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 371 of PCT/US2021/039947 filed 06/30/2021, which claims benefit of US provisional 63/046,565 filed 06/30/2020.
Claim 80 recites the limitation “wherein introducing to a cell of said porcine mammal said nuclease comprising a gene-binding moiety significantly reduces viral load in the cell.” The phrase “viral load” has no support in the US provisional 63/046,565. The phrase “viral load” first appears in the claims filed 08/28/2023 (claim 80). “Viral load” refers to the level of virus in the blood, which is distinct from “replication of a virus,” as recited in claim 61 (Burrell et al., Fenner and White’s Medical Virology, 2017: 39-55, p 54, col 1, para 1).
Therefore, the effective filing date of claim 80 is 08/28/2023. The effective filing date of claims 61-97 and 81-82 is 06/30/2020.
Election/Restrictions
Applicant’s election without traverse of the following species in the reply filed on 12/09/2025 is acknowledged:
Species 1: the Cas Type enumerated in claims 68-69 as the Type II Cas polypeptide;
Species 2: the sequence enumerated in claim 73 as SEQ ID NO: 1; and
Species 3: the sequence enumerated in claim 74 as SEQ ID NO: 25.
Claims 70-71 are withdrawn from consideration as being drawn to a non-elected species of Cas polypeptide (species 1). Claims 61-69 and 72-82 are examined on the merits herein.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). See sequences without sequence identifiers in Example 4 (para 81) and Example 5 (para 86).
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Rejections - 35 USC § 112(b)
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.
Claim 73 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 73 refer to Tables 1-5 and 7-9 in the specification. MPEP 2173.05(s) states “Where possible, claims are to be complete in themselves. Incorporation by reference to a specific figure or table “is permitted only in exceptional circumstances where there is no practical way to define the invention in words and where it is more concise to incorporate by reference than duplicating a drawing or table into the claim. Incorporation by reference is a necessity doctrine, not for applicant’s convenience.” Ex parte Fressola, 27 USPQ2d 1608 (bd. Pat. App. & Inter. 1993) (citation omitted).” The information from Tables 1-5 and 7-9 pertinent to the claims does not qualify as an exception to circumstance. The sequence identifiers of the sequences listed in Tables 3-5 and 7-9 and the gene names listed in Tables 1-2 can be incorporated into the claims. Therefore, claim 73 is rejected as failing to particularly point out and distinctly claim the subject matter in a manner consistent with 35 USC 112(b).
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 72 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 72 is drawn to “The method of claim 61, wherein said virus is African swine fever virus (ASFV).” Claim 61 recites the limitation, “wherein said virus belongs to the family Asfarviridae” (line 4-5). Claim 72 does not further limit the subject matter of claim 61 because ASFV is the sole member of the family Asfarviridae (Hübner (Scientific Reports, 2018, 8(1): 1449), Introduction, para 2). Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 112(a)
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 73 and 74 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
From M.P.E.P. § 2163, the analysis of whether the specification complies with the written description requirement calls for the examiner to compare the scope of the claim with the scope of the description to determine whether applicant has demonstrated possession of the claimed invention from the standpoint of one of skill in the art at the time the application was filed. For inventions in emerging and unpredictable technologies, or for inventions characterized by factors not reasonably predictable which are known to one of ordinary skill in the art, more evidence is required to show possession.
For claims drawn to a genus, possession may be shown (for example) through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. A “representative number of species” means that the species which are adequately described are representative of the entire genus, and is an inverse function of the skill and knowledge in the art. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus. See, e.g., Eli Lilly. If a representative number of adequately described species are not disclosed for a genus, the claim to that genus must be rejected as lacking adequate written description under 35 U.S.C. 112, para. 1.
Regarding claim 73: Claim 73 is drawn to the method of claim 61, wherein said nuclease is configured to bind at least 5 consecutive nucleotides of the sequence set forth in SEQ ID NO: 1, or a variant having at least 80%, 90%, 95%, or 99% sequence identity thereto.
Claim 73 recites a structure: a nuclease configured to bind at least 5 consecutive nucleotides of the sequence set forth in SEQ ID NO: 1, or a variant having at least 80%, 90%, 95%, or 99% sequence identity thereto. Claim 73 also recites a function: wherein binding of said nuclease to at least 5 consecutive nucleotides of the sequence set forth in SEQ ID NO: 1, or a variant having at least 80%, 90%, 95%, or 99% sequence identity thereto results in inhibiting infection of or reducing replication of a virus in a porcine mammal in need thereof. Thus, the claim is drawn to a method comprising the use of a very large genus of nucleic acid molecules comprising the claimed function.
Neither the specification nor the prior art establishes a structure-function relationship wherein a nuclease configured to bind at least 5 consecutive nucleotides of the sequence set forth in SEQ ID NO: 1, or a variant having at least 80%, 90%, 95%, or 99% sequence identity thereto, would be capable of functioning as claimed with any degree of predictability.
The instant specification appears to only have one nucleic acid capable of providing for the claimed function of inhibiting infection of or reducing replication of a virus: full-length SEQ ID NO:1 (p 25-26, Table 3). The sequence set forth in SEQ ID NO: 1 is 2796 base pairs. The specification does not disclose any other embodiments which would be encompassed within the pending claim. The specification does not identify what changes, mutations, fragments, etc. could be made to SEQ ID NO: 1, and that would be encompassed by the structural limitations of claim 73, and would predictably result in the claimed function of inhibiting infection of or reducing replication of a virus. As such, the instant specification does not provide a sufficient representative sampling of structures that are as little as 80% identical to the sequence set forth in SEQ ID NO: 1 that are capable of providing for said function as claimed.
The prior art is unpredictable. The sequence set forth in SEQ ID NO: 1 is the DNA polymerase G1211R of the African swine fever virus (p 25-26, Table 3). The closest prior art, Hübner (Scientific Reports, 2018, 8(1): 1449) teaches a method for inhibiting African swine fever virus replication in a porcine cell line by CRISPR/Cas9 targeting of the viral p30 gene, wherein the viral DNA polymerase (G1211R) of ASFV was targeted. However, Hübner does not teach a method for targeting a sequence with 80% identity to the DNA polymerase G1211R of the African swine fever virus. Thus, the prior art cannot be relied upon for making up for the deficit of the instant specification with regard to a sufficient representative number of species that are as little as 80% identical to the sequence set forth in in SEQ ID NO: 1 that provide for said function as claimed.
Accordingly, neither the specification nor the prior art establishes a known structure-function relationship wherein the genus of structures that are as little as 80% identical to the sequence set forth in SEQ ID NO: 1 are capable of providing for the function as claimed with any predictability. In this case, the skilled artisan would not have reasonable concluded at the time of the invention that application was in possession of the invention as claimed.
Regarding claim 74: Claim 73 is drawn to the method of claim 61, wherein said gene-binding moiety of said nuclease comprises a heterologous RNA polynucleotide configured to hybridize to said at least one essential gene of said virus, wherein said heterologous RNA polynucleotide comprises at least one, at least two, or at least three targeting sequences, wherein said targeting sequence comprises at least 17 consecutive nucleotides of the sequence set forth in SEQ ID: 25, or a variant having at least 80%, 90%, 95%, or 99% sequence identity thereto.
Claim 74 recites a structure: a targeting sequence comprises at least 17 consecutive nucleotides of the sequence set forth in SEQ ID: 25, or a variant having at least 80%, 90%, 95%, or 99% sequence identity thereto. Claim 74 also recites a function: wherein a targeting sequence comprises at least 17 consecutive nucleotides of the sequence set forth in SEQ ID: 25, or a variant having at least 80%, 90%, 95%, or 99% sequence identity thereto results in inhibiting infection of or reducing replication of a virus in a porcine mammal in need thereof. Thus, the claim is drawn to a method comprising the use of a very large genus of nucleic acid molecules comprising the claimed function.
Neither the specification nor the prior art establishes a structure-function relationship wherein a targeting sequence comprises at least 17 consecutive nucleotides of the sequence set forth in SEQ ID: 25, or a variant having at least 80%, 90%, 95%, or 99% sequence identity thereto, would be capable of functioning as claimed with any degree of predictability.
The instant specification appears to only have one nucleic acid capable of providing for the claimed function of inhibiting infection of or reducing replication of a virus: full-length SEQ ID NO: 25 (p 37, Table 4). The sequence set forth in SEQ ID NO: 25 is 20 base pairs. The specification does not disclose any other embodiments which would be encompassed within the pending claim. The specification does not identify what changes, mutations, fragments, etc. could be made to SEQ ID NO: 25, and that would be encompassed by the structural limitations of claim 74, and would predictably result in the claimed function of inhibiting infection of or reducing replication of a virus. As such, the instant specification does not provide a sufficient representative sampling of structures that are as little as 80% identical and that have as few as 17 base pairs out of a possible 20 nucleic acids present in the sequence set forth in SEQ ID NO: 25 that are capable of providing for said function as claimed.
The prior art is unpredictable. The sequence set forth in SEQ ID NO: 25 is the targeting sequence (reverse complement) of DNA polymerase G1211R (p 37, Table 4). The closest prior art, Hübner (Scientific Reports, 2018, 8(1): 1449) teaches a method for inhibiting African swine fever virus replication in a porcine cell line by CRISPR/Cas9 targeting of the viral p30 gene, wherein the viral DNA polymerase (G1211R) of ASFV was targeted. However, Hübner does not teach a method for targeting a sequence as little as 80% identical and that have as few as 17 base pairs out of a possible 20 nucleic acids present in the sequence set forth in SEQ ID NO: 25. Thus, the prior art cannot be relied upon for making up for the deficit of the instant specification with regard to a sufficient representative number of species that are as little as 80% identical and that have as few as 17 base pairs out of a possible 20 nucleic acids present in the sequence set forth in SEQ ID NO: 25 that provide for said function as claimed.
Accordingly, neither the specification nor the prior art establishes a known structure-function relationship wherein the genus of structures that are as little as 80% identical and that have as few as 17 base pairs out of a possible 20 nucleic acids present in the sequence set forth in SEQ ID NO: 25 are capable of providing for the function as claimed with any predictability. In this case, the skilled artisan would not have reasonable concluded at the time of the invention that application was in possession of the invention as claimed.
Claims 61-69 and 72-82 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention without undue or unreasonable experimentation. See Mineral Separation v. Hyde, 242 U.S. 261, 270 (1916). The key word is 'undue,' not experimentation.' " (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all these factors are considered, a sufficient number are discussed below so as to create a prima facie case.
The breadth of the claims:
Claims 61-69 and 72-82 are directed to a method for inhibiting infection of or reducing replication of a virus in a porcine mammal in need thereof, comprising introducing to a cell of said porcine mammal a nuclease comprising a gene-binding moiety, wherein said gene binding moiety is configured to bind at least one essential gene of said virus, or any combination thereof, wherein said virus belongs to the family Asfarviridae, wherein said at least one essential gene of said virus encodes Topoisomerase II or a fragment thereof, RNA helicase or a fragment thereof, or DNA polymerase or a fragment thereof.
The nature of the invention:
Broadly, the nature of all claims, which depend from claim 61, is a method for inhibiting infection of or reducing replication of a virus in a porcine mammal in need thereof. The claims are directed to an in vivo method, wherein the subject is a porcine mammal.
The state of the prior art:
Art published prior to the 06/30/2020 effective filing date suggest a method for reducing replication of a virus in vitro, comprising introducing to a porcine cell a nuclease comprising a gene-binding moiety, wherein said gene binding moiety is configured to bind at least one essential gene of said virus, or any combination thereof, wherein said virus belongs to the family Asfarviridae, wherein said at least one essential gene of said virus encodes a DNA polymerase or a fragment thereof. However, the prior art does not disclose, or suggest, an in vivo method for inhibiting infection of or reducing replication of an Asfarviridae virus in a porcine mammal in need thereof.
Hübner (Scientific Reports, 2018, 8(1): 1449; cited in IDS filed 12/20/2023), the closest prior art, teaches a method for inhibiting African swine fever virus (ASFV) replication in vitro in a wild boar (Sus scrofa) lung cell line (p 5, Methods, para 1) by CRISPR/Cas9 targeting of the viral p30 gene (Abstract), wherein the viral DNA polymerase (G1211R) of ASFV was targeted (Results, para 2). ASFV is a member of the family Asfarviridae (Introduction, para 2). Importantly, the method taught in Hübner is an in vitro method. Hübner teaches that the method taught therein “can now be applied to create Cas9 and guide RNA-expressing pigs by transgenesis” (Discussion, para 1). However, Hübner does not reduce to practice the method taught therein to inhibiting infection of or reducing replication of a virus in a porcine mammal in vivo, and stresses the steps required to apply the method to an in vivo model. Hübner writes, “We will attempt to generate pigs expressing ASFV-specific CRISPR/Cas9 systems. Since swine fibroblasts are in general hardly infectable by ASFV (results not shown), CRISPR/Cas9-expressing transgenic animals have to be generated first, before their macrophages as the natural ASFV host cells can be tested for susceptibility. If they exhibit significantly reduced levels of ASFV replication upon in vitro infection, the respective animals will be tested for resistance by challenge infections with virulent ASFV” (Discussion, para 1).
Liu (International Journal of Molecular Sciences, 2022, 23(13): 7331) offers a review of advancements of CRISPR/Cas genome editing tools and their applications in animal breeding, including its applications in improving disease resistance (Abstract). Liu teaches that the use of CRISPR/Cas as a designed immune system to eliminate or inhibit the replication of animal viruses has been tested in animal cells, including in the experiments taught in Hübner et al., 2018 regarding ASFV. Liu teaches although Cas9 or Cas13d and sgRNAs could be integrated into the genome of transgenic animals to breed antiviral animals, or delivered to animal cells as novel antivirus agents, “there seem to be many hurdles to overcome before its realization” (p 5, para 2-3). Liu teaches that these hurdles include 1) the need to evaluate therapeutic potential in vivo, and the need to conduct a detailed analysis of the host immune response to the Cas9 and Cas13 proteins and multiple sgRNAs for the prediction of potential side effects associated with this antiviral therapy, and 2) the need to evaluate the potential off-target activity of CRISPR/Cas9-based antiviral therapy (p 5, para 3).
Zhang (Journal of Genetics and Genomics, 2021, 48(5): 347-360) offers a review of advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research (Abstract). Zhang teaches that the method taught in Hübner et al., 2018 to suppress ASFV replication in vitro “can potentially be used to produce transgenic pigs that inhibit ASFV infection with Cas9/sgRNA knock-in, thereby conferring resistance to ASFV pathogens in pigs. These attempts emphasize the potential application of CRISPR/Cas9 technology for the rapid development of ASFV vaccines and disease-resistant pigs” (p 352, col 2). Importantly, Zhang emphasizes that the in-vitro method taught in Hübner has potential, and not guaranteed success, for in-vivo use. Zhang further teaches that off-target effects must be considered in undertaking in-vivo CRISPR/Cas9-based gene editing (p 356, col 1, para 3 – col 2, para 1)
The level of one of ordinary skill:
One of ordinary skill in the art is a research scientist, such as a virologist or molecular biologist, holding a postgraduate degree or equivalent experience.
The level of predictability in the art:
The prior art, namely Hübner (Scientific Reports, 2018, 8(1): 1449), recognized a method for inhibiting ASFV replication in vitro in a porcine cell line by CRISPR/Cas9 targeting of the viral p30 gene, wherein the viral DNA polymerase (G1211R) of ASFV was targeted. However, although Hübner discussed the potential application of the in vitro results to an in vivo model, Hübner stressed unpredictability in applying the method to an in vivo model. Among other things, Hübner teaches that 1) swine fibroblasts are in general hardly infectable by ASFV, thereby requiring the generation of CRISPR/Cas9-expressing transgenic animals, before their macrophages as the natural ASFV host cells can be tested for susceptibility, and 2) if they exhibit significantly reduced levels of ASFV replication upon in vitro infection, the respective animals need to be tested for resistance by challenge infections with virulent ASFV (Discussion, para 1).
Liu (International Journal of Molecular Sciences, 2022, 23(13): 7331), reviewing the state of the art in 2022, teaches that the unpredictability of applying CRISPR/Cas genome editing tools in vivo include 1) the need to evaluate therapeutic potential in vivo, and the need to conduct a detailed analysis of the host immune response to the Cas9 and Cas13 proteins and multiple sgRNAs for the prediction of potential side effects associated with this antiviral therapy, and 2) the need to evaluate the potential off-target activity of CRISPR/Cas9-based antiviral therapy (p 5, para 3).
Zhang (Journal of Genetics and Genomics, 2021, 48(5): 347-360), reviewing the state of the art in 2021, teaches that in-vitro methods of CRISPR/Cas9-based gene editing have potential, and not guaranteed success, for in-vivo use. Zhang further teaches that off-target effects must be considered in undertaking in-vivo CRISPR/Cas9-based gene editing (p 356, col 1, para 3 – col 2, para 1)
Therefore, at the time the invention was made, there was a high level of unpredictability regarding the application of an in-vitro, CRISPR/Cas-based method for inhibiting ASFV replication in a porcine cell line to an in-vivo method for inhibiting infection or reducing replication of a virus in a porcine mammal.
Working examples and the amount of guidance:
The instant specification offers descriptions of DNA constructs, CRISPR/Cas9 construct, and sgRNAs, as well as working examples of in-vitro transfection experiments on human HEK293 cells. However, there are no working examples of in-vitro transfection experiments on porcine cells or in vivo experiments demonstrating a method for inhibiting infection of or reducing replication of a virus in a porcine mammal in need thereof, as claimed.
Example 1 (p 163) recites that the “direct prevention of ASFV using genome editing in the animal to target the virus early in its development of an infection can be accomplished by delivery of a DNA construct such as described in the specification as well as in FIGURE 1, FIGURE 2, FIGURE 3 and FIGURE 5,” but Example 1 merely shows DNA constructs designed to target genes for cleavage that are involved early in the replication cycle of ASFV and thereby disrupt the gene and stop replication, and does not show the use of said DNA constructs for genome editing in vivo. Likewise, Example 2 (p 163-165) shows the construction of a CRISPR/Cas9 construct targeting DNA polymerase and Topoisomerase II of ASFV for direct gene targeting, but does not demonstrate the use of said construct for genome editing in vivo. Example 3 (p 165-167) shows the selection of sgRNAs capable of targeting multiple genes in the ASFV genome through the targeting of conserved sites within multigene families, but is not directed to an in vivo example.
Examples 4 and 5 (p 167-168 and p 168-169, respectively) describe in-vitro transfection experiments, wherein each vector described in Table 5 was transfected into cultured HEK293 cells using a standard Lipofectamine 3000 protocol (para 81, 85). In Example 4, the expression of Cas9 endonuclease was verified by Western blotting and RT-PCR following transfection (para 81). In Example 5, each targeted gene was inserted into a lentiviral vector and transformed into E. coli for production of model plasmid DNA, then lentiviruses bearing the corresponding genes were co-transfected alongside the targeting plasmids (para 85). Viral gene targeting was assessed by a heteroduplex formation assay 48 hours after co-transfection (para 86). Importantly, the cells used in for transfection in Examples 4 and 5 are HEK293, which are immortalized human embryonic kidney cells, not porcine cells.
The specification does not offer working examples directed to inhibiting infection of or reducing replication of a virus in a porcine mammal in need thereof, as recited in independent claim 61, or more broadly, any in-vivo working examples, or even any in-vitro examples directed to porcine cells. Moreover, the specification fails to provide guidance in correlating or applying data from the in-vitro assays on human HEK293 cells to the in-vivo method of inhibiting infection or reducing replication of a virus in a porcine mammal, as claimed.
The quantity of experimentation necessary:
Based on the content of the disclosure, undue experimentation is required to use the invention as claimed. As stated above, the experimental examples disclosed in the specification are in-vitro transfection experiments on human HEK293 cells. However, the claims are drawn to an in-vivo method for inhibiting infection of or reducing replication of a virus in a porcine mammal in need thereof.
First, the transfection experiments conducted on HEK293 cells in Examples 4-5 of the specification must be validated in porcine cells. Subsequently, the in-vitro data must be validated in vivo; that is, it is necessary to establish that the method for reducing replication of ASFV in vitro is effective to inhibit infection or reduce replication of ASFV in a porcine mammal in vivo. Given the changes in environments between in vitro and in vivo conditions, CRISPR/Cas-based gene editing methods do not necessarily replicate in vivo. Additionally, the safety of CRISPR/Cas-based gene editing methods, including the potential for off-target effects, must be evaluated for in-vivo applications. Given that a method for reducing replication of ASFV was only established in vitro at the time the invention was made, the process to apply this method to inhibit infection or reduce replication of ASFV in a porcine mammal in vivo requires significant experimentation.
In conclusion, the disclosure does not support a method for inhibiting infection of or reducing replication of a virus in a porcine mammal in need thereof, comprising introducing to a cell of said porcine mammal a nuclease comprising a gene-binding moiety, wherein said gene binding moiety is configured to bind at least one essential gene of said virus, or any combination thereof, wherein said virus belongs to the family Asfarviridae, wherein said at least one essential gene of said virus encodes Topoisomerase II or a fragment thereof, RNA helicase or a fragment thereof, or DNA polymerase or a fragment thereof, as recited in independent claim 61. The evidence provided in the instant specification, in light of the teachings available in the art, does not enable one skilled in the art to make and use the claimed invention without undue or reasonable experimentation. Therefore, the methods recited in claims 61-69 and 72-82 are not enabled.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Risa Takenaka whose telephone number is (571)272-0149. The examiner can normally be reached M-F, 12-7 EST.
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/RISA TAKENAKA/Examiner, Art Unit 1632
/TITILAYO MOLOYE/Primary Examiner, Art Unit 1632