DETAILED ACTION
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-4, in the reply filed on 8/18/26 is acknowledged.
Claims 5-8 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Claim Rejections - 35 USC § 112-2nd paragraph
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 1-4 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is vague and indefinite because it is unclear what is meant by a “Chlamydia knock-out.” Chlamydia is a bacterium, and the claim does not make it clear that it is the gene that expresses GrgA that is the gene that is knocked out. A Chlamydia sp. not expressing GrgA does not necessarily mean the GrgA gene was knocked out in as gene expression has many different factors. The claim wording is confusing and not fully descriptive. The claim also provides no structure for the GrgA gene. It is also taught that the C.trachomatis generated in the specification has conditional GrgA deficiency which is not instantly conveyed. The claims do not provide sufficient description in the claims to satisfy the Statute's requirement of adequately describing and setting forth the inventive concept. While the specification can be used to provide definitive support, the claims are not read in a vacuum. Rather, the claim must be definite and complete in and of itself. Limitations from the specification will not be read into the claims. The claims as they stand are incomplete and fail to provide adequate structural properties to allow one to identify what is being claimed. Accordingly, the claim is being interpreted to encompass any Chlamydia sp. with deletions/mutations that does not express GrgA or expresses an extremely diminished level of GrgA as described in the instant specification.
Appropriate clarification and/or correction is required.
Claim 2 is vague and indefinite due to the phrase “and the like.” The metes and bounds of this term cannot be readily understood. The use of the term “and the like” does not precisely define the metes and bounds. Like what? Other Chlamydia? Other Gram-negative bacteria? While the specification can be used to provide definitive support, the claims are not read in a vacuum. Rather, the claim must be definite and complete in and of itself. Limitations from the specification will not be read into the claims. The claims as they stand are incomplete and fail to provide adequate structural properties to allow one to identify what is being claimed. Appropriate correction is required.
Claim Rejections - 35 USC § 112-Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-4 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.
The instant claims recite:
1. (Original) A Chlamydia knock-out that does not express GrgA.
2. (Originial) The Chlamydia knock-out of claim 1 wherein the Chlamydia is selected from the group consisting of C. trachomatis, C. pneumoniae, C. psittaci, C. muridarum, C. suis, Chlamydophila. abortus, Chlamydophila felis, Chlamydophila pecorum, C. ibidis, C. avium, C. gallinacea, and the like.
3. (Original) The Chlamydia knock-out of claim 2 wherein the Chlamydia is selected from the group consisting of C. trachomatis, C. pneumoniae, and C. psittaci.
4. (Original A vaccine comprising the Chlamydia knock-out of claim 1 and a pharmaceutically acceptable carrier.
To fulfill the written description requirements set forth under 35 USC § 112, first paragraph, the specification must describe at least a substantial number of the members of the claimed genus, or alternatively describe a representative member of the claimed genus, which shares a particularly defining feature common to at least a substantial number of the members of the claimed genus, which would enable the skilled artisan to immediately recognize and distinguish its members from others, so as to reasonably convey to the skilled artisan that Applicant has possession the claimed invention. Applicants have not described the genus of any Chlamydia knockout that does not express GrgA such that the specification might reasonably convey to the skilled artisan that Applicants had possession of the claimed invention at the time the application was filed.
Paragraph [0034] teaches that Applicants attempted but failed to disrupt grgA through group II intron (Targetronᵀ) insertional mutagenesis. Previously, saturated chemical mutagenesis also failed to generate grgA-null mutants. The specification has shown that using Targetron vectors containing spectinomycin-resistance gene-bearing group II introns specific for multiple grgA insertion sites. Whereas they did obtain spectinomycin-resistant chlamydiae, diagnostic PCR analysis detected the intron but not within grgA. Taken together with the earlier unsuccessful attempts to generate grgA-null mutants using chemical mutagenesis it was found that grgA is an essential gene and, as such, not amenable to conventional mutagenesis approaches. The data shown in the specification demonstrate the successful disruption of chromosomal grgA in L2/cgad-peig, in which expression, and thus function, of GrgA depends on ATC-induced GrgA expression from pGrgA-DOPE, e.g., a recombinant C. trachomatis comprising a plasmid- complemented, chromosomal grgA-disrupted L2/cgad-peig. This does not provide adequate written description for the full breadth of the claimed Genus.
Applicants teach that they developed and applied a novel genetic tool that they termed DOPE (dependence on plasmid-mediated expression) and showed that GrgA is necessary for RB-to-EB differentiation during the late developmental cycle and is further required for optimal RB growth. They generated a specific recombinant Chlamydia trachomatis grgA conditional knockout which leads to slower C. trachomatis growth and lack of RB-to_EB growth.
Paragraph [0050] teaches that DOPE offers several advantages over technologies developed for studying essential genes that rely on downregulation using deactivated CRSPR-associate d proteins (dCas) and complementation using constitutive expression from a transformed plasmid. Because specific genes are disrupted by an intron, DOPE is devoid of the "off-target" effects of CRISPR interference. More importantly, DOPE lacks the nonspecific toxicity of dCas9. With a modified, carefully calibrated, and tunable anhydrotetracycline (ATC)-inducible system, DOPE allows for studying essential genes such as GrgA that are toxic when constitutively expressed from a recombinant plasmid.
Specification at paragraph [0051] Derivation of CtL2 with conditional GrgA knock-out (L2/cgad-peig) through DOPE was attempted. The grgA gene was disrupted using type II intron insertional mutagenesis to further investigate the role of GrgA in chlamydial physiology. However, multiple attempts resulted in two transformants whose grgA remained intact. These negative results, together with the failures of saturated chemical mutagenesis studies and transposon mutagenesis to produce grgA-null mutants, suggest the possibility that that grgA is an essential gene. We then attempted to knock down GrgA expression through CRISPR transcription interference using deactivated Cas9 from multiple sources but discovered that the CRISPR/dCas9 systems are toxic in Chlamydia in the absence any guide RNA.
[0062] The chlamydiae to be used for vaccines according to this invention are produced using ATC in the culture medium to induce GrgA expression. ATC is removed from the vaccine preparations and is not present in the subject so that when the vaccines are administered to the subject, expression is GrgA is turned off. This prevents continued infection and cause elevated levels of released immunogenic but non-infectious RB.
[0090] In summary, the grgA gene was disrupted in the Chlamydia trachomatis chromosome using a group II intron bearing aadA, which confers resistance to spectinomycin. The disruption was made possible only in Chlamydia transformed with a plasmid carrying grgA, in which the intron target site was synonymously mutated. Hence, this gene-targeting strategy is referred to as DOPE (dependence on plasmid-mediated expression). The ATC-inducible expression system was reengineered in the DOPE plasmid to drastically diminish the GrgA expression level, which allowed for effective complementation of chromosomal grgA disruption without excessive GrgA overexpression-mediated growth inhibition.
The specification teaches the construction of a shuttle vector named pGrgA-DOPE, which encodes an anhydrotetracycline (ATC)-inducible grgA allele (plasmid-encoded inducible grgA or peig) (Fig. S1). In addition to the replication origin, they kept all eight genes encoded by the wild-type C. trachomatis plasmid in the shuttle vector. This is because Pgp1, 2, 6, and 8 are essential for plasmid maintenance, while Pgp3 and 4 are a virulence determinant and regulator of certain chromosomal genes, respectively. Compared to the native chromosomal grgA allele containing a Targetron-insertion site between nucleotides 67 and 68, the grgA allele in pGrgA-DOPE carries a His-tag sequence and four synonymous point mutations around the intron-targeting site, rendering peig resistant to the Targetron designed for this site.
Since ATC-induced GrgA overexpression previously caused C. trachomatis growth inhibition, Applicants employed a weakened ribosomal binding site identified via a green fluorescence protein reporter (Fig. S2A through D) in pGrgA-DOPE to drive the expression of His-tagged GrgA (His-GrgA). They also removed a region that contained potential alternative −35 and −10 promoter elements between the ATC-inducible promoter and the ribosomal binding site from pGrgA-DOPE (Fig. S2E).
They next transformed L2/cg-peig with the aforementioned Targetron plasmid carrying an aadA-containing group II intron that targets the insertion site between nucleotides 67 and 68 in grgA. ATC was employed to induce GrgA expression from pGrgA-DOPE while selecting for chromosomal mutants carrying an intron-disrupted grgA using spectinomycin. PCR analysis confirmed the genotypes of L2/cg, L2/cg-peig, and the plasmid-complemented, chromosomal grgA-aadA-disrupted L2/cgad-peig (Fig. 1A and B; Table S1). Sanger sequencing confirmed the nucleotide sequences surrounding the intron target site in L2/cg and L2/cg-peig, and the grgA-intron joint regions in L2/cgad-peig (Fig. 1C). Importantly, western blotting detected a time-dependent GrgA loss in L2/cgad-peig following ATC withdrawal (Fig. 1D). By 2 h post-ATC withdrawal, His-GrgA was nearly undetectable. Collectively, the data demonstrates the successful disruption of chromosomal grgA in L2/cgad-peig, in which expression, and thus function, of GrgA depends on ATC-induced GrgA expression from pGrgA-DOPE.
Accordingly, this experiment does not provide full written description for the breadth of the instant claims, e.g., any Chlamydia knockout that does not express GrgA. The specification only provides written description for Chlamydia trachomatis with the disruption of chromosomal grgA in L2/cgad-peig, the plasmid-complemented, chromosomal grgA-aadA-disrupted L2/cgad-peig, and is noted includes drastically diminished expression of GrgA.
With the written description of a genus, merely drawing a fence around a perceived genus is not a description of the genus. One needs to show that one has truly invented the genus, i.e., that one has conceived and described sufficient representative species encompassing the breadth of the genus. Otherwise, one has only a research plan, leaving it to others to explore the unknown contours of the claimed genus. See Ariad, 598 F.3d at 1353 (The written description requirement guards against claims that "merely recite a description of the problem to be solved while claiming all solutions to it and . . . cover any compound later actually invented and determined to fall within the claim's functional boundaries."). Abbvie Deutschland GmbH & Co. v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 U.S.P.Q.2d 1780, 1790, 2014 BL 183329, 12 (Fed. Cir. 2014).
To fulfill the written description requirements set forth under 35 USC § 112, first paragraph, the specification must describe at least a substantial number of the members of the claimed genus, or alternatively describe a representative member of the claimed genus, which shares a particularly defining feature common to at least a substantial number of the members of the claimed genus, which would enable the skilled artisan to immediately recognize and distinguish its members from others, so as to reasonably convey to the skilled artisan that Applicant has possession the claimed invention. Applicants have not described the genus of claimed Chlamydia knockouts that don’t express GrgA such that the specification might reasonably convey to the skilled artisan that Applicants had possession of the claimed invention at the time the application was filed.
The purpose of the "written description" requirement is broader than tomerely explain how to "make and use"; the applicant must convey with reasonableclarity to those skilled in the art that, as of the filing date sought, he or she was inpossession of the invention. The invention is, for purposes of the "writtendescription" inquiry, whatever is now claimed. See Vas-Cath, Inc. v. Mahurkar,935 F.2d 1555, 1563-64, 19 USPQ2d 1111, 1117 (Federal Circuit, 1991).Furthermore, the written description provision of 35 USC § 112 is severable fromits enablement provision; and adequate written description requires more than amere statement that it is part of the invention and reference to a potential methodfor isolating it. The nucleic acid itself is required. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. V. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016. Possession may be shown in a variety of ways including description of an actual reduction to practice, or by showing the invention was 'ready for patenting' such as by disclosure of drawings or structural chemical formulas that show that the invention was complete, or by describing distinguishing identifying characteristics sufficient to show that the applicant was in possession of the claimed invention. Moreover, because the claims encompass a genus of variant species, an adequate written description of the claimed invention must include sufficient description of at least a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics sufficient to show that Applicant was in possession of the claimed genus. However, factual evidence of an actual reduction to practice has not been disclosed by Applicant in the specification; nor has Applicant shown the invention was "ready for patenting" by disclosure of drawings or structural chemical formulas that show that the invention was complete; nor has Applicant described distinguishing identifying characteristics sufficient to show that Applicant were in possession of the claimed invention at the time the application was filed. 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'" (Id. at 1106); accordingly, it follows that an adequate written description of a genus cannot be achieved in the absence of a disclosure of at least one species within the genus.
It is noted that MPEP 2111.01 states that "[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow." The Court of Appeals for the Federal Circuit has recently held that a "written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." University of California v. Eli Lilly and Co., 1997 U.S. App. LEXIS 18221, at *23, quoting Fires v. Revel, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993). To fully describe a genus of genetic material, which is a chemical compound, applicants must (1) fully describe at least one species of the claimed genus sufficient to represent said genus whereby a skilled artisan, in view of the prior art, could predict the structure of other species encompassed by the claimed genus and (2) identify the common characteristics of the claimed molecules, e.g., structure, physical and/or chemical characteristics, functional characteristics when coupled with a known or disclosed correlation between function and structure, or a combination of these (paraphrased from Enzo Biochemical).
University of Rochester v. G.D. Searle & Co. (69 USPQ2d 1886 (2004)) specifically points to the applicability of both Lilly and Enzo Biochemical to methods of using products, wherein said products lack adequate written description. While in University of Rochester v. G.D. Searle & Co. the methods were held to lack written description because not a single example of the product used in the claimed methods was described, the same analysis applies wherein the product, used in the claimed methods, must have adequate written description (see Enzo paraphrased above).
The specification does not describe the structure for a representative number of knock out Chlamydia sp. with the required functionality in the claim.
Because the art is unpredictable and the scope of the claim includes numerous structural variants, and the genus is highly variant because a significant number of structural differences between genus members is permitted. The specification does not describe any members of the claimed genus by complete structure. One of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the genus, and thus, that the applicant was not in possession of the claimed genus. The claimed subject matter is not supported by an adequate written description because a representative number of species has not been described.
Based on the lack of knowledge and predictability in the art, those of ordinary skill in the art would not conclude that the applicant was in possession of the claimed genus.
"Possession may not be shown by merely describing how to obtain possession of members of the claimed ,genus or how to identify their common structural features" (See University of Rochester, 358 F.3d at 927, 69 USPQ2d at 1895). A definition by function, as we have previously indicated, does not suffice to define the genus because it is only an indication of what the .gene does (function), rather what it is (structure), see University of California v. Eli Lilly & Co., 43 USPQ2d 1938, thus above claims lack adequate written description.
Applicant is referred to the revised guidelines concerning compliance with the written description requirement of U.S.C. 112, first paragraph, published in the Official Gazette and also available at www.uspto.gov
Claim Rejections - 35 USC § 112-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 1-4 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.
The instant claims recite:
1. (Original) A Chlamydia knock-out that does not express GrgA.
2. (Originial) The Chlamydia knock-out of claim 1 wherein the Chlamydia is selected from the group consisting of C. trachomatis, C. pneumoniae, C. psittaci, C. muridarum, C. suis, Chlamydophila. abortus, Chlamydophila felis, Chlamydophila pecorum, C. ibidis, C. avium, C. gallinacea, and the like.
3. (Original) The Chlamydia knock-out of claim 2 wherein the Chlamydia is selected from the group consisting of C. trachomatis, C. pneumoniae, and C. psittaci.
4. (Original A vaccine comprising the Chlamydia knock-out of claim 1 and a pharmaceutically acceptable carrier.
The instant specification teaches a recombinant Chlamydia trachomatis transformed with plasmid pGrgA-DOPE to cause disruption of chromosomal grgA (e.g., L2/cgad-peig) and a Targetron plasmid carrying an aadA-containing group II intron that targets the insertion site between nucleotides 67 and 68 in grgA, expression, and thus function, of GrgA depends on ATC-induced GrgA expression from pGrgA-DOPE.
The instant specification fails to teach any other Genus/species, than C. trachomatis or GrgA in C.trachomatis. The specification also does not enable the breadth of any Chlamydia knock-out that does not express GrgA, as it is shown in the instant specification that since GrgA is an essential gene, normal means to create a knockout do not work. It also fails to provide working examples of any Chlamydia knock-out GrgA as a vaccine.
The instant Specification at paragraph [0051] teaches derivation of CtL2 with conditional GrgA knock-out (L2/cgad-peig) through DOPE was attempted. The grgA gene was disrupted using type II intron insertional mutagenesis to further investigate the role of GrgA in chlamydial physiology. However, multiple attempts resulted in two transformants whose grgA remained intact. These negative results, together with the failures of saturated chemical mutagenesis studies and transposon mutagenesis to produce grgA-null mutants, suggest the possibility that that grgA is an essential gene. We then attempted to knock down GrgA expression through CRISPR transcription interference using deactivated Cas9 from multiple sources but discovered that the CRISPR/dCas9 systems are toxic in Chlamydia in the absence any guide RNA.
Claim 4 recites the use of the Chlamydia as a vaccine, which could not contain toxicity to be effective and safe. Accordingly, the instant specification fails to enable vaccines comprising any Chlamydia knockout that does not expresss GrgA as a vaccine. There are no results for immunization experiments or challenge experiments. It is well known in the prior art that vaccines against Chlamydia are highly unpredictable. See paragraph [0005] which teaches there is no Chlamydia vaccine for human use and the efficacy and safety of animal Chlaymdia vaccines remain uncertain. The instant specification has not provided results with any of the multitude of knockout Chlamydia sp. vaccines encompassed in instant claim 4.. Given the inherent unpredictability of combination vaccines combined with the very large and divergent group of GrgA knockouts of any type and from any species recited in the claims, including the ones that retain toxicity as described in the instant specification, it would take one skilled in the art undue experimentation to make and/or use the invention.
Genentech Inc. v. Novo Nordisk A/S (CAFC) 42 USPQ2d 1001 clearly states: “Patent protection is granted in return for an enabling disclosure of an invention, not for vague intimations of general ideas that may or may not be workable. See Brenner v. Manson, 383 U.S. 519, 536, 148 USPQ 689, 696 (1966) (stating, in context of the utility requirement, that "a patent is not a hunting license. It is not a reward for the search, but compensation for its successful conclusion.") Tossing out the mere germ of an idea does not constitute enabling disclosure. While every aspect of a generic claim certainly need not have been carried out by an inventor, or exemplified in the specification, reasonable detail must be provided in order to enable members of the public to understand and carry out the invention.”
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1 and 4 is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by and Fan et al "GrgA:a Multifunctional Transcription Factor that Control Chlamydial Gene Expression", National Institute of Allergy and Infectious Diseases, 2019, 1 page; provided by Applicants; priority date as recited in PCT International examination PCT/US23/16633 ; provided by Applicants).
Fan discloses a Chlamydia knock-out that does not express GrgA (a novel chlamydia-specific transcription factor termed GrgA that controls chlamydial growth examine the effects of GrgA gene knockout (a Chlamydia knock-out that does not express GrgA); abstract). Although Fan fails to explicitly disclose a vaccine comprising the Chlamydia knock-out of claim 1 and a pharmaceutically acceptable carrier. The term “vaccine” is an intended use only. A recitation of the intended use of the claimed invention must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim. A “pharmaceutically acceptable carrier” reads on water and therefore would be inherent in the preparation of the mutants.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1-4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bao et al (PNAS, 10/1/2012, 109(42): 16870-16875) in view of Griffais et al (US 2011/0159041; provided by Applicants).
As recited in the 112, second paragraph rejection above, the claims are being interpreted to encompass any Chlamydia sp. with deletions/mutations that does not express GrgA or expresses an extremely diminished level of GrgA as described in the instant specification.
Bao et al report the identification and characterization of a previously undocumented Chlamydia trachomatis transcription factor, designated GrgA (general regulator of genes A). They demonstrated in vitro that GrgA is a DNA-binding protein that can stimulate transcription from a range of σ66-dependent promoters. They further show that GrgA activates transcription by contacting the NCR of the primary σ factor of C. trachomatis, σ66. The findings suggest GrgA serves as an important regulator of σ66-dependent transcription in C. trachomatis. Furthermore, because GrgA is present only in chlamydiae, the findings highlight how non-conserved regions of the bacterial RNA polymerase can be targets of regulatory factors that are unique to particular organisms.
Bao identified amino acid residues in GrgA that were required for sequence-nonspecific DNA binding. They constructed a series of GrgA mutants with deletions of 50–82 amino acid. GrgA mutants lacking amino acids 1–64 (Δ1–64), 65–113 (Δ65–113), 166–206 (Δ166–206), or 207–288 (Δ207–288) all exhibited DNA binding activities that were identical to that of the wild-type GrgA protein. In contrast, the GrgA mutant lacking amino acid residues 114–165 (Δ114–165) was severely compromised in its ability to bind DNA (Fig. 3A and Fig. S4C), suggesting that residues 114–165 comprise a portion of GrgA that is essential for sequence-nonspecific DNA binding. Noticeably, this region is rich in positively charged lysine and arginine residues (Fig. S4D), which may mediate the binding of GrgA to the negatively charged DNA. Furthermore, the lysine/arginine-rich sequence and a following region appeared to form a helix-turn-helix (Fig. S4D), a structural motif characteristic of DNA binding proteins.
Next, they assessed the effects of the various deletions in GrgA on the ability of GrgA to activate transcription from the defA promoter. Transcription activation was essentially that of the wild-type GrgA for the Δ65–113, Δ166–206, and Δ207–288 mutants. In contrast, the Δ114–165 mutant, which could not bind DNA, suffered a significant (∼75%) loss of transcription activation activity compared with wild-type GrgA (Fig. 3B), suggesting that the ability of GrgA to bind DNA is important for GrgA-dependent transcription activation. In addition, the Δ1–64 mutant, which retained a strong DNA binding activity (Fig. 3A and Fig. S4C), also displayed a significant (∼65%) loss of transcription activation activity (Fig. 3B). These results indicate that DNA binding is necessary but not sufficient for GrgA to efficiently activate transcription.
The Identification of GrgA as a defA Promoter-Binding Protein with Transcription Activation Activity. PDF catalyzes the removal of the N-formyl group from the leading methionine of newly synthesized proteins. Most bacterial proteins require the deformylation and subsequent removal of the N-terminal methionine to function properly. Furthermore, even for the small proportion of proteins that can function while carrying the N-formyl methionine, deformylation is necessary for the initiation of regulated degradation. Thus, PDF is a potential therapeutic and/or prophylactic target for infectious diseases. Accordingly, small inhibitors of PDF have shown effectiveness against a variety of pathogens, including chlamydiae both in vitro and in vivo.
Bao recites that their goal was to identify additional chlamydial proteins that might serve as potential therapeutic targets. Bao concludes that because GrgA can stimulate transcription in vitro from several promoters that control the expression of genes that are critical for chlamydial growth, GrgA likely represents a promising antichlamydial target. Although Bao fails to explicitly disclose a vaccine comprising the Chlamydia knock-out of claim 1 and a pharmaceutically acceptable carrier. The term “vaccine” is an intended use only. A recitation of the intended use of the claimed invention must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim. A “pharmaceutically acceptable carrier” reads on water and therefore would be inherent in the preparation of the mutants. However, Bao does not specifically recite making the mutants and expressing in a Chlamydia bacterium.
Griffais is in the field of modulating bacterial infections comprising vaccines (Abstract), and teaches a vaccine comprising Chlamydia (modifications of the polypeptide make it capable of modulating, regulating, inhibiting or inducing the expression of a gene of Chlamydia, in particular of Chlamydia trachomatis and its variants, or one of its associated micro-organisms, wherein the associated micro-organisms are: the genus Chlamydia, which is composed of four species: Chlamydia psittaci, Chlamydia pecorum, Chlamydia pneumoniae and Chlamydia trachomatis; and/or capable of modulating the replication cycle of Chlamydia, in the host cell and/or organism, allowing for incorporation into vaccine compositions; Paras. [0004], [0125]-[0127]) and a pharmaceutically acceptable carrier (a pharmaceutically acceptable vehicle or carrier; Para. [0374]). Further, the term “vaccine” is an intended use only. A recitation of the intended use of the claimed invention must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim. A “pharmaceutically acceptable carrier” reads on water and therefore would be inherent in the preparation of the mutants. Accordingly, it would have been prima facie obvious to express the mutants disclosed by Bao in host cells as described by Griffais as doing so would allow for recombinant expression of these proteins and/or provided for attenuated cells for use in treatment.
Prior art not presently relied upon:
Zhang et al (PLoS One. 2019 Mar 1;14(3):e0212874)
GrgA as a potential target of selective antichlamydials. In summary, we have unequivocally established that R51G GrgA is both necessary and sufficient for the low level of benzal-N-acylhydrazones (BAH) resistance in the Chlamydia variant MCR. These findings and the facts that GrgA is a Chlamydia-specific protein and plays important roles in chlamydial transcription indicate GrgA as a promising selective therapeutic/prophylactic target, even though it is unclear whether GrgA is a direct target of BAH or regulates BAH susceptibility without directly interacting with BAH. In addition to the high selectivity, the ultralow rate of BAH resistance in chlamydiae is another super attractive feature for developing BAH compounds as therapeutic/prophylactic agents.
Forsyth et al (US 2002/00227)
Forsyth discloses a method of determining whether a bacterial gene in a bacterial cell is essential to its growth or development (the method serves as a test for essentiality of the targeted gene, e.g., applied to essential genes, only cells with a wild type allele on the chromosome will be obtained, wherein growth or development would be would be measured in the fact that the null allele would be lethal, i.e., a method of determining whether a bacterial gene in a bacterial cell is essential to its growth or development; Para. [0123]), comprising: (a) determining the growth or development of the bacterial cell when the gene is intact and expressed (culturing of the strain, comprising the desired gene, in growth medium lacking chloramphenicol but containing sucrose is used to select for such recombinative resolution, i.e., determining the gene is intact and expressed, wherein sucrose is toxic per se if the plasmid has lost both the plasmid sequences from the chromosome and the autonomously replicating plasmid, i.e., determining the growth or development of the bacterial cell when the gene is intact and expressed; Paras. [0027], [0122]); (b) disrupting the gene (a nucleic acid sequence complementary to at least a portion of within an operon in a sequence comprising a proliferation-required gene whose activity or expression is inhibited by an antisense nucleic acid, i.e., disrupting the gene; Para. [0018]) and determining the growth or development of the bacterial cell when the gene is not functional (expression of said nucleic acid in a microorganism, wherein the nucleic acid comprises and inhibitory effect, i.e., when the gene is not functional, is capable of inhibiting the proliferation, i.e., growth or development, of a microorganism; Paras. [0011], [0027]); (c) introducing into the bacterial cell with the bacterial gene disrupted, a plasmid that contains an inducible version of the bacterial gene (expressing, i.e., inducible version of the bacterial gene, the coding sequence of an entire gene identified as required for bacterial proliferation or a fragment thereof operably linked to a promoter in an expression vector, i.e., plasmid; Para. [0127]) and determining the growth or development of the bacterial cell when the bacterial gene is induced and when the bacterial gene is not induced (growth inhibition may be measured by monitoring optical density of a culture growth solution, i.e., determining the growth or development of the bacterial cell, wherein the determining step may comprise determining whether said compound inhibits the growth of said sensitized cell, i.e., when the gene comprising an antisense nucleic acid may be transcribed from an inducible promoter is induced, to a greater extent than said compound inhibits the growth of a nonsensitized cell, i.e., when the said gene is not induced; Para. [0039]).
Wurihan et al GrgA controls Chlamydia trachomatis growth and development by regulating expression of transcription factors Euo and HrcA
July 2020
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