg. 4, col. 1, para 1].
Regarding claim 22, Shao teach that to perform one-step integration targeting the δ sites on yeast chromosomes, DNA mixture of individual cassettes together with the fragment, δ1-hisG-ura3-hisG, digested from pdδUB with BamHI and XhoI, was used to transform S. cerevisiae [pg. 2, col. 2, para 2].
Regarding claim 24-25, Shao teachings of a one-step DNA assembly of 3, 5, and 8 genes and the PCR analysis of the genes, is a teaching where the nucleotide sequences of the two or more DNA fragments outside the homologous region are open reading frames (ORFs) encoding polypeptides and are devoid of homologous sequences to one another and within itself [Figs. 1-3].
Regarding claims 29-31, Shao used a comprehensive set of PCR comprising pairs of primers to confirm the correct assembly and integration 2-4 days after the cells were transformed with the fragments [pg. 4, col. 2]. Shao teach that for the three gene assembly, for example, assembly efficiency was 80-100% for both plasmid-based DNA assembly and chromosome based DNA assembly, thereby teaching wherein undesired crossing out and/or crossing over occurs in at most 0-20% [pg. 4, col. 2]. Shao teaches that only the strains carrying the whole D-xylose utilization pathway either assembled into the vector or integrated onto the chromosome were able to grow on D-xylose where they reached an OD600 of more than 4, thereby teaching 100% assembly efficiency (0% undesired crossing over) over at least 2 cell divisions [pg. 5, col. 1, para 2; Fig. 1c].
Regarding claim 34, Shao does not teach repeatedly recombinantly engineering the Leishmania cell. However, it would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to repeatedly recombinantly engineering the Leishmania cell for the advantage of continuous production of large-scale recombinant proteins.
Regarding claim 1, Shao do not teach where the fragment assembly occurs in Leishmania tarentolae.
Regarding claims 1, 32-33, and 35, Basile teach that Leishmania tarentolae, has been suggested as candidate for heterologous genes expression because of its ability to produce mammalian like complex type N-glycosylation, easy genetic manipulation, straightforward adaptation to large-scale production, and minimal nutrition requirement [pg. 277, col. 1, para 2]. Basile teach that, therefore, Leishmania tarentolae is a promising host for large-scale recombinant protein production for both pharmaceutical purpose and structural studies [pg. 277, col. 1, para 2].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to substitute the cell of Shao with the Leishmania tarentolae cell of Basile for the advantage of generating mammalian like recombinant DNA that can be used to produce large-scale recombinant proteins.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Shao (Shao et al. Nucleic Acids Research, 2009, Vol. 37, No. 2) in view of Basile (Basile et al. Mol Biotechnol (2009) 43:273–278), as applied to claims 1-4 and further in view of Niimi (Niimi et al. Methods in Molecular Biology, vol. 824 2011) and MiBlitz (MiBlitz et al. Molecular and Biochemical Parasitology 107 (2000) 251–261).
The teachings of Shao and Basile are discussed above as applied to claim 1-4 and similarly apply to claim 6.
Shao and Basile wherein the two or more DNA fragments are integrated at the start of the 18S coding region (Ssu-Poll).
Basile teach that small ribosomal subunit RNA gene is strongly transcribed by RNA polymerase I [Fig. 1].
Niimi teaches that one of the main advantages of a L. tarentolae expression system is the mammalian-type posttranslational modification of target proteins [abstract]. Nimii teaches that in a L. tarentolae expression system, integration of the expression cassette into the chromosomal small subunit rRNA locus (ssu) enables the generation of large numbers of transcripts for constitutive expression. The expression cassette of pLEXSY-2 expression vectors for L. tarentolae is flanked by two fragments of the small subunit rRNA locus (5 ¢ and 3 ¢ ssu) for homologous recombination [pg. 309, para 2]..
MiBlitz teach the construction of a DNA cassette for integration into a genomic small sub-unit rRNA locus of Leishmania mexicana by homologous recombination [abstract]. MiBlitz teaches that amplification of a 1.3 Kb fragment was only possible if integration of the DNA cassette into a 18S rRNA gene occurred [pg. 255, col. 2, last paragraph – pg. 256, col. 1, para 1; Fig. 1-4].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the method as taught and suggested by Shao and Basile so that the two or more DNA fragments are integrated at the start of the 18S coding region (Ssu-Poll) of the Leishmania tarentolae cell for the advantage of integration and gene amplification. One of ordinary skill would be motivated to make the modification since Basile Niimi and MiBlitz teaches that DNA integration can occur in the ssu-locus encoding the 18S rRNA of a Leishmania cell. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Shao Niimi, and MiBlitz teach using homologous recombination to insert a DNA fragment into a genome while Basile Niimi, and MiBlitz teach recombinantly engineering Leishmania cells.
Claims 19-20, 26-28 and 36 are rejected under 35 U.S.C. 103 as being unpatentable over Shao (Shao et al. Nucleic Acids Research, 2009, Vol. 37, No. 2) in view of Basile (Basile et al. Mol Biotechnol (2009) 43:273–278), as applied to claim 1 and 33 and further in view of Cheo (US 2004/0229229 A1).
The teachings of Shao and Basile are discussed above as applied to claim 1 and 33 and similarly apply to claims 19-20, 26-28 and 36.
Shao and Basile do not teach where the 5'/3’ homologous region of the first DNA fragment and the 3'/5’ homologous region of the second DNA fragment have at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. Shao and Basile do not teach expressing multiple copies of the same polypeptide where the expression level of the polypeptide increases. Shao and Basile do not teach kits comprising the components and cells for multiple fragment assembly.
Cheo teaches novel methods using such recombination sites to join or link multiple nucleic acid molecules or segments and more specifically to clone such multiple segments (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, seventy-five, one hundred, two hundred, etc.) into one or more vectors [0042]. Cheo teach methods for performing homologous recombination between nucleic acid molecules comprising (a) mixing at least a first nucleic acid molecule which comprises one or more recombination sites with at least one target nucleic acid molecule, wherein the first and target nucleic acid molecules have one or more homologous sequences; and (b) causing the first and target nucleic acid molecules to recombine by homologous recombination [0015, 0157].
Regarding claims 19-20, Cheo teach that the nucleic acid segments which facilitate homologous recombination will share at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) in their regions of homology [0130].
Regarding claims 26-28, Cheo teaches that the multiple nucleic acid segments are inserted into another nucleic acid molecules, such as a vector, where the segments may be the same or different and encode expression products [0162, 0189; Fig. 10, 20]. Cheo teach that when the nucleic acid segments encode expression products, vectors of the invention may be used to amplify the copy number or increase expression of encoded products, for example, by providing multiple binding sites for proteins which activate transcription or by the expression of multiple copies of nucleic acid molecules which encode factors involved in the regulation of transcription [0162].
Regarding claim 36, Chao teaches kits for joining, or replacing nucleic acid segments, these kits comprising at least one component selected from the group consisting of (1) one or more recombination proteins or compositions comprising one or more recombination proteins, and (2) at least one nucleic acid molecule comprising one or more recombination sites (preferably a vector having at least two different recombination specificities) [00195]. Choa teaches that the kit also comprise additional nucleic acid molecules comprising additional recombination sites, one or more host cells; and instructions for using the kit components [0195].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the cell in the method as taught and suggested by Shao and Basile such that the multiple DNA fragments share at least 70% sequence identity in their homology regions and encode the same polypeptide thereby increasing the expression level of the polypeptide. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Shao and Cheo teach using such recombination sites to join or link multiple nucleic acid molecules or segments, and Cheo teaches that increase expression of encoded products of the invention can be the result of expressing multiple copies of nucleic acid molecules. It also would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to assemble the components of the methods and cells into a kit for the purpose of carrying out the method and particularly for use in creating the product nucleic acid molecules of the
invention or other linked molecules and/or compounds.
Conclusion
No claims allowed.
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/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
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