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 .
Priority
The instant application is a national stage entry of PCT application PCT/US2022/029601, filed 11/15/2023 under 35 USC 371. Acknowledgement is made of the applicant’s claim for benefit to prior-filed U.S. provisional patent application 63/189,771, which was filed 05/18/2021.
Election/Restrictions
Applicant's election with traverse of group I, claims 1, 2, 4, 6-8, 10, 12, 14, 17, 20, 23, 27, 29, 34, 37, 42 and 63, drawn to a viral vector production system, in the reply filed on 06/26/2026 is acknowledged. The traversal is on the ground(s) that: the pending claims are relate to viral vector production systems for viral vector production that allow one to have increased control over expression of a payload molecule, and related methods of using viral vector production systems for this purpose. While the reference Wilson et al. does not teach or suggest this concept (Remarks, p2).
Applicant’s argument is found persuasive. Specifically, the shared technical feature of groups I and II is a viral vector production system comprising: (a) an engineered cell comprising a viral vector production component comprising one or more heterologous polynucleic acids that collectively encode the gene products of a viral vector; (b) a heterologous nucleic acid sequence encoding for a first expression cassette, wherein the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a regulatory RNA; and (c) a transfer polynucleic acid comprising a central nucleic acid sequence flanked, on the 5' and 3' end, by a nucleic acid sequence of a viral terminal repeat. Wilson et al. mainly teach a dual AAV expression system but not production system (see Abstract). When Wilson et al. teach the production of AAV (see figure 1, as well as parag 0053), the production system is comprising a producer cell line that stably supplies rep and cap (reads on (a) of the technical feature) is transfected with a construct encoding the transgene flanked by ITRs (reads on (c) of the technical feature). Wilson et al. do not teach b) a constitutive promoter operably linked to a nucleic acid sequence encoding a regulatory RNA as recited in the technical feature. Therefore Wilson et al. fails to anticipate the technical feature. The reference of Wilson et al. is withdrawn.
However, the shared technical feature of group I and II is still not a special technical feature as it does not make a contribution over the prior art Xie et al. (US 2020/0199622 A1, cited in IDS). Xie et al. teach a method for controlling or improving recombinant virus production yield comprising: introducing into a host cell a first nucleic acid comprising a transgene; introducing into the host cell a second nucleic acid capable of expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of the transgene (parag 0004). In some embodiments, a host cell is a viral vector packaging cell (parag 0006). In some embodiments, a first nucleic acid is a lentiviral transfer plasmid, an adeno-associated virus (AAV) vector, an adenovirus (Ad) vector, or a retroviral vector (parag 0007). Xie et al. teach a recombinant lentiviral transfer vector encoding one or more transgenes of interest flanked by long terminal repeat (LTR) sequences. This teaching reads on “a viral vector production system comprising (c) a transfer polynucleic acid comprising a central nucleic acid sequence flanked, on the 5' and 3' end, by a nucleic acid sequence of a viral terminal repeat”. Xie et al. teach in some embodiments, a second nucleic acid expresses a short hairpin RNA (shRNA), miRNA, or an amiRNA (parag 0012). Xie et al. teach example of shRNA cassettes targeting Apob and driven by the H1 promoter (parag 0016, as well as figure 2A). This teaching reads on “(b) a heterologous nucleic acid sequence encoding for a first expression cassette, wherein the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a regulatory RNA”. Xie et al. also teach a schematic depiction of 3rd generation lentiviral vector production, four constructs (packaging plasmid, Rev encoding plasmid, Env encoding plasmid, transgene encoding plasmid) are transfected into a permissive cell line (e.g., HEK293) to produce the vectors (parag 0017, and figure 3). This teaching reads on “(a) an engineered cell comprising a viral vector production component comprising one or more heterologous polynucleic acids that collectively encode the gene products of a viral vector”. Thus groups I and II lack unity of invention a posteriori.
The requirement is still deemed proper and is therefore made FINAL. Accordingly, claims 1, 2, 4, 6-8, 10, 12, 14, 17, 20, 23, 27, 29, 34, 37, 42 and 63 have been considered on the merits. Claims 62 and 82 are withdrawn from consideration pursuant 37 CFR 1.142(b).
Claim Objections
Claim 27 is objected to because of the following informalities:
Claim 27 recites abbreviations “shRNA” and “amiRNA”. Abbreviations need to be spelled out at the first encounter in the claims.
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, 6-8, 10, 12, 14, 17, 20, 23, 27, 29, 34, 37, 42 and 63 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.
Claims 1, 42 and 63 recite “the gene product of a viral vector” render instant claims indefinite. The phrase “the gene product of a viral vector” is not defined by the claim, the specification does not provide the definition or clarification. Under the broadest reasonable interpretation (BRI), “the gene product of a viral vector” means any protein (i.e., transgene) encoded by any viral vector. Since the specification provides the definition of the term "viral vector production component", which refers to one or more polynucleic acids that collectively encode the gene products required for generation of viral vectors in a recombinant host cell (p10, L11-13), the recitation “a viral vector production component comprising one or more heterologous polynucleic acids that collectively encode the gene products of a viral vector” in the claims makes it unclear what the engineered cell comprises, thus the claim scope is indefinite. Moreover, the phrase “encode the gene products of a viral vector” refers to the gene products of any viral vector, since the gene products are not necessarily from “the viral vector production system”.
Claims 2, 4, 6-8, 10, 12, 14, 17, 20, 23, 27, 29, 34 and 37 depend from, at least, claim 1, and thus inherit the deficiency and are rejected on the same basis.
Claims 1, 2, 6, 7, 42 and 63 recite “central nucleic acid” render instant claims indefinite. The term “central nucleic acid” is not defined by the claim, the specification does not provide the definition or clarification, therefore PHOSITA would not be readily appraised of the meaning of “central nucleic acid” as recited.
Claims 4, 8, 10, 12, 14, 17, 20, 23, 27, 29, 34 and 37 depend from, at least, claim 1, and thus inherit the deficiency and are rejected on the same basis.
Claim 7 recites “the central nucleic acid sequence of the transfer polynucleic acid sequence comprises a second expression cassette” renders instant claim indefinite. The independent claim 1 only refers a heterologous nucleic acid sequence encoding for “a first expression cassette” in (b), but not refer any expression cassette in the transfer polynucleic acid. It is not clear whether claim 7 means the central nucleic acid sequence of the transfer polynucleic acid sequence comprising a (second) expression cassette compare to the first expression cassette in claim 1(b) (that is, the central nucleic acid sequence of the transfer polynucleic acid sequence only has one expression cassette), or the central nucleic acid sequence of the transfer polynucleic acid sequence comprises two expression cassettes.
Claim 8 depends from claim 7, and thus inherit the deficiency and are rejected on the same basis.
Claim Interpretation
Claim 1 recites “a viral vector production component”. The specification provides the definition: the term "viral vector production component" refers to one or more polynucleic acids that collectively encode the gene products required for generation of viral vectors in a recombinant host cell (p10, L11-13). The claim is interpreted in light of the definition in the Specification. Moreover, the recitation “a viral vector production component comprising one or more heterologous polynucleic acids that collectively encode the gene products of a viral vector” in 1 (a) is interpreted as a viral vector production component comprising one or more heterologous polynucleic acids that collectively encode the gene products required for generation of viral vectors in a recombinant host cell.
As stated above, claim 1 and the depending claims are indefinite by the term “central nucleic acid”. In the interest of compact prosecution, claim 1 (c) is interpreted as a transfer polynucleic acid comprising a nucleic acid sequence flanked, on the 5' and 3' end, by a nucleic acid sequence of a viral terminal repeat.
Claim 7 is indefinite. In the interest of compact prosecution, claim 7 is interpreted that the transfer polynucleic acid sequence comprising an expression cassette comprises a nucleic acid sequence encoding a payload molecule operably linked to both a promoter and a target nucleic acid sequence that complements the regulatory RNA encoded by the first expression cassette.
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.
(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 1-2, 4, 6-8, 14, 17, 20, 23, 27, 29 and 42 are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Xie et al. (US 2020/0199622 A1, cited in IDS), as evidenced by Monroe (Plasmids 101, published in 2014) and Wessagowit et al. (J Dermatol Sci. 2005 Nov;40(2):73-84).
Xie et al. teach methods for improving titer and yield of viral vector production. In some embodiments, the methods comprise transient silencing of transgene expression during packaging of a viral vector (Abstract).
Regarding claim 1, Xie et al. teach a method for controlling or improving recombinant virus production yield comprising: introducing into a host cell a first nucleic acid comprising a transgene; introducing into the host cell a second nucleic acid capable of expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of the transgene (parag 0004). In some embodiments, a host cell is a viral vector packaging cell (parag 0006). In some embodiments, a first nucleic acid is a lentiviral transfer plasmid, an adeno-associated virus (AAV) vector, an adenovirus (Ad) vector, or a retroviral vector (parag 0007). Xie et al. teach a recombinant lentiviral transfer vector encoding one or more transgenes of interest flanked by long terminal repeat (LTR) sequences (parag 0053). This teaching reads on “(a system comprising) (c) a transfer polynucleic acid comprising a central nucleic acid sequence flanked, on the 5' and 3' end, by a nucleic acid sequence of a viral terminal repeat” as recited in instant claim. Xie et al. teach in some embodiments, a second nucleic acid expresses a short hairpin RNA (shRNA), miRNA, or an amiRNA (parag 0012). Xie et al. teach example of shRNA cassettes targeting Apob, driven by the H1 promoter, or targeting the Firefly luciferase gene (Flue), driven by the U6 promoter, were inserted adjacent to the mTR (m-R and m-F), within the intron (Intron-R and Intron-F), or adjacent to the wtTR (Wt-R and Wt-F) (parag 0016, as well as figure 2A). This teaching reads on “(b) a heterologous nucleic acid sequence encoding for a first expression cassette, wherein the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a regulatory RNA” as recited in instant claim. Xie et al. also teach a schematic depiction of 3rd generation lentiviral vector production. In this embodiment, four constructs (packaging plasmid, Rev encoding plasmid, Env encoding plasmid, transgene encoding plasmid) are transfected into a permissive cell line (e.g., HEK293) to produce the vectors (parag 0017, and figure 3). This teaching reads on “(a) an engineered cell comprising a viral vector production component comprising one or more heterologous polynucleic acids that collectively encode the gene products of a viral vector” as recited in instant claim. Xie et al. further teach in examples, i.e., cells were infected with a lentiviral vector expressing either GFP (Lenti-GFP), Lenti-GFP-GR80, or Lenti-GFP-GR80 that was packaged during transient gene expression silencing by a plasmid expressing Lenti-GFP-GR80-specific shRNA (parag 0081). Herein the vector of Lenti-GFP-GR80 reads on (c) as recited in instant claim, the vector with Lenti-GFP-GR80-specific shRNA reads on (b) as recited in instant claim, and the packaging cell (with the packaging plasmid, Rev encoding plasmid, Env encoding plasmid which are necessary for lentivirus production) reads on (a) as recited in instant claim. Therefore Xie et al. anticipate instant claim.
Regarding claim 2, Xie et al. teach introducing into a host cell a first nucleic acid comprising a transgene (parag 0003). It is evidenced by that Monroe that a vector or plasmid which can insert a foreign gene (transgene) is necessary having a Multiple Cloning Site (MCS) which allowing for the easy insertion of DNA.
Regarding claim 4, following the discussion above, Xie et al. teach in figure 7, three tandem repeat of artificial target sites which can complement the regulatory RNA (amiR plasmid which is encoded by the first expression cassette) (also see parag 0021).
Regarding claim 6, following the discussion above, Xie et al. teach in figure 7, the nucleic acid sequence comprises a promoter.
Regarding claims 7-8, following the discussion above, Xie et al. teach in figure 7, the nucleic acid encoding a payload (i.e., the un-packageable gene) operably linked to a promoter and a target nucleic acid sequence that complements the regulatory RNA encoded by the first expression cassette (three artificial target sites), as recited in instant claim 7, and the 3' UTR of the payload comprises a target nucleic acid sequence that complements the regulatory RNA encoded by the first expression cassette (three artificial target sites), as recited in instant claim 8.
Regarding claim 14, Xie et al. teach figure 2A, a schematic of self-complementary AAV (scAAV) plasmids comprising a CMV enhancer/chicken β-actin promoter (CB), an EGFP reporter gene, and a beta-globin polyA sequence (PA). shRNA cassettes targeting Apob, driven by the H1 promoter, or targeting the Firefly luciferase gene (Fluc), driven by the U6 promoter, were inserted adjacent to the mTR (m-R and m-F), within the intron (Intron-R and Intron-F), or adjacent to the wtTR (Wt-R and Wt-F) (parag 0016). This teaching reads on an expression cassette encoding a constitutive promoter operably linked to shRNA, and the expression cassette further comprises a nucleic acid sequence encoding a selectable marker EGFFP.
Regarding claims 17 and 20, following the discussion above, Xie et al. teach in some embodiments, a first nucleic acid is a lentiviral transfer plasmid, an adeno-associated virus (AAV)
vector, an adenovirus (Ad) vector, or a retroviral vector (parag 0007). A host cell further comprises one or more accessory plasmids. In some embodiments, one or more accessory plasmids are a packaging plasmid, an Env encoding plasmid, a Rev encoding plasmid, a Rep encoding plasmid, or a Cap encoding plasmid (parag 0013).
Regarding claim 23, following the discussion above, Xie et al. teach a method for controlling or improving recombinant virus production yield comprising: introducing into a host cell a first nucleic acid comprising a transgene; introducing into the host cell a second nucleic acid capable of expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of the transgene (parag 0004). In some embodiments, a mammalian cell is a human cell, for example a HEK293T cell (parag 0005). In some embodiments, a second nucleic acid expresses a short hairpin RNA, miRNA, or an amiRNA (parag 0012). This teaching reads on “(iv) the engineered cell is derived from a HEK293 cell” and “(v) the regulatory RNA is an shRNA or an amiRNA” as recited in instant claim.
Regarding claim 27, following the discussion above, Xie et al. teach in some embodiments, a second nucleic acid expresses a short hairpin RNA (shRNA), miRNA, or an amiRNA (parag 0012).
Regarding claim 29, following the discussion of claims 14 and 27, Xie et al. teach in figure 2A, a schematic of self-complementary AAV (scAAV) plasmids comprising a CMV enhancer/chicken β-actin promoter (CB), an EGFP reporter gene, and a beta-globin polyA sequence (PA). shRNA cassettes targeting Apob, driven by the H1 promoter, or targeting the Firefly luciferase gene (Fluc), driven by the U6 promoter, were inserted adjacent to the mTR (m-R and m-F), within the intron (Intron-R and Intron-F), or adjacent to the wtTR (Wt-R and Wt-F). It is evidenced by Wessagowit et al. that 5’ splice site of an intron is the intron donor site, and the 3’ splice site of an intron is the intron acceptor site (p75, left column). Therefore the insert of shRNA within the intron reads on the nucleic acid sequence encoding for the selectable marker comprises an intron having, from 5' to 3': (i) an intron donor site; (ii) a nucleic acid sequence encoding for the shRNA or amiRNA; and (iii) an intron acceptor site, as recited in instant claim.
Regarding claim 42, following the discussion above, Xie et al. teach a method for controlling or improving recombinant virus production yield comprising: introducing into a host cell a first nucleic acid comprising a transgene; introducing into the host cell a second nucleic acid capable of expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of the transgene (parag 0004). In some embodiments, a host cell is a viral vector packaging cell (parag 0006). In some embodiments, a first nucleic acid is a lentiviral transfer plasmid, an adeno-associated virus (AAV) vector, an adenovirus (Ad) vector, or a retroviral vector (parag 0007). Xie et al. teach a recombinant lentiviral transfer vector encoding one or more transgenes of interest flanked by long terminal repeat (LTR) sequences (parag 0053). Xie et al. further in examples, i.e., cells were infected with a lentiviral vector expressing either GFP (Lenti-GFP), Lenti-GFP-GR80, or Lenti-GFP-GR80 that was packaged during transient gene expression silencing by a plasmid expressing Lenti-GFP-GR80-specific shRNA (parag 0081). Herein the vector of Lenti-GFP-GR80 reads on (c) as recited in instant claim, the vector with Lenti-GFP-GR80-specific shRNA reads on (b) as recited in instant claim, and the packaging cell (with the packaging plasmid, Rev encoding plasmid, Env encoding plasmid which are necessary for lentivirus production) reads on (a) as recited in instant claim.
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.
Claims 1-2, 4, 6-8, 10, 14, 17, 20, 23, 27, 29 and 42 are rejected under 35 U.S.C. 103 as being unpatentable over Xie et al. (US 2020/0199622 A1, cited in IDS) in view of Lambeth et al. (BMC Mol Biol. 2010 Oct 11;11:77), as evidenced by as evidenced by Monroe (Plasmids 101, published in 2014) and Wessagowit et al. (J Dermatol Sci. 2005 Nov;40(2):73-84).
Xie et al. teach methods for improving titer and yield of viral vector production. In some embodiments, the methods comprise transient silencing of transgene expression during packaging of a viral vector (Abstract).
Regarding claim 1, Xie et al. teach a method for controlling or improving recombinant virus production yield comprising: introducing into a host cell a first nucleic acid comprising a transgene; introducing into the host cell a second nucleic acid capable of expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of the transgene (parag 0004). In some embodiments, a host cell is a viral vector packaging cell (parag 0006). In some embodiments, a first nucleic acid is a lentiviral transfer plasmid, an adeno-associated virus (AAV) vector, an adenovirus (Ad) vector, or a retroviral vector (parag 0007). Xie et al. teach a recombinant lentiviral transfer vector encoding one or more transgenes of interest flanked by long terminal repeat (LTR) sequences (parag 0053). This teaching reads on “(a system comprising) (c) a transfer polynucleic acid comprising a central nucleic acid sequence flanked, on the 5' and 3' end, by a nucleic acid sequence of a viral terminal repeat” as recited in instant claim. Xie et al. teach in some embodiments, a second nucleic acid expresses a short hairpin RNA (shRNA), miRNA, or an amiRNA (parag 0012). Xie et al. teach example of shRNA cassettes targeting Apob, driven by the H1 promoter, or targeting the Firefly luciferase gene (Flue), driven by the U6 promoter, were inserted adjacent to the mTR (m-R and m-F), within the intron (Intron-R and Intron-F), or adjacent to the wtTR (Wt-R and Wt-F) (parag 0016, as well as figure 2A). This teaching reads on “(b) a heterologous nucleic acid sequence encoding for a first expression cassette, wherein the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a regulatory RNA” as recited in instant claim. Xie et al. also teach a schematic depiction of 3rd generation lentiviral vector production. In this embodiment, four constructs (packaging plasmid, Rev encoding plasmid, Env encoding plasmid, transgene encoding plasmid) are transfected into a permissive cell line (e.g., HEK293) to produce the vectors (parag 0017, and figure 3). This teaching reads on “(a) an engineered cell comprising a viral vector production component comprising one or more heterologous polynucleic acids that collectively encode the gene products of a viral vector” as recited in instant claim. Xie et al. further teach in examples, i.e., cells were infected with a lentiviral vector expressing either GFP (Lenti-GFP), Lenti-GFP-GR80, or Lenti-GFP-GR80 that was packaged during transient gene expression silencing by a plasmid expressing Lenti-GFP-GR80-specific shRNA (parag 0081). Herein the vector of Lenti-GFP-GR80 reads on (c) as recited in instant claim, the vector with Lenti-GFP-GR80-specific shRNA reads on (b) as recited in instant claim, and the packaging cell (with the packaging plasmid, Rev encoding plasmid, Env encoding plasmid which are necessary for lentivirus production) reads on (a) as recited in instant claim. Therefore Xie et al. anticipate instant claim.
Regarding claim 2, Xie et al. teach introducing into a host cell a first nucleic acid comprising a transgene (parag 0003). It is evidenced by that Monroe that a vector or plasmid which can insert a foreign gene (transgene) is necessary having a Multiple Cloning Site (MCS) which allowing for the easy insertion of DNA.
Regarding claim 4, following the discussion above, Xie et al. teach in figure 7, three tandem repeat of artificial target sites which can complement the regulatory RNA (amiR plasmid which is encoded by the first expression cassette) (also see parag 0021).
Regarding claim 6, following the discussion above, Xie et al. teach in figure 7, the nucleic acid sequence comprises a promoter.
Regarding claims 7-8, following the discussion above, Xie et al. teach in figure 7, the nucleic acid encoding a payload (i.e., the un-packageable gene) operably linked to a promoter and a target nucleic acid sequence that complements the regulatory RNA encoded by the first expression cassette (three artificial target sites), as recited in instant claim 7, and the 3' UTR of the payload comprises a target nucleic acid sequence that complements the regulatory RNA encoded by the first expression cassette (three artificial target sites), as recited in instant claim 8.
Regarding claim 14, Xie et al. teach figure 2A, a schematic of self-complementary AAV (scAAV) plasmids comprising a CMV enhancer/chicken β-actin promoter (CB), an EGFP reporter gene, and a beta-globin polyA sequence (PA). shRNA cassettes targeting Apob, driven by the H1 promoter, or targeting the Firefly luciferase gene (Fluc), driven by the U6 promoter, were inserted adjacent to the mTR (m-R and m-F), within the intron (Intron-R and Intron-F), or adjacent to the wtTR (Wt-R and Wt-F) (parag 0016). This teaching reads on an expression cassette encoding a constitutive promoter operably linked to shRNA, and the expression cassette further comprises a nucleic acid sequence encoding a selectable marker EGFFP.
Regarding claims 17 and 20, following the discussion above, Xie et al. teach in some embodiments, a first nucleic acid is a lentiviral transfer plasmid, an adeno-associated virus (AAV)
vector, an adenovirus (Ad) vector, or a retroviral vector (parag 0007). A host cell further comprises one or more accessory plasmids. In some embodiments, one or more accessory plasmids are a packaging plasmid, an Env encoding plasmid, a Rev encoding plasmid, a Rep encoding plasmid, or a Cap encoding plasmid (parag 0013).
Regarding claim 23, following the discussion above, Xie et al. teach a method for controlling or improving recombinant virus production yield comprising: introducing into a host cell a first nucleic acid comprising a transgene; introducing into the host cell a second nucleic acid capable of expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of the transgene (parag 0004). In some embodiments, a mammalian cell is a human cell, for example a HEK293T cell (parag 0005). In some embodiments, a second nucleic acid expresses a short hairpin RNA, miRNA, or an amiRNA (parag 0012). This teaching reads on “(iv) the engineered cell is derived from a HEK293 cell” and “(v) the regulatory RNA is an shRNA or an amiRNA” as recited in instant claim.
Regarding claim 27, following the discussion above, Xie et al. teach in some embodiments, a second nucleic acid expresses a short hairpin RNA (shRNA), miRNA, or an amiRNA (parag 0012).
Regarding claim 29, following the discussion of claims 14 and 27, Xie et al. teach in figure 2A, a schematic of self-complementary AAV (scAAV) plasmids comprising a CMV enhancer/chicken β-actin promoter (CB), an EGFP reporter gene, and a beta-globin polyA sequence (PA). shRNA cassettes targeting Apob, driven by the H1 promoter, or targeting the Firefly luciferase gene (Fluc), driven by the U6 promoter, were inserted adjacent to the mTR (m-R and m-F), within the intron (Intron-R and Intron-F), or adjacent to the wtTR (Wt-R and Wt-F). It is evidenced by Wessagowit et al. that 5’ splice site of an intron is the intron donor site, and the 3’ splice site of an intron is the intron acceptor site (p75, left column). Therefore the insert of shRNA within the intron reads on the nucleic acid sequence encoding for the selectable marker comprises an intron having, from 5' to 3': (i) an intron donor site; (ii) a nucleic acid sequence encoding for the shRNA or amiRNA; and (iii) an intron acceptor site, as recited in instant claim.
Regarding claim 42, following the discussion above, Xie et al. teach a method for controlling or improving recombinant virus production yield comprising: introducing into a host cell a first nucleic acid comprising a transgene; introducing into the host cell a second nucleic acid capable of expressing an interfering nucleic acid, wherein the interfering nucleic acid specifically inhibits expression of the transgene (parag 0004). In some embodiments, a host cell is a viral vector packaging cell (parag 0006). In some embodiments, a first nucleic acid is a lentiviral transfer plasmid, an adeno-associated virus (AAV) vector, an adenovirus (Ad) vector, or a retroviral vector (parag 0007). Xie et al. teach a recombinant lentiviral transfer vector encoding one or more transgenes of interest flanked by long terminal repeat (LTR) sequences (parag 0053). Xie et al. further in examples, i.e., cells were infected with a lentiviral vector expressing either GFP (Lenti-GFP), Lenti-GFP-GR80, or Lenti-GFP-GR80 that was packaged during transient gene expression silencing by a plasmid expressing Lenti-GFP-GR80-specific shRNA (parag 0081). Herein the vector of Lenti-GFP-GR80 reads on (c) as recited in instant claim, the vector with Lenti-GFP-GR80-specific shRNA reads on (b) as recited in instant claim, and the packaging cell (with the packaging plasmid, Rev encoding plasmid, Env encoding plasmid which are necessary for lentivirus production) reads on (a) as recited in instant claim.
Regarding claim 10, Xie et al. do not teach the first expression cassette comprises a tandem repeat of the nucleic acid sequence encoding the regulatory RNA and/or wherein the first expression cassette comprises a nucleic acid sequence of two or more distinct regulatory RNAs. However, this was disclosed by Lambeth et al. at the time of instant invention.
Lambeth et al. teach a set of active siRNAs and delivered them using validated and proven expression systems without extensive method specific optimization (p2, right column).
Regarding claim 10, Lambeth et al. teach schematic representation of the strategies used for combinatorial RNAi (see figure 1), wherein all the combinatorial RNAi strategies including the multiple promoter/shRNA, the long hairpin RNA and miRNA-embedded construct read on the nucleic acid sequence of two or more distinct regulatory RNAs as recited in instant claim.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Xie et al.’s method for controlling or improving recombinant virus production yield comprise transient silencing of transgene expression by regulatory RNAs during packaging of a viral vector, and use two or more distinct regulatory RNAs as taught by Lambeth et al.. The skilled artisan would have been motivated to use two or more distinct regulatory RNAs since Lambeth et al. teach combinatorial RNAi such as multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets (Abstract). There would be a reasonable expectation of success of using two or more distinct regulatory RNAs, since Lambeth et al. teach the strategies used for combinatorial RNAi (see i.e., figure 1).
Claims 1-2, 4, 6-8, 12, 14, 17, 20, 23, 27, 29 and 42 are rejected under 35 U.S.C. 103 as being unpatentable over Xie et al. (US 2020/0199622 A1, cited in IDS) in view of Xie et al. (Mol Ther. 2020 Feb 5;28(2):422-430, “Xie2020” hereinafter), as evidenced by Monroe (Plasmids 101, published in 2014) and Wessagowit et al. (J Dermatol Sci. 2005 Nov;40(2):73-84).
The teaching of Xie et al. is set forth above.
Regarding claim 12, Xie et al. do not teach the first expression cassette further comprises a nucleic acid sequence encoding a gene product of the viral vector production component. However, this was disclosed by Xie2020 at the time of instant invention.
Xie2020 reports that embedding the guide strand of a small silencing RNA into an artificial microRNA (miRNA) scaffold derived from mouse miRNA-33 ensures rAAV genomic integrity and reduces off-targeting by 10-fold, while maintaining effective in vivo target gene repression in mice (Abstract).
Regarding claim 12, Xie2020 packaged constructs harboring shRNAmiR-33 within the intron or
shRNA next to the mutant terminal repeat or within the intron into AAV capsids (p423, right column). AAV capsid is a nucleic acid sequence encoding a gene product of the viral vector production component.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Xie et al.’s method for controlling or improving recombinant virus production yield comprise transient silencing of transgene expression by regulatory RNA such as shRNA or amiRNA during packaging of a viral vector, and place the shRNA or amiRNA in one of the vectors comprising viral vector production component as taught by Xie2020. The only difference between instant claim and Xie et al.’s method is instant claim place regulatory RNA such as shRNA with a nucleic acid sequence encoding a gene product of the viral vector production component. Given that Xie2020 teach shRNA can be placed within the intron into AAV capsids, one of ordinary skill in the art would have substituted Xie et al.’s location of shRNA, and place the shRNA within the intron into AAV capsids depends on their research preference. This simple substitution of one known element for another known element is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Claims 1-2, 4, 6-8, 14, 17, 20, 23, 27, 29, 34, 37, 42 and 63 are rejected under 35 U.S.C. 102(3) as being unpatentable over Xie et al. (US 2020/0199622 A1, cited in IDS)in view of Hsu et al. (US 2020/0199556 A1, published in 2020, cited in IDS), as evidenced by Monroe (Plasmids 101, published in 2014) and Wessagowit et al. (J Dermatol Sci. 2005 Nov;40(2):73-84).
The teaching of Xie et al. is set forth above.
Regarding claims 34 and 37, Xie et al. do not teach the regulatory RNA is a Cas13 guide RNA. However, this was disclosed by Hsu et al. at the time of instant invention.
Hsu et al. teach CRISPR/Cas methods and compositions for targeting RNA molecules, which can be used to detect, edit, or modify a target RNA (Abstract).
Regarding claims 34 and 37, Hsu et al. teach comparing the CasRx (Cas13d from Ruminococcus flavefaciens strain XPD3002) and shRNA for the RNA interference: CasRx fusions knocked down a diverse set of 14 endogenous mRNAs and lncRNAs, consistently achieving >90% knockdown with favorable efficiency relative to RNA interference, dCas9-mediated CRISPR interference, and other members of the Cas13 superfamily. Additionally, CasRx interference is markedly more specific than spacer-matching shRNAs, with no detectable off-target changes compared with hundreds for RNA interference (parag 0378). This teaching indicates that CasRx and CasRx guide RNA mediated a more efficient and more specific RNA interference than shRNA, and it can be a substitute of shRNA for the same function.
Regarding claim 63, following the discussion above, Xie et al. teach a host cell comprises one or more accessory plasmids. In some embodiments, one or more accessory plasmids are a packaging plasmid, an Env encoding plasmid, a Rev encoding plasmid, a Rep encoding plasmid, or a Cap encoding plasmid (parag 0013), reads on (a) in instant claim, Xie et al. teach a recombinant lentiviral transfer vector encoding one or more transgenes of interest flanked by long terminal repeat (LTR) sequences (parag 0007). This teaching reads on (d) in instant claim. Xie et al. teach a vector comprising shRNA or amiRNA. Similarly, Hsu et al. also teach Cas13d and CAS13d guide RNA in AAV system, i.e., in figure 12C, a vector encoding dCasRX. Regarding dCasRX guide RNA, Hsu et al. teach in some examples, the guide nucleic acid molecule is operably linked to a promoter or expression control element (parag 0451), reads on (b) and (c) in instant claim.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Xia et al.’s method for controlling or improving recombinant virus production yield comprise transient silencing of transgene expression by shRNA or amiRNA during packaging of a viral vector, and use Cas 13d guide RNA for the same function in the viral production system as taught by Hsu et al.. The only difference between instant claims and Xie et al.’s method is instant claims use Cas 13d guide RNA as the regulatory RNA. Given that Hsu et al. teach CasRx and CasRx guide RNA also conduct RNA interference, and mediated a more efficient and more specific RNA interference than shRNA, one of ordinary skill in the art would have substituted Xie et al.’s shRNA or amiRNA, and use Cas13d and Cas 13d guide RNA for the same function with a better efficiency and specificity. This simple substitution of one known element (use Cas13d and Cas 13d for transient silencing of transgene) for another known element (use shRNA or amiRNA for transient silencing of transgene) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Conclusion
No claims are allowed.
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/Q.G./Examiner, Art Unit 1633
/FEREYDOUN G SAJJADI/Supervisory Patent Examiner, Art Unit 1699
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