DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendments to the claims and arguments filed on August 25,2025 have been received and entered. Claims 26 and 46 have been amended, while claims 1-25, 38-42 remain canceled. Claims 26-37, 43-48 are pending in the instant application.
Priority
This application is a continuation of US application no 17830011 filed on June 1, 2022 which is a continuation of US application no 17/590,613 filed on 02/01/2022, which is a continuation of 17/366,290 filed on 07/02/2021, which is a continuation of 16/800,444 filed on 02/25/2020, which is a continuation of 16/601,144 filed on 10/14/2019, which claims priority from US provisional 62/864,432 filed on 06/20/2019, US provisional 62/830,654 filed on 04/08/2019, US provisional of 62/746,497 filed on 10/16/2018.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 08/25/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Election/Restrictions
Applicant’s election of claims 33-37, 45-48 in the reply filed on January 26, 2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)). Applicant’s election of stem cell as species of cells for claim is acknowledged. However, upon further consideration election of species requirement between different species of cell was withdrawn.
Claims 26-37, 43-48 are under consideration.
Withdrawn-Claim Rejections - 35 USC § 101
Claims 46-48 are rejected under 35 U.S.C. 101 and section 33(a) of the America Invents Act as being directed to or encompassing a human organism. Applicant’s argument is found persuasive, therefore, previous rejection of claims 46-48 are hereby withdrawn Applicants’ arguments with respect to the withdrawn rejections are thereby rendered moot. .
Maintained- Claim Rejections - 35 USC § 112-scope of enablement
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 26-37, 43-45 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for
An in vitro method of providing a mammalian cell comprising an exogenous nucleic acid molecule comprising a transgene integrated to an endogenous gene in a mammalian cell, the method comprising:
introducing into an isolated mammalian cell in vitro
(a) a CRISPR-Cas nuclease that cleaves a target nucleic acid sequence in an intron of the endogenous gene to provide a cleavage site in the intron;
(b) a guide RNA (gRNA) encoding the target nucleic acid sequence in the intron of the endogenous gene; and
(c) an exogenous transgene comprising from 5’ to 3’ a first homology arm, a first splice acceptor sequence; a first coding sequence; a first terminator sequence; a second terminator reverse complement; a second coding sequence reverse complement; a second splice acceptor reverse complement, and a second homology arm; wherein the exogenous transgene is operably linked to the promoter of the endogenous gene
wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, and the second coding sequence is operably linked to the second splice acceptor and second terminator; and wherein
the first homology arm comprises a sequence homologous to a first splice acceptor site in the intron of the endogenous gene and is upstream of the cleavage site;
the second homology arm comprises a sequence homologous to the intron of the endogenous gene that is downstream of the cleavage site; and
wherein the transgene integrates into the cleavage site in the intron of the endogenous gene of the isolated mammalian cell.
The specification does not reasonably provide enablement for the following: providing a in vivo or in utero cell comprising an exogenous nucleic acid molecule; 2) introducing the nucleic acid by any other mean to provide a cell with said exogenous nucleic acid molecule; or a transgene that does not comprise all the component list above. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
In determining whether Applicant’s claims are enabled, it must be found that one of skill in the art at the time of invention by applicant would not have had to perform “undue experimentation” to make and/or use the invention claimed. Such a determination is not a simple factual consideration, but is a conclusion reached by weighing at least eight factors as set forth in In re Wands, 858 F.2d at 737, 8 USPQ 1400, 2d at 1404. Such factors are: (1) The breadth of the claims; (2) The nature of the invention; (3) The state of the art; (4) The level of one of ordinary skill in the art; (5) The level of predictability in the art; (6) The amount of direction and guidance provided by Applicant; (7) The existence of working examples; and (8) The quantity of experimentation needed to make and/or use the invention.
The office has analyzed the specification in direct accordance to the factors outlines in In re Wands. MPEP 2164.04 states: “[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection.” These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform “undue experimentation” to make and/or use the invention and therefore, applicant’s claims are not enabled.
Nature of the Invention
The claims are directed to method for providing a cell with an exogenous nucleic acid molecule comprising a transgene, wherein said transgene comprises, from 5' to 3' orientation:(a) a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement, wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, wherein the second coding sequence is operably linked to the second splice acceptor and second terminator, wherein said method comprises introducing a nucleic acid comprising said transgene into said cell, thereby providing said cell with said exogenous nucleic acid molecule. The dependent claims limit, wherein said introducing step comprises lipid-mediated transfer, electroporation, direct injection or calcium phosphate co-precipitation of said nucleic acid into said cell. Claims further limits the introducing step comprises viral vector-mediated transfer of said nucleic acid into said cell. Claim 43 limits the cells to a K562 cell, a CHO cell, an HEP-G2 cell, a BaF-3 cell, a COS cell, a CV-1 cell, an HuTu80 cell, an NTERA2 cell, an NB4 cell, an HL-60 cell, a HeLa cell, or an HEK293 cell. Claim 45 limits the cells are an induced pluripotent stem cell, a mesenchymal stem cell, an hematopoietic stem cell, a liver stem cell, a skin stem cell, or a neuronal stem cell. Claims are also directed to a cell comprising an exogenous nucleic acid, wherein said cell is in culture and wherein said exogenous nucleic acid molecule comprises a transgene comprising, from 5' to 3' orientation:(a) a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement, wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, wherein the second coding sequence is operably linked to the second splice acceptor and second terminator.
Breadth of the claims
The claims broadly embrace providing an in vitro, in vivo or in utero method of providing any cell with an exogenous nucleic acid molecule comprising a transgene. The phrase transgene is defined in the specification as a sequence of nucleic acids that can be transferred to an organism or cell. The transgene may comprise a gene or sequence of nucleic acids not normally present in the target organism or cell (see para. 40). The claims recite “introducing” a nucleic acid comprising said transgene into any cell, which suggests that the ultimate intended use is for providing a cell with exogenous nucleic acid molecule is prepare or provide in vivo, in utero or ex-vivo genetically modified cells with the claimed transgene integrated to any site in the genome. The claims recite a transgene that is integrated into a target site. This specification provides guidance of using a CRISPR-Cas gene editing system. However, the claims do not recite the administration of a gRNA and/or CRISPR /Cas to introduce targeted insertion of transgene into the cells to provide a cell comprising an exogenous nucleic acid at any specific site. The claims also recite a transgene that is intended to introduce or fix mutations in the 5’ region of a gene of interest or a specific exon that uses any first and second splice donor sequence located anywhere in the transgene, a first and second coding sequence operably linked to a first and second promoter, respectively, located anywhere in the transgene. All actual constructs of present application comprise the following elements in the following 5’ to 3’ order (cf. Examples 1-5 and Fig. 1-7) SA 1-CDS1 -T1 -T2-CDS2-SA2 or SA 1-CDS1 -BT-CDS2-SA2. It is relevant to note that at the 5’ end of SA 1 and the 3' end of SA2 there are always target sites (TS), i.e. a nucleic acid to which a rare-cutting endonuclease or CRISPR-associated transposase will bind (page 15, line 9-11), or homology arms (HA) (page 18, lines. 6-13) to be found (cf. Fig. 1-7). The guidance provided in the specification requires each of said elements and the order of said elements in specific configuration that are essential for integration of gene. The claims as such requires only requires providing a cell comprising the exogenous nucleic acid molecule with no functional requirement or any level of expression. The disclosure provided by the applicant, in view of prior art, must encompass a wide area of knowledge to a reasonably comprehensive extent. In other word each of these, aspect must be shown to a reasonable extent so that one of the ordinary skills in the art would be able to practice the invention without any undue burden being on such Artisan.
Guidance of the Specification and the Existence of Working Examples
The specification discloses novel approaches for correcting mutations found at the 5′ end of genes. The method is based in part on the design of bimodule, bidirectional transgenes compatible with integration through multiple repair pathways. The transgenes described herein can be integrated into genes by the homologous recombination pathway, the non-homologous end joining pathway, or both the homologous recombination and non-homologous end joining pathway, or through transposition. Further, the outcome of integration in any case (HR, NHEJ forward, NHEJ reverse; transposition forward, or transposition reverse) can result in precise correction/alteration of the target gene's protein product. The transgenes described herein can be used to fix or introduce mutations in the 5′ region of genes-of-interest. The methods are particularly useful in cases where precise editing of genes is necessary, or where the mutated endogenous gene being targeted cannot be ‘replaced’ by a synthetic copy because it exceeds the size capacity of standard vectors or viral vectors. The methods described herein can be used for applied research (e.g., gene therapy) or basic research (e.g., creation of animal models, or understanding gene function) (see para. 7 of the specification. The specification discloses a method for integrating a transgene into an endogenous gene. The method can include delivery of a transgene, where the transgene harbors a first and second splice donor sequence, a first and second coding sequence, and one bidirectional promoter or a first and second promoter (FIG. 1, see below). In another aspect, the transgene can also include a first and second terminator. In some embodiments, the first and second terminators can be replaced with a single bidirectional terminator.
PNG
media_image1.png
795
531
media_image1.png
Greyscale
The method further includes administering a rare-cutting endonuclease targeted to a site within the endogenous gene. The result of the method is that the transgene is integrated with the endogenous gene, and regardless of the orientations (e.g., forward or reverse) the integration will result in a precise modification of the amino acid sequence of the protein produced from the endogenous gene (FIGS. 3 and 4).
PNG
media_image2.png
218
835
media_image2.png
Greyscale
The method can include the use of any suitable rare-cutting endonuclease, including CRISPR, TAL effector nuclease, zinc-finger nuclease, or mega nuclease. The specification contemplates a method that can be used to alter the 3' end of the endogenous ATXN3 gene or CACNA1A gene. In specific embodiments, the target for integration of the transgenes described herein can be intron 9 of the ATXN3 gene or intron 46 of the CACNA1A gene (see para. 9). Applicant example disclose designing transgene to be inserted within intron 9 or the junction of intron 9 and exon 10 of the ATXN3 gene and all transgenes were designed to insert at least one splice acceptor and at least one functional coding sequence for exons 10 and 11 of the ATXN3 gene. The first plasmid, designated pBA1135, comprised a left and right homology arm with sequence homologous to the 3' end of intron 9 and 5' end of intron 10 (i.e., successful gene targeting would result in removal of exon 10 and replacement with the cargo sequence within pBA1135). Between the homology arms, from 5' to 3', was a splice acceptor (splice acceptor from ATXN3 intron 9), coding sequence for exons 10 and 11 of ATXN3, SV40 terminator, reverse BGH terminator, reverse coding sequence for exons 10 and 11 (codon adjusted), and reverse splice acceptor. The results show that the described transgenes comprising bidirectional partial coding sequences can be integrated into genomic DNA through multiple different repair pathways (see example 1). It is noted that example 2 describes transfecting HEK293 cells with each of the plasmid constructs and combinations thereof using lipofectamine. Two days post transfection, DNA is extracted and assessed for mutations and targeted insertions within the CACNA1A gene. Nuclease activity is analyzed using the Cel-I assay or by deep sequencing of amplicons comprising the CRISPR/Cas12a target sequence. Successful integration of the transgene is analyzed using PCR (FIG. 5) (example 2). Example 3 shows targeted integration of DNA in the ATXN3 gene in HEK293 cells (see fig. 7). Example 4 discloses targeted integration of DNA in the ATXN3 gene using Cas12k transposases in an HEK293 cells.
In view of foregoing teaching of the instant specification as discussed above, the specification generally contemplates in vitro or in vivo transgene integration of a nucleic acid molecule using a gene editing system. However, the specification does not provide specific guidance to such an in vivo method. The specification also describes specific guidance to the use of a CRISPR-Cas system for gene editing. The specification also provide description of what appears to be a novel transgene that allows for precise gene editing in of the exon of interest in an endogenous gene that potentially replaces the exon of interest with a desired sequence variant. The specification teaches that this approach is useful for gain of function mutations that need to be repaired or mutated. However, the claims do not recite all the elements that the specification describe as necessary for the function of the claimed transgene in the method of providing a cells comprising a nucleic acid molecule integrated into the genome of the cell as described by the specification, particularly homologous arms and the orientation of the components of the splice donor sites and coding sequences. Thus, the instant specification does not enable the breadth of the claims.
State of the Art and Predictability of the Art
Regarding the breadth of the claims encompassing in vitro, in utero or in vivo CRISPR-Cas gene delivery to a cell. The art teaches that, “CRISPR/Cas9 holds tremendous promise for the treatment of human disease…. However, these applications face technical hurdles as they are brought to patients, with challenges including safe and effective in vivo delivery of CRISPR/Cas9 components…Recent reports have shown some success in using LNPs to deliver CRISPR/Cas9 components, but editing efficiencies have fallen short of clinically relevant levels.” See p. 2227, col 1 paragraph 1 and col 2, second to last paragraph) (see Fin et al. Cell Reports 22:2227-2235, 2018, IDS). In view of foregoing, it is apparent that Fin discloses several obstacles to in vivo CRISPR-Cas gene editing that render this type of gene therapy unpredictable. Further, Fin teaches that there are issues of effectively delivery of the CRISPR system to a target cell and editing efficiency that have short of what is needed for clinical use or effective function of the introduced transgene that render this technology unpredictable. Guo (Cell Research, 25: 767-768, 2015, IDS) notes “mosaic mutations and off-target effects caused by CRISPR/Cas9 have led to concerns about the efficiency and specificity of this new technique in non-human primates and other large animals” (page 767, left column, para.1). Mosaic mutation may result from the prolonged expression of Cas mRNA, however, Cas9 protein also leads to mosaic mutations. Mosaic mutations may affect generation of animal models of genetic human diseases” (page 767, right column, last paragraph). Therefore, Guo describes functional deficiencies that hinder the specific target integration and mosaic mutations that can unpredictably alter transgene expression and potentially other non-intended endogenous genes.
Further, a CRISPR-Cas gene delivery, not only requires targeting specificity of the Cas nuclease but is also requires fidelity of the delivery to a cell in a specific organ in the body. The claims broadly recite “administer…a transgene…”. However, the claim only requires administering the nucleic acid molecule by any mean without any expression of any coding sequence. The specification fails to provide any guidance for such deliveries in vivo or in utero to a cell. The specification fails to describe any routes of administration to the body or to the cell that predictably arrive at the integration into a mammalian cell in vivo. Thus, the specification does not enable such an administration. The art at the time of the invention fails to provide any guidance to a means of delivering other than HR or a CRISPR/Cas system to a specific target site via administering in any way the transgene and Cas to effectively integrate that transgene into an endogenous gene of a mammalian cell. As such, the art fails to supplement the short comings of the specification. Since the specification only provides general guidance to an in vivo CRISPR-Cas gene editing for gene delivery method, the ordinary artisan would look to the specification of predictable guidance to do the claimed method in an in vivo manner. However, the art upon which the ordinary artisan would be rely, fails short of that predictable guidance and further implies that such in vivo method are unpredictable lacking a reasonable expectation of success. As such, the art at the time of the effective filing fails short of the requisite enabling guidance to supplement the shortcomings of the specification.
The art teaches a requirement for gRNA to impart the specificity of guidance of the Cas9 to the specific target site. Konstantakos (Nucleic Acid Research 50(7):3616-3637, 2020, IDS) states, “Compared with previous gene editing technologies, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which bind to DNA sequences by protein-DNA recognition and require substantial protein engineering, the CRISPR–Cas9 system requires only changing the guide sequence”. Page 33616, sentence bridging col 1 and 2. Thus, the success and specificity a CRISP-Cas arrival at a target site in fully dependent upon a gRNA encoding a target nucleic acid sequence to guide the Cas to the target site. As such, gRNA with specificity for a target site is requisite to the function of the instantly claimed invention. In the instant case, neither the specification nor the art provides a CRISPR-Cas gene editing gene delivery system that does not utilize a gRNA, neither the specification nor the art enables such embodiment. Further, since the specificity of Cas to cleave at a target site is imparted by a gRNA with a sequence encoding the sequence of that target site as described by the art above, neither the specification nor the art is enabling for the use of any gRNA with any specificity for any target sequence. Regarding a transgene that comprising any splice donor site, any coding sequence operably linked to promoters in any order or orientation: The prior art fails to elucidate a transgene comprising a first and second donor sequence and two coding sequences operably linked to two promoters wherein the first and second doing sequences differ in nucleic acid sequence but encode the same amino acid. As such, the teachings of the art fail to supplement the shortcomings of the specification.
The specification clearly states the intended use is for the fix of monogenic, gain of function mutation associated diseases. The specification also teaches that for this to occur the transgene must be introduced in the one particular orientation which requires homologous directed recombination and thus homologous arms flanking the transgene. Further, for the intended “fix” or replacement to occur the splice donor sequence need to be position flanking the two coding sequences operably linked to the promoter and the first one needs to be a reverse complemented splice donor site. As such, the breadth of the claimed transgene, while the ordinary artisan has the ability to make it as claimed, is not enabled for the recited intended uses described by the specification. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of cells, different rare-cutting endonuclease and modifications made at any targeted site within exons and/or intron of genus of endogenous gene recited in the claim are unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). An artisan of skill would have required undue experimentation to practice the method as Claimed at the time of filing of this application as supported by the observations in the art record.
The Amount of Experimentation Necessary
Since neither the specification nor the art provide enabling guidance to the make and use of the claimed in vivo or in utero cell comprising a transgene into any site including at an endogenous gene by administering the transgene and without integrating the transgene via HR (through the homology arms) or by NHEJ forward direction or NHEJ reverse direction (through direct integration of a viral or non-viral vector within a targeted double-strand break). CRISP-Cas-gRNA systems that have great specificity and fidelity would need to be achieved. The routes of administering the Cas and the transgene that predictably target the transgene, CRISPR-Cas, and gRNA to a target cell in the massive multitude of cells in the human, then means of getting all three factors successfully into the cell, means to effectively get all three factors into the nucleus, and means to specific target the Cas to a finite nucleic acid long sequence in a vast genome in an effective amount with enough fidelity to arrive at an effective less of integration into enough target cells. This level of discovery experimentation goes far beyond the use of routine optimization using the disclosed invention, but rather would require the development of new techniques, methods, and products. As such, the level of experimentation would be considered undue. An artisan would have to perform undue experimentation to make and use the invention, without reasonable expectation of success.
Response to arguments
As an initial matter, applicant’s argument pertaining to rejection of claims 45-48 is found persuasive, therefore, rejection of claims 45-48 is hereby withdrawn .
To the extent applicant’s argument pertains to introducing the transgene of the invention into any cell in vivo, instant rejection is maintained for the reasons of record.
Applicants disagree with the rejection arguing para. [0058] of Applicant’s specification teaches splice acceptors; para. [0059] teaches coding sequences; and para. [0060] teaches terminators. In addition, Applicant's specification fully describes and enables multiple ways for delivering transgenes and nucleases to cells. Applicants’ arguments have been fully considered, but are not found persuasive.
In response, it is noted that applicant’s argument and reliance that instant specification multiple ways of delivering transgene and nuclease in vivo or in utero is not persuasive because none of the claim require delivering nuclease. Thus, applicant’s argument is not commensurate with the scope of the claim.
Applicant continue to argue that instant specification teaches b “lipid-mediated transfer, electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer” at para. [0031]. For additional methods, see paras. [0080] and [0081]. Moreover, Applicant’s specification provides numerous examples of cells in culture and means to transfect such cells. Applicant provide a number of publications showing exemplary cells that include “oocytes, K562 cells, CHO (Chinese hamster ovary) cells, HEP-G2 cells, BaF-3 cells, Schneider cells, COS cells (monkey kidney cells expressing $SV40 T-antigen), CV-1 cells, HuTu80 cells, NTERAZ cells, NB4 cells, HL-60 cells and HeLa cells, 293 cells (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59), and myeloma cells like SP2 or NSO (see, e.g., Galfre and Milstein (1981) Meth. Enzymol. 73(B):3 46. Applicant conclude that a person of ordinary skill in the art reading Applicant's specification at the time of filing would have appreciated that the presently presented claims are fully enabled. Applicants’ arguments have been fully considered, but are not found persuasive.
In response, as discussed above in the body of the rejection, instant specification is not enabling for an in vivo method that require a high degree of specificity and targeting. In the instant case, none of the claims recite any elements or method steps that require nuclease, or targeting elements, that would specifically target the administered transgene and endonuclease to the target cells in vivo in need of these genetic modification. Applicant provided citations are all to an in vitro method, however, claims are not so limited. As discussed in the body of the rejection such targeting elements are required to overcome the unpredictability of delivering gene and coding sequences. As such, the breadth of the claims as is lacking such requirement for such targeting and delivery structures lacks enablement. Further, in a post filing publication, Fin et al teach an optimized guide sequence, a specific sgRNA chemical modification pattern, and a biodegradable ionizable lipid, these LNPs meet all of the criteria essential for CRISPR/Cas9 delivery system. The art of record further teaches specificity, off-target, and mosaic mutation issues that render CRISPR and use of another nuclease unpredictable. In view of foregoing, the breadth of the claims continues to lack predictable enabling guidance and amendment to the claims and the argument are not sufficient to overcome the rejection of record.
Withdrawn-Claim Rejections - 35 USC § 103
Claims 26-37, 43-48 are rejected under 35 U.S.C. 103 as being unpatentable over Jaskula-Ranga et al (WO/2018/009534, dated 01/11/2018, EFD07/5/2016, IDS), Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018) and Harrington et al (US6740503), Sheng et al (Canadian Journal of Microbiology, 2014, 445-454, IDS) /Ryu et al (Plant Molecular Biology 54: 489-502, 2004, IDS)/ Jarvis (US 2020/0231974, 7/23/2020, IDS) . In view of Applicants’ amendment of base claim 1, introducing the limitation “wherein the first coding sequence encodes an amino acid sequence, and the second coding sequence encodes the same amino acid sequence as the first coding sequence”, that is not taught by the combination of references., the previous rejection is rendered moot and hereby withdrawn. The claims are however subject to new rejections over the prior art of record, as set forth below.
New-Claim Rejections - 35 USC § 103- necessitated by amendments
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 26-27, 29, 32-37, 43-48 are rejected under 35 U.S.C. 103 as being unpatentable over Jaskula-Ranga et al (WO/2018/009534, dated 01/11/2018, EFD07/5/2016, IDS), Sheng et al (Canadian Journal of Microbiology, 2014, 445-454, IDS), Vogl (Nature Communication, 2018, 3589, 1-13), Harrington et al (US6740503), and Terai et al (Bioinformatics, 2017, 33(11), 1613–1620).
Instant rejection is applied to the breadth of the claims and to the extent claims require an in vitro method of integrating a transgene into an endogenous gene. There is requirement of expression of any coding sequence.
With respect to claims 26, 32, 34, Jaskula-Ranga teaches a method of providing a cell having altered gene expression (claim 122, page 57 line 9-25), said method comprising introducing into the cell a construct comprising two coding sequences, a bidirectional promoter and an endonuclease (Claims 1, 15, page 3 line 25-31, page 4, line 15-26, page 5 line. 7, page 27 lines 8-20, page 40 line. 32-pahe 41 line. 8, example 6). It is further disclosed that the construct may comprises at least one that includes 2 terminator sequences (see claim 55-56). Jaskula-Ranga teaches that nuclease is a CRISPR and wherein the genome-targeted nuclease is Cas9 protein (see claim 66, page 8 line 22, and page 41 line 26-27). Jaskula-Ranga shows potential configurations for HDR delivery and targeting within an intronic region (see figure 24). Jaskula-Ranga teaches that nuclease system may include CRISPR associated (Cas) 9 CRISPR-Cpf-1 (Cas12a) (see page 73, lines 13, page 29, line 20).
Regarding claim 27, 29, Jaskula-Ranga teaches introducing the nucleic acid molecule by lipofection or using lipid-mediated transfer or injection of said nucleic acid into said cell (see page 64, lines 3-15 and claims in ‘534).
Regarding claim 32, Jaskula-Ranga teaches transgene is harbored on a viral vector selected from AAV or lentivirus vector (Claim 3, page 4 lines. 27-28, page 27 lines. 20-21, page. 43 line 25). It is further disclosed that wild type AAV is -4.7 kb and recombinant AAV can be stuffed up to 5.2kb., Jaskula-Ranga further teaches that transgene may also be harbored on a non-viral vector (see page 63, lines 25-28, page 64, lines 3-5).
With respect to claims 33-37, 48, Jaskula-Ranga teaches that the coding sequence
epitope tags include histidine (His) tags, Myc tags, reporter protein including luciferase, green
fluorescent protein (GFP) (see page 52, lines 27-page 53).
Regarding claim 43, 46-47 Jaskula-Ranga teaches that cell may be a CHO cells (see page 66, line 21).
While Jaskula-Ranga teaches introducing into the cell a construct comprising at least one coding sequences (encompass 2 coding sequence), a bidirectional promoter and an endonuclease may comprises at least one that includes 2 terminator sequence (Claims 1, 15, page 3 line 25-31, page 4 , line 15-26, page 5 line. 7, page 27 lines 8-20, page 40 line. 32-pahe 41 line. 8, example 6, claims 55-56) but differ from claimed invention by not disclosing a transgene comprising two (specie acceptor-coding sequence-terminator) cassettes arranged in tail-to-tail orientation.
Before the effective filing date of instant application, The deficiency is cured by Sheng who teaches a method of introducing and integrating a into cells and expressing the polypeptide (GFP) in cells, said method comprising a constructs comprising two coding sequences encoding green fluorescent protein (figure 2). Sheng et al teaches a first coding sequence, a first terminator sequence, a second terminator reverse complement a second coding sequence of GFP reverse complement that is located 3' of the first, is highly complementary to the first (figure 2 page 446, col. 2 to page 447, col. 1) (limitation of claims 33-34).
PNG
media_image3.png
285
1343
media_image3.png
Greyscale
Sheng differs from claimed invention by not disclosing that GFP sequence is operably linked to a promoter and GFP GFP reverse complement sequence is operably linked to reverse complement promoter.
Vogl teaches use of bidirectional promoter on the opposite strand showing these promoters could independently drive expression of the coding sequence (see fig.4 in Vogl), while Harrington provided guidance with respect to use of splice acceptor sequence at 5' end of exon and/or 3’ end of intron boundary that could be selected by a person of ordinary skill in the art. Harrington teaches that a splice site is useful as each transcriptional regulatory sequence is operably linked to a separate splice site, and the transcriptional regulatory sequence/splice pairs may be in inverse orientation relative to each other (i.e., the first transcriptional regulatory sequence may be integrated into the host cell genome in an orientation that is inverse relative to the orientation in which the second transcriptional regulatory sequence has integrated into the host cell genome). The two opposing transcriptional regulatory sequence/splice sites can be separated (see col. 4 lines 24-36, col. 46 lines 28-39, 56-63, col. 48 lines. 34-43, col. 51 lines 30-37, col. 60 line57-64, col. 61 lines 8-16, col. 61 lines. 34-36, Fig. 11). The combination of reference differs from claimed invention by not explicitly disclosing that the first and second coding sequence is different but encodes the same protein.
Terai cures the deficiency by disclosing a method, which is based on multi-objective genetic algorithm, is intended to design a set of CDSs whose nucleotide sequences are as different as possible and whose codon usage frequencies are as highly adapted as possible to the host organism. We show that our method not only successfully designs a set of intended CDSs, but also provides insight into the trade-off between nucleotide differences among gene copies and codon usage frequencies.
It would have been prima facie obvious for a person of ordinary skill to combine the teachings of prior art to modify the method of Jaskula-Ranga of integrating a transgene into an endogenous gene for expression of by introducing at a target site within an intron of an endogenous gene of a cell by using a bidirectional construct containing first coding sequence for GFP and first terminator and a second terminator reverse complement and a second coding sequence for second coding sequence (GFP) reverse complement that are positioned 20in opposite orientation or a construct with bi-directional terminator as suggested in Sheng, before the effective filing date of the instant invention. Said modification amounting to combining prior art elements according to known methods to yield predictable results. The limitation of using splice acceptor sequence upstream of two coding sequence would be obvious to a person of ordinary skill such that it could be positioned for divergent transcription to function as evident from the teaching of Harrington. One of ordinary skill in the art would be motivated to do so because this would allow the construct to integrate in (i) both forward and reverse direction and would enable simultaneous independent and stable expression of the same protein and (ii) bidirectional terminator sequence would prevent interference between two transcription units. Absent any requirement of any specific level of expression or any unexpected advantage of using construct in a tail to tail configuration, one of skill in the art would have been expected to have a reasonable expectation of success particularly since prior art successfully reported integrating a transgene into an endogenous gene by administering (i) a transgene comprising 2 splice acceptor sequence, 5222 bidirectional construct; (ii) two coding sequences encoding the same polypeptide as in Sheng in view of Terai, wherein the second sequence is located 3' of the first, and comprises splicing acceptor sites upstream of the first and second coding sequence as in Sheng in view of Harrington. It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d--, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007, (citing KSR, 82 USPQ2d at 1396, http: www uspto.gov/web/offices/dcom/bpai/prec/fd071925. pdf).
Claims 26-32, 43-48 are rejected under 35 U.S.C. 103 as being unpatentable over Jaskula-Ranga et al (WO/2018/009534, dated 01/11/2018, EFD07/5/2016, IDS), Sheng et al (Canadian Journal of Microbiology, 2014, 445-454, IDS), Vogl (Nature Communication, 2018, 3589, 1-13), Harrington et al (US6740503), and Terai et al (Bioinformatics, 2017, 33(11), 1613–1620) as applied above and further in view of Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018).
The teaching of Jaskula-Ranga, Sheng, Vogl, Harrington et al (US6740503), and Terai have been discussed above and relied in same manner. The combination of references differs from claimed invention by not disclosing (i) different introducing steps as set forth in claims 27-32 and (ii) the cells may be any one set forth in claims 43-45.
With respect to claims 27-32, Carlo teaches that the nucleic acid molecule may be introduced into cell via any methods known in the art, such as, e.g., viral, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, microinjection, and nanoparticle-mediated delivery (see para. 690).
Regarding 43-45, Carlo teaches that the cells may include hepatocytes, colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cell, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells (see para, 674).
It would have been prima facie obvious for a person of ordinary skill to combine the teachings of prior art to modify the method of Jaskula-Ranga of integrating a transgene into an endogenous gene for expression of by introducing at a target site within an intron of an endogenous gene of a cell as disclosed in Carlos by using a bidirectional construct containing first coding sequence for GFP and first terminator and a second terminator reverse complement and a second coding sequence for second coding sequence (GFP) reverse complement that are positioned 20in opposite orientation or a construct with bi-directional terminator as suggested in Sheng, before the effective filing date of the instant invention. Said modification amounting to combining prior art elements according to known methods to yield predictable results. The limitation of using splice acceptor sequence upstream of two coding sequence would be obvious to a person of ordinary skill such that it could be positioned for divergent transcription to function as evident from the teaching of Harrington. One of ordinary skill in the art would be motivated to do so because this would allow the construct to integrate in (i) both forward and reverse direction and would enable simultaneous independent and stable expression of the same protein and (ii) bidirectional terminator sequence would prevent interference between two transcription units. Absent any requirement of any specific level of expression or any unexpected advantage of using construct in a tail to tail configuration, one of skill in the art would have been expected to have a reasonable expectation of success particularly since prior art successfully reported integrating a transgene into an endogenous gene by administering (i) a transgene comprising 2 splice acceptor sequence, 5222 bidirectional construct; (ii) two coding sequences encoding the same polypeptide as in Sheng in view of Terai, wherein the second sequence is located 3' of the first, and comprises splicing acceptor sites upstream of the first and second coding sequence as in Sheng in view of Harrington. It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d--, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396) (available at http: www. uspto.gov/web/offices/dcom/bpai/prec/fd071925.pdf).
Examiner’s note: Applicant should note that Applicant's arguments and reliance on arguments and/or evidence filed during the prosecution of the prior applications that relied in part on declaration filed under 37 CFR 1.130, 1.131 and 1.132,do not automatically become a part of this application. Where it is desired to rely on an earlier-filed affidavit or declaration, the applicant should make the remarks of record in this application and include a copy of the original affidavit or declaration filed in the prior application (see MPEP 201.06(c) IX).
Withdrawn- Double Patenting
Claims 26-37, 43-48 were rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No.11091756 and Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018).
Claims 26-37, 43-48 were rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10, of USP.12054706 and Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018).
Claims 26-37, 43-48 were rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6, of USP. 11993770 and Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018).
Claims 26-37, 43-48 were rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-22 of U.S. Patent No. 11365407 and Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018).
Claims 26-37, 43-48 were rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No.11254930 in view Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018).
Applicants’ terminal disclaimer dated August 25, 2025, the previous rejections are rendered moot and hereby withdrawn.
Maintained-Double Patenting
Claims 26-37, 43-48 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 18-39, of copending Application No.18526794 and Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018). Although the claims at issue are not identical, they are not patentably distinct from each other because they are directed to similar nucleic acid configuration. In the instant method as claimed specifically encompass the transgene claimed in ‘794.
Claims 26-37, 43-48 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 18-20, 23-27, 29-30,34-37, 39, of copending Application No.18526772 and Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018). Although the claims at issue are not identical, they are not patentably distinct from each other because they are directed to similar nucleic acid configuration. For instance, in the instant case, claims are directed to method for providing a cell with an exogenous nucleic acid molecule comprising a transgene, wherein said transgene comprises, from 5' to 3' orientation:(a) a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement, wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, wherein the second coding sequence is operably linked to the second splice acceptor and second terminator, wherein said method comprises introducing a nucleic acid comprising said transgene into said cell, thereby providing said cell with said exogenous nucleic acid molecule. In the instant method as claimed specifically encompass the transgene claimed in ‘772.
Claims 26-37, 43-48 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 18-39, of copending Application No.18526758 and Carlo et al (US20200040362, dated 02/06/2020, EFD, 06/28/2018). Although the claims at issue are not identical, they are not patentably distinct from each other because they are directed to similar nucleic acid configuration. For instance, in the instant case, claims are directed to method for providing a cell with an exogenous nucleic acid molecule comprising a transgene, wherein said transgene comprises, from 5' to 3' orientation:(a) a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement, wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, wherein the second coding sequence is operably linked to the second splice acceptor and second terminator, wherein said method comprises introducing a nucleic acid comprising said transgene into said cell, thereby providing said cell with said exogenous nucleic acid molecule.
Claims 26-37, 43-48 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 18, 24-26, of copending Application No.18526826. Although the claims at issue are not identical, they are not patentably distinct from each other because they are directed to similar nucleic acid configuration. For instance, in the instant case, claims are directed to method for providing a cell with an exogenous nucleic acid molecule comprising a transgene, wherein said transgene comprises, from 5' to 3' orientation:(a) a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement, wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, wherein the second coding sequence is operably linked to the second splice acceptor and second terminator, wherein said method comprises introducing a nucleic acid comprising said transgene into said cell, thereby providing said cell with said exogenous nucleic acid molecule.
Response to arguments
While Applicant has requested that the rejection be held in abeyance until allowable subject matter can be identified, a request of abeyance does not overcome or address an issue of obvious double patenting be