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 .
Application Status
This action is written in response to applicant’s correspondence received on 6/9/2025. Claims 1-8, 10-16, 18-22, 24-25, 27-32, 41-42, 95, and 107-111 are pending. Claims 1, 24, 32, 41-42, and 95 have been amended. Claims 9, 17, 23, 26, 33-40, 43-94, and 96-106 have been cancelled. Claims 109-111 have been newly added All pending claims are currently under examination.
Any rejection of record in the previous office actions not addressed herein is withdrawn. New grounds of rejection are presented herein that were not necessitated by applicant’s amendment of the claims since the office action mailed 12/09/2024. Therefore, this action is not final.
Claim Objections – New Objections
Claims 1, 3, 41, 42 are objected to because of the following informalities:
Regarding claim 1, claim 1 recites “withing” which should be changed to “within” (second-to-last line).
Claim 1 recites “complementary nucleic sequence” which should be amended to say “complementary nucleic acid sequence” (line 22).
Claim 3 recites “andany” which should be changed to “and any” (line 4).
Claim 41 recites “wherein the change is state comprises” which should read “wherein the change in state comprises” (line 11).
Claim 42 recites “wherein the change is state comprises” which should read “wherein the change in state comprises” (line 10).
Appropriate correction is required.
Claim Rejections - 35 USC § 112 – New Rejections
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 1-8, 10-16, 18-22, 24-25, 27-32, 41, 42, 95, and 107-111 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.
Regarding claim 1, claim 1 recites “insertion of the action gene behind the selected native promoter.” Recitation of the action gene location being “behind” the native promoter is unclear, as the positional relationship and therefore location of the action gene relative to the native promoter is unclear with the present language. For instance, it is unclear if the term “behind” is meant to mean upstream or downstream of the promoter. It is recommended that language which is conventional in the art be used to clarify the position of the action gene and its relationship to the promoter (e.g., insertion of the action gene “downstream” or “upstream” of the native promoter, or “insertion of the action gene so that it is under the control of the native promoter”).
Furthermore, claim 1 recites “and wherein measurable average cell death comprising a cfu count reduction of at least 50% in the synthetic microorganism occurs withing about 240 minutes or less following exposure to the second environmental condition” (final four lines of claim 1). Claim 1 is drawn to a method, and it is unclear if a re-exposure of the second environmental condition to the synthetic microorganism is required for the method, or if the recited claim language is only meant to describe an attribute of the synthetic microorganism.
Claims 2-8, 10-16, 18-22, 24-25, 27-32, and 107-108 depend from claim 1 and do not resolve this 112(b) issue and are therefore also rejected.
Regarding claim 15, claim 15 recites “the subject,” however no subject is previously recited in claim 15 or from the claims from which it depends. Recitation of “the subject” therefore lacks proper antecedent basis. Claim 16 also recites “the subject” without proper antecedent basis. Claim 16 depends from claim 15 and does not resolve this issue and is also rejected.
Regarding claim 18, claim 18 recites that the measurable average cell death occurs within a range of preset times. Claim 18 is dependent from claim 1, which is directed to a method of preparing a synthetic microorganism. It is unclear if claim 18 is merely reciting characteristics of the synthetic microorganism of claim 1, or if the “exposure to the second environmental condition” recited in claim 18 is meant to be an additional method step. Claims 19-22 depend from claim 18 and do not resolve this issue and are therefore also rejected.
Regarding claim 24, claim 24 is recited to depend from claim 23. However, claim 23 has been canceled; the claim dependency and limitations of claim 24 are therefore unclear. Claim 25 depends from claim 24 and does not resolve this 112(b) issue and is therefore also rejected.
Regarding claim 25, claim 25 recites “selecting from the group consisting of” and recites a list of gens with associated SEQ ID numbers, followed by “and a substantially identical nucleotide sequence.” This claim is unclear because it is unclear to what the nucleotide sequence is meant to be “substantially identical.” The list includes a group consisting of SEQ ID numbers and “a substantially identical nucleotide sequence” but does not clarify that the substantially identical nucleotide sequence is in reference to one of the previously recited SEQ ID numbers. It is recommended that the claim language be clarified to indicate that the option from the list which includes a “substantially identical nucleotide sequence” refers to a sequence that is substantially identical to one of the recited SEQ ID numbers recited within the list recited in the claim (e.g., “substantially identical nucleotide sequence thereof”).
Regarding claim 27, claim 27 is recited to depend from claim 26. However, claim 26 has been canceled; the claim dependency and limitations of claim 27 are therefore unclear.
Furthermore, claim 27 recites “the inducible first promoter gene.” It is unclear what the “first” promoter gene is in reference to. If claim 27 is meant to depend from claim 1, claim recites “one or more” candidate native inducible genes, and also a selected native inducible promoter. It is unclear which of these is meant to be “the inducible first promoter gene.” Claim 27 therefore lacks proper antecedent basis.
Regarding claim 28, claim 28 recites “the control arm” however, no “control arm” is recited previously in the claim or claim 1. Recitation of “the control arm” therefore lacks proper antecedent basis. Claims 29-32 depend from claim 28 and do not resolve this 112(b) issue and are therefore also rejected.
Regarding claim 41, claim 41 recites “wherein the change is state comprises wherein the second environmental condition comprises” (lines 11-12). It is unclear what the change of state is meant to comprise in this phrase, as it is unclear if recitation of “wherein the second environmental condition comprises” is redundant or unneeded in this claim. Furthermore, no “second environmental condition” is recited previously in the claim; “the second environmental condition” therefore lacks proper antecedent basis. It appears that the phrase “wherein the second environmental condition comprises” may be irrelevant and confuses the claim language of the presently recited claim 41. However, it should also be noted that “the second environmental condition” is recited in the final line of claim 41, and any potential amendment to the claim should retain consistency throughout the claim.
Regarding claim 42, claim 42 recites “wherein the change is state comprises wherein the second environmental condition comprises” (lines 11-12). It is unclear what the change of state is meant to comprise in this phrase, as it is unclear if recitation of “wherein the second environmental condition comprises” is redundant or unneeded in this claim. Furthermore, no “second environmental condition” is recited previously in the claim; “the second environmental condition” therefore lacks proper antecedent basis. It appears that the phrase “wherein the second environmental condition comprises” may be irrelevant and confuses the claim language of the presently recited claim 42. However, it should also be noted that “the second environmental condition” is recited in the final line of claim 42, and any potential amendment to the claim should retain consistency throughout the claim.
Claims 109-110 depend from claims 41 and 42, respectively, and do not resolve these 112(b) issues and are therefore also rejected.
Regarding claim 95, claim 95 recites “wherein the microorganism exhibits a measurable average cell death,” (third-to-last line). However, multiple “mircroorganisms” are previously recited in the claim, including a “synthetic microorganism” and a “target microorganism,” both of which are exposed to a fluid or environment of interest. Recitation of “the microorganism” therefore lacks proper antecedent basis because it is unclear to which microorganism is being referred.
Claim 111 depends from this claim and does not resolve the 112(b) issue and is therefore also rejected.
112(d) – New Rejection
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.
Claims 7 and 14 are 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.
Regarding claim 7, claim 7 appears to recite characteristics of the synthetic microorganisms which are already recited in claim 1. Claim 7 therefore does not appear to further limit claim 1.
Regarding claim 14, claim 14 recites that the pass through strain is an E. coli strain or a yeast strain. Claim 14 depends from claim 11, which recites that the pass through strain is E. coli. Claim 14 therefore broadens the scope of claim 11, and therefore does not further limit the subject matter of the claim from which it depends.
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.
112(a) Maintained (Partially)
(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 11-16 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.
Claims 11-16 recite “a first synthetic nucleic acid sequence encoding a DNA methylation enzyme derived from the target microorganism”. Claim 13 further recites that “the methylation enzyme in the pass through strain allows the pass through strain to impart a methylation pattern on the plasmid DNA similar enough to the methylation pattern of the target microorganism, to enable or enhance efficiency of transformation of the target strain with the plasmid propagated in the pass through strain”. The applicant is therefore claiming a methylation enzyme which provides a methylation pattern onto plasmid DNA to enhance or enable the transformation of said plasmid DNA into the target microorganism. This claim is problematic because the genus of methylation enzyme claimed, wherein the methylation imparts a beneficial methylation pattern onto the plasmid DNA while inside the pass through microorganisms, is not represented by the species of methylation enzymes offered in the specification.
Concerning the specification, the specification offers one strain of E. coli which is used as a pass through organism, wherein the methylation pattern of the target Staphylococcus organism is alleged to be impart on the plasmid DNA shuttle vector in the E. coli strain to enhance the transformation efficiency of the plasmid DNA (paragraphs 463, 525, and Example 5). Thus, the applicant offers one example of a pass through organism with methylation enzymes which impart methylation patterns onto plasmid DNA to improve transformation efficiency.
Monk (Monk IR et al. Front Cell Infect Microbiol. 2012 Apr 12;2:49) is a research article focused on overcoming barriers associated with the genetic manipulation of Staphylococcus aureus (throughout). Monk teaches that “while bypassing the type IV barrier allows us to transform DNA directly into wild-type Staphylococci, the efficiency of plasmid transfer is still low and for some applications borderline for selection (e.g., transferring pVW01ts into S. epidermidis RP62a or direct integration of plasmids at phage att sites mediated by integrase). Bypassing the type I RM barrier would require
the decoration of plasmid with the methylation pattern determined by the hsdMS genes in the strain to be transformed. There is a paucity of information on the properties of the type I RM systems in S. aureus,” page 3, right column, second paragraph. Thus, Monk teaches that there is a lack of information regarding RM systems in S. aureus, and therefore a lack of information regarding methylation enzymes required to overcome such barriers. Monk further teaches in the same paragraph that “the term plasmid artificial modification (PAM) has been coined to describe pre-methylation of plasmid DNA in an E. coli strain which expresses the target strain’s modification and
specificity genes.” In the following paragraph on page 3, Monk teaches that only two groups have used such technology, and that there is only one example of this technique being applied to a type I RM system. There is therefore unpredictability and a paucity of knowledge concerning methylation enzymes which, when expressed in pass through organisms, can be used to efficiently enhance or enable transformation of said plasmids to target organisms. Thus, the applicant’s limited examples of successful methylation enzymes present in a pass through organism, which render increased transformation efficiencies of plasmid DNA when entering into a target organism, do not fully represent this unpredictable genus. The applicants were therefore not in possession of the claimed invention at the time of filing.
Response to Arguments
The Applicant’s arguments have been considered but are not persuasive with respect to claims 11-16. The Applicant has amended the independent claims in order to obviate the original 112(a) rejections. However, dependent claims 11-16 also suffered from an additional 112(a) which was neither addressed by claim amendment or argument. Thus, the original 112(a) rejection of claims 11-16 is maintained (see above).
Claim Rejections - 35 USC § 102 – New rejection not necessitated by amendment
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.
Claims 41, 42, and 109-110 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Starzl (US 2019/0169623 A1, published 6/6/2019).
Regarding claim 41, Starzl teaches a synthetic microorganism comprising a first molecular modification inserted into the genome of S. aureus, where the modification is a cell death action gene that is sprA1, where the first recombinant nucleotide encoding the sprA1 action gene is operatively associated with an endogenous first regulator region or an iron-regulated pathway comprising a native inducible promoter (paragraph 116, Figure 22). Furthermore, Starzl teaches the kill switches can be endogenously regulated, which reasonably includes such a kill switch operatively associated with an endogenous first regulatory region comprising a native first promoter (paragraph, 424). Starzl teaches that the native inducible promoter controlling the sprA1 gene is isdB (paragraph 116, Figure 22). Starzl teaches that isdB was at least 3-fold enhanced for transcriptional activation in a second condition compared with a first condition, where the second condition was serum and/or blood exposure (Figure 13A-13B). Starzl teaches that isdB is an iron-regulated gene (paragraph 82). Thus, Starzl teaches the recited structure of the organism recited in claim 41. With regards to the claim limitation that the synthetic microorganism has measurable reduction of cfu of at least 50% within 240 minutes after exposure to the second environment, recitation of these claim limitations is simply an inherent property of the structure taught by Starzl, above. Thus, the synthetic microorganisms taught by Starzl (above) comprise these inherent properties.
Regarding claim 42, Starzl teaches a synthetic microorganism comprising a first molecular modification inserted into the genome of S. aureus, where the modification is a recombinant nucleotide comprising a regulatory region (promoter) or an iron-regulated pathway, e.g., the isdB gene, per paragraph 82 of Starzl, operably associated with a cell death action gene that is endogenous (sprA1) (paragraph 116, Figure 22). Starzl teaches that the native inducible promoter controlling the sprA1 (an endogenous action gene/kill switch of S. aureus) gene is isdB (paragraph 116, Figure 22). Starzl teaches that isdB was at least 3-fold enhanced for transcriptional activation in a second condition compared with a first condition, where the second condition was serum and/or blood exposure (Figure 13A-13B). Starzl teaches that isdB is an iron-regulated gene (paragraph 82). Thus, Starzl teaches the recited structure of the organism recited in claim 42. With regards to the claim limitation that the synthetic microorganism has measurable reduction of cfu of at least 50% within 240 minutes after exposure to the second environment, recitation of these claim limitations is simply an inherent property of the structure taught by Starzl, above. Thus, the synthetic microorganisms taught by Starzl (above) comprise these inherent properties.
Note that the term “endogenous” recited in the above claims broadly encompasses the introduction of, for instance, the endogenous isdB promoter controlling the sprA1 cell death gene into the chromosome, as taught by Starzl in paragraph 116, Figure 22, and/or Figure 11D. The isdB and sprA1 nucleotides are “endogenous” to the S. aureus microorganisms taught by Starzl, and the fact that they are introduced on a cassette into the genome is irrelevant, as such constructs still read on the recited claim limitations (Starzl, paragraph 116, Figure 22). Furthermore, given that Starzl teaches the broad genus of Staph aureus as a target microorganism (paragraph 79), and that such constructs can be integrated into the genome (paragraph 370), a practitioner could immediately envision the insertion of constructs such as those taught in paragraph 116 into a S. aureus genome. Additionally, Starzl teaches that such kill switches can be endogenous regulated (paragraph 424).
Regarding claims 109-110, Starzl teaches that the inducible promoter can be isdB (paragraph 82).
Claim Rejections - 35 USC § 103 – New Rejection, not necessitated by amendment
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-8, 10-16, 18-22, 24-25, 28-32, 95, and 107-108, and 111 is/are rejected under 35 U.S.C. 103 as being unpatentable over Starzl (US 2019/0169623 A1, published 6/6/2019) in view of Angelichio (Angelichio MJ et al. Infect Immun. 2002 Dec;70(12):6518-23). The rejection is further evidenced by Lowe (Lowe AM et al. Mol Microbiol. 1998 Mar;27(5):967-76).
Regarding claim 1, Starzl is a patent document which teaches the durable introduction of microorganisms into subjects (Title, Abstract, and throughout). Starzl teaches a method of preparing a synthetic microorganism comprising selecting a native inducible promoter gene in a target microorganism for targeted insertion of a synthetic nucleic acid sequence comprising an action gene (e.g., paragraphs 565, “method of preparing a synthetic S. aureus”). Starzl teaches the selection of a native inducible promoter gene in a target microorganism (Example 2, paragraphs 678-693). Starzl teaches the targeted insertion of an action gene (e.g., paragraph 411, where “kill switch” is an action gene). Starzl teaches comparing the relative RNA transcript levels of one or more candidate native inducible promoter genes in the target microorganism when grown in a first environmental condition compared with a second environmental condition (Example 2, Table 12). Starzl teaches that the native inducible gene can be isdB (paragraph 82, Figure 13A). Starzl teaches that the fold-increase is at least 10-fold in the second environment (e.g, serum) for a selected native inducible gene (e.g., isdB, growth in serum, where the fold change is over 100, Figure 13A). Starzl teaches transforming the target microorganism in the presence of a plasmid, where the plasmid comprises a synthetic nucleic acid sequence comprising an action gene flanked by upstream and downstream homology arms comprising complementary sequences to a nucleic acid sequence, for the targeted insertion of the construct into the genome of the target microorganism (e.g., Figure 11D). Starzl teaches that the target microorganism is S. aureus, where the action gene is the cell death gene sprA1 (throughout, e.g., paragraphs 79-80). Starzl teaches that measurable average cell death comprising a cfu count reduction of at least 50% in the synthetic microorganism occurs within 240 minutes or less following exposure of the second environment (e.g., paragraph 73).
In addition, with regards to the limitation that the action gene is flanked with homologous regions to be targeted “behind” a native selected inducible promoter, Starzl teaches that the kill switch (i.e., action genes such as sprA1) can be “endogenously regulated” which reasonably includes a selected native inducible promoter (paragraph 424).
Starzl therefore teaches the essential principles of design of the presently recited method, including the specific inducible promoters (e.g., isdB), bacteria (S. aureus), and kill switches (sprA1), above, as well as a method of determining and selecting a candidate inducible promoter based on transcriptional activation in two separate environments including blood and serum (Example 2 or Starzl). Furthermore, Starzl teaches the integration of a construct comprising the selected inducible promoter controlling the expression of the action gene directly into the chromosome, where such a construct is integrated on a nucleic acid comprising up- and downstream homology arms (Figure 11D, paragraph 54). Furthermore, Starzl teaches that such action genes/kill switches can be “endogenously regulated” which reasonably includes their expression from endogenous promoters (i.e., “behind” native promoters, paragraph 424).
Starzl, while teaching the principle design on the present method as well as chromosomal integration strategies, and teaching that such kill switches as they teach can be “endogenously regulated” and therefore operatively associated with an endogenous promoter (“behind,” i.e., integrated into the genome under control of such promoters) does not explicitly reduce to practice integrating the action gene into the chromosome “behind” the selected native promoter, within certain interpretations of the phrase “behind.”
As an initial matter, as discussed above in the 112(b) rejection, the term “behind” is unclear in this context. Arguably, Starzl does teach the integration of the action gene “behind” the native promoter because, in its broadest sense, “behind” is not clearly defined and does not clearly impart structural limitations to the claim. Broadly speaking, Starzl can be interpreted to teach this limitation simply by an embodiment where the construct of Figure 11D is integrated into the chromosome, where the construct is “behind,” for instance, “upstream” of a native promoter.
Furthermore, it can be reasonably argued that because Starzl teaches a method of identifying an upregulated promoter/gene in a target microorganism, and also that the design and aim of Starzl is to insert a sprA1 kill switch under the control of said upregulated gene/promoter, a practitioner of ordinary skill could immediately envision inserting the kill switch under the control of the inducible promoter already existing within the target microorganism (i.e., the native inducible promoter), because Starzl teaches finding these promoters within the target microorganism. This is further supported by the fact that Starzl teaches that the kill switches can be “endogenously regulated,” where a reasonable interpretation of this term includes insertion of the kill switches downstream (“behind”) of an endogenous promoter (paragraph 424). Thus, a practitioner could reasonably conceive of inserting the kill switch into the target which Starzl has taught to identify, as such an inducible promoter would inherently be known to be upregulated and identified following the teachings and methods of Starzl. In other words, a practitioner could envision including the kill switch under control of the gene which they have just identified using the methods of Starzl, as it would be understood that such a gene that has been identified would have the properties desired by the method of Starzl and already exist within the target microorganism (i.e., upregulation after exposure to a second condition), particularly in light of the teachings of Starzl at paragraph 424 where Starzl teaches that such kill switches can be integrated downstream of a native/endogenous promoter (“endogenously regulated”).
Furthermore, even if it were argued that Starzl does not reduce to practice such a method of integrating a gene downstream of a native/endogenous promoter, the integration of a gene downstream of a native bacterial inducible promoter is already a known method, as taught by in vivo expression technology methods taught by Angelichio (Title, Abstract, and throughout). For instance, Angelichio teaches that a candidate gene can be introduced and expressed from a native promoter using homologous recombination (Figure 1 and page 6518, right column, second paragraph). Furthermore, Angelichio teaches that such methods of inserting a desired gene into the chromosome of a target microorganism to be under the control of a promoter has been used in S. aureus with great success, and has therefore been reduced to practice and is a predictable method of integrating a gene into a desired locus/promoter (page 6521, right column, first paragraph, final sentence, and here references Lowe). Furthermore, as Angelichio here references Lowe, Lowe is also included as an evidentiary reference which also teaches that such gene introduction strategies as IVET are more realistic with regards to the actual genetic expression patterns of a bacteria when inside of a host, i.e., when going from a first environmental condition to a second environmental condition, a desired attribute of the methods taught by Starzl (e.g., Discussion, first paragraph of Lowe). Thus, Angelichio/Lowe teach that a gene can be chromosomally inserted into an inducible gene/promoter of S. aureus, that such methods reliably reflect in vivo expression patterns of microorganisms, and that such methods have been reduced to practice. The result is therefor predictable.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the insertional construct taught by Starzl to be downstream/behind a native promoter, as taught by such methods of Angelichio and Lowe because such a combination is the simple substitution of one known prior art element for another with predictable results. In the present case, a practitioner would substitute the chromosomal integration of a construct including the native promoter and kill switch into the chromosome of S. aureus (Figure 11D, Starzl) for the chromosomal integration of the kill switch directly under control of identified native promoter (as taught by Angelichio/Lowe), where the practitioner is simply substituting one known chromosomal integration strategy for another, where furthermore the kill switch would ultimately be under control of the same promoter. Furthermore, the result is predictable because such integration strategies, where a candidate gene is integrated under the control of a native promoter, is already known and reduced to practice in S. aureus – the host organism taught by Starzl - as taught by Angelichio/Lowe.
Regarding claim 2, Starzl teaches at least a 20-fold change in the second environment (Figure 13A, which shows over a 150-fold change of isdB in serum).
Regarding clam 3, Starzl teaches the time period is 45 minutes (Figure 13A).
Regarding claim 4, Starzl teaches that the microorganism is capable of durably colonizing a first niche (paragraph 346).
Regarding claim 5, Starzl teaches the microorganism can colonize a dermal and/or mucosal niche (paragraph 76).
Regarding claim 6, Starzl teaches that the first environment can be blood, serum, or plasm (e.g., paragraph 321).
Regarding claim 7, Starzl/Angelichio/Lowe teaches that the microorganism can comprise a first molecular modification inserted into the genome of a target microorganism, the modification comprising a first recombinant nucleotide comprising the action gene operatively associated with an endogenous first regulatory region comprising a native inducible promoter, where the native promoter imparts conditionally high level gene transcription in response to a second environment of at least 10-fold compared to the first environment (see rejection of claim 1 for a rejection of these claim limitations, also Figure 11D, and paragraph 381 of Starzl).
Regarding claim 8, Starzl teaches that the synthetic microorganism can also comprise a virulence block (e.g, paragraph 54).
Regarding claim 10, Starzl teaches that the plasmid can be derived from a shuttle vector capable of being used as a pass-through microorganism and the target microorganism (e.g., paragraph 92 and Figure 2).
Regarding claim 11, Starzl teaches that the pass-through strain can be E. coli (paragraph 488). Starzl teaches that it is known that the methylation pattern is important in order to impart a viable plasmid into a target organism, and therefor teaches a motivation to include a methylation enzyme from the target organism in the pass-through strain (paragraph 626). Starzl teaches that it is possible to create a synthetic organism capable of having a unique methylation pattern based on the fact that methylation genes (i.e., methylation enzymes) responsible for such methylation patterns of DNA are known in bacteria (paragraph 626). Starzl teaches that the E. coli pass through strain can comprise an “expression clamp” (e.g., an antitoxin gene capable of targeting the toxin gene/cell death gene, per paragraphs 445 and 488). Given the motivational teaching of Starzl to include a methylation enzyme to faithfully impart a correct methylation pattern onto DNA, it would be obvious to include such an enzyme in the pass-through strain, as Starzl teaches the benefits and feasibility of such an approach (paragraph 626).
Regarding claim 12, Starzl teaches that the “expression clamps” (i.e., antisense antitoxin RNA to target the cell death gene) is designed to help propagate and suppress leaky expression of the toxin (paragraph 376).
Regarding claim 13, Starzl teaches E. coli strain IM08B, the embodiment recited in claim 13 encompassing the claim limitations (paragraph 92).
Regarding claim 14, Starzl teaches that the pass through strain is E. coli (paragraph 92).
Regarding claim 15, Starzl teaches multiple examples of antimicrobial agent-resistant microorganisms such as S. aureus, which can cause bacteremia, and can be the target microorganism (e.g., paragraph 279, paragraph 271, paragraph 341).
Regarding claim 16, Starzl teaches that S. aureus strains can cause SSTI (e.g., paragraph 136).
Regarding claim 18, Starzl teaches that the cell death can occur within 180 minutes (paragraph 379).
Regarding claim 19, Starzl teaches the microorganism can colonize a dermal and/or mucosal niche (paragraph 76).
Regarding claim 20, Starzl teaches that the second condition can be an increase in blood/serum (e.g., paragraph 373).
Regarding claim 21, Starzl teaches that the measurable average cell death can be measured as a reduction in cfu of at least 50% following a defined period of time (e.g., paragraph 55).
Regarding claim 22, Starzl teaches that S. aureus can cause SSTI (e.g., paragraph 136).
Regarding claim 24, Starzl teaches that the action gene can be the sprA1 toxin gene, where the gene includes at least a 5’ portion UTR for antitoxin binding (paragraph 468, which teaches that sprA1 comprises a 5’UTR for the antitoxin gene; by teaching the sprA1 toxin gene, Starzl inherently teaches the 5’UTR of this gene).
Regarding claim 25, Starzl teaches that the kill switch can comprise sprA1 SEQ ID NO 274 (paragraph 80). As shown below, instantly recited SEQ ID NO 3 is a 100% match of the first 117 basepairs of SEQ ID NO 274 of Starzl: SEQ ID NO 274 of Starzl therefore comprises instantly recited SEQ ID NO 3:
PNG
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198
918
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Greyscale
SEQ ID NO 274 (Starzl, top) aligned with instant SEQ ID NO 3 (bottom)
Regarding claim 28, Starzl teaches that the method further comprises inserting a second molecular modifier into the genome of the target, where the second modification comprises an anti-action regulator gene (sRNA) specific for the action gene, where the regulator gene is associated with a second regulatory region comprising a second promoter which is conditionally active in the first environment but not induced, induced less than 1.5 fold, or is repressed after exposure to the second environment for at least 120 minutes (e.g., paragraph 458, Figure 1, paragraph 430, paragraph 427, paragraph 451).
Regarding claim 29, Starzl teaches that the regulator (e.g., antitoxin sprA1) can hybridize with a portion of the action gene (e.g., paragraph 468).
Regarding claim 30, Starzl teaches the regulator can be the sprA1 antitoxin (e.g., paragraph 468, Figure 1).
Regarding claim 31, SEQ ID NO: 5 is the sprA1 antitoxin gene (page 13 of response filed 4/14/2023). Starzl, by teaching the sprA1 antitoxin gene, inherently teaches the sequence of the gene (paragraph 83).
Regarding claim 32, claim 32 recites the native sprA1 promoter expressing a native gene (a sprA1 toxin); thus, given that Starzl teaches S. aureus as a target microorganism, such a target organism would naturally encode the sprA1 expressed from the promoter PsprA1 (paragraph 79).
Regarding claim 95, Starzl teaches a method of preparing a synthetic microorganism comprising a genomically stable, genomically incorporated kill switch modification, comprising identifying a target microorganism that is a S. aureus strain (paragraph 490), selecting a fluid of interest such as blood or serum (e.g., paragraph 503), mapping at least part of the target microorganism genome for KS integration (e.g., Figure 11D, which comprises left and right homology arms, which necessarily means that at least part of the genome is mapped for complementarity for integration), finding an upregulated gene after exposure to an environment of interest (see Example 2, paragraphs 678-693, where upregulated promoters are methodologically discovered), where the inducible promoter is associated with an iron-regulated gene (e.g., isdA in Example 2, an iron-regulated gene per paragraph 82 of Starzl). Starzl teaches identifying a native or non-native toxin gene to the target microorganism (paragraph 110, Figure 16). Starzl teaches creating a plasmid comprising the inducible promoter controlling the cell death gene, transforming the plasmid into the target microorganism, inducing the promoter, and screening/testing for performance (paragraph 488-489). Starzl teaches selecting a lethal candidate toxin gene for genomic integration into the target microorganism under the control of the upregulated promoter (e.g., paragraph 116, Figure 22). With regards to the limitation that the toxin gene is “near the gene or promoter region” in the target microorganism, this claim limitation broadly encompasses simply integrating the cassettes taught by Starzl into the genome (paragraph 116, Figure 22), as Starzl teaches that such genomic integrations can occur anywhere in the genome, and furthermore no specific requirements are recited in the claim limitations otherwise (e.g., the toxin gene is not recited to be required to be under control of the gene or promoter of the target microorganism). Starzl teaches that measurable average cell death of a cfu of 50% reduction occurs within 240 minutes after exposure to the second condition (paragraph 55).
Regarding claim 107, claim 107 merely recites an inherent feature of the microorganisms rendered obvious by the combination of Starzl, Angelichio, and Lowe; given that the creation of such target microorganisms are already taught with motivation in the art (see rejections of claims 1-4, above), any inherent characteristic of said microorganisms can not be considered novel given what would flow naturally from the teachings to generate such organisms.
Regarding claim 108, Starzl teaches the durability of the synthetic microorganism for at least 2 weeks (paragraph 346).
Regarding claim 111, Starzl teaches that the inducible promoter can be isdB (paragraph 82).
Claims 27 is rejected under 35 U.S.C. 103 as being unpatentable over Starzl (US 2019/0169623 A1, published 6/6/2019) in view of Angelichio (Angelichio MJ et al. Infect Immun. 2002 Dec;70(12):6518-23) as applied to claims 1-8, 10-16, 18-22, 24-25, 28-32, 41, 42, 95, and 107-111 above, and further in view of GenBank CP053101 isdB (published 5/11/2020).
Regarding claim 27, Starzl teaches the isdB gene and promoter (paragraph 82, Figure 13A). Starzl therefore inherently teaches the sequence of the isdB promoter and gene. GenBank CP053101 isdB teaches the isdB gene (page 2), and also the sequence of the gene (pages 2-3). Nucleotides 961-1935 of GenBank CP053101 isdB are 99% identical to SEQ ID NO 20 (page 4), and GenBank CP053101 isdB is thus “substantially identical” to SEQ ID 20 of the present application. The isdB sequence SEQ ID NO 20 was therefore known in the art, as taught by GenBank CP053101 isdB.
It would be obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Starzl, Angelichio/Lowe, and GenBank CP053101 isdB to arrive at the presently recited invention because such a combination is the simple combination of known prior art elements with predictable results. In the present case, the combination amounts to the combination of a method taught by Starzl with the promoter isdB identified by Starzl, which GenBank CP053101 isdB teaches is a known sequence matching SEQ ID NO: 20. A practitioner would have a reasonable expectation of success because isdB is a promoter in Staphylococcus and Starzl teaches that their methods are compatible with Staphylococcus, specifically the isdB gene (paragraph 82).
Response to Arguments
The Applicant’s arguments filed 6/9/2025 have been fully considered. The Applicant’s arguments with regards to the original 103 rejections, which were premised on the combination of Falb, Malachowa, and Sayed 1 and 2, are persuasive. Thus, the 103 rejection has been withdrawn. However, upon further search and consideration, a new 103 rejection in light of Starzl I provided above The Application stands rejected under 103 for the reasons detailed in the above 103 rejections.
Conclusion
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/D.C.R./Examiner, Art Unit 1635
/KIMBERLY CHONG/Primary Examiner, Art Unit 1636