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 .
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on July 28, 2025. Claims 1-5 and 8-11 are pending. Claims 4-5 and 8 are withdrawn. Claims 1-3 and 9-11 are currently examined.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION. —The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
(Previous rejection-withdrawn) Claims 1-3, 6-7 and 9-11 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly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.
This rejection is withdrawn in view of the amendments filed on July 28, 2025.
(New Rejection-necessitated by amendment) Claims 1-3 and 9-11 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.
The base claim 1 recites “one or more barcoded host organism libraries” that render the claims indefinite. It is unclear if the “barcoded “is a naturally occurring genetic sequence or an artificially engineered one. Further, it is not clear if the organism contains a single barcode or if various genes in the organism contain a barcode. Also, the base claim 1 recites “barcode abundance” and “gene fitness” that render the claims indefinite. It is not clear how to decide or acquire the “barcode abundance” and what the metes and bounds of the “gene fitness”.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(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.
(New Rejection-necessitated by amendment) Claims 1-3 and 9-11 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 amendments to the base claim 1 introduce new matter.
The amended base claim 1 recites phrases “determining barcode abundance for each gene of a barcoded host organism in a culture prior to infecting by a bacteriophage’ and “log2 (barcode abundance after the infecting/barcode abundance prior to the infecting”, where the “prior to infecting” were not disclosed in the originally filed specification. The instant specification discloses a barcode abundance in time 0/time-zero (See e.g., Fig. 1), but did not disclose” prior to” time zero. Therefore, the base claim 1 introduces new matter.
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.
(previous rejection-withdrawn) Claims 1-3, 6-7, 9 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Bohm et al. (Mol Microbiol. 2018 May;108(3):288-305) in view of Price et al. (Nature. 2018 May;557(7706):503-509) as evidenced by BiTeSiZeBio (https://bitesizebio.com/45131/a-beginners-guide-to-how-blunt-end-cloning-works/).
This rejection is withdrawn in view of the amendments filed on July 28, 2025.
(New Rejection-necessitated by amendment)) Claims 1-3, 9 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Bohm et al. (Mol Microbiol. 2018 May;108(3):288-305) as evidenced by Price et al. (Nature. 2018 May;557(7706):503-509).
The amended base claim 1 is directed to a method for screening for gene function for bacteriophage infection, the method comprising: (a) providing one or more barcoded host organism libraries, (b) determining barcode abundance for each gene of a barcoded host organism in a culture prior to infecting by a bacteriophage, (c) infecting the culture with the bacteriophage, (d) determining barcode abundance for each gene of the barcoded host organism in the culture after infecting by the bacteriophage, and (e) determining a gene fitness for each gene, wherein the gene fitness equals log2 (barcode abundance after the infecting/barcode abundance prior to the infecting)
Bohm et al. describes the genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screenings, and teaches that a random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection (See Title and Abstract). Bohm et al. further teaches the transposon library construction, barcoding and assay for P22 infection by stating the EZ-Tn5TM <KAN-2> (http://www.lucigen.com) being modified to contain an N18 barcode directly adjacent to an Illumina Read 1 sequence and the barcode associated with each unique Tn5 insertion at each position in the genome was then determined (See page 300, right column, paragraph 2).
Accordingly, Bohm et al. teaches a high throughput method for screening the gene function for a bacteriophage comprises the “(a) providing one or more barcoded host organism libraries” such as Salmonella Typhimurium. Bohm et al. teaches the claimed steps (b)-(e) by stating that they compared the genetic profile of mutants with no infection at ‘time zero’ (T0), and after 90 min of further growth (T90-control), to the genetic profile of mutants present after 90 min of infection (T90-infected). The quantitative pattern of Tn5 insertions was measured by PCR of a region that contains the barcode unique to each Tn insertion, followed by Illumina sequencing of the amplified DNA, as previously described (See page 290, left column, paragraph 1), which indicated that determining barcode abundance by PCR before infection and after infection as claimed in (b)-(d). Bohm et al. also teaches that the differences in the relative abundance of transposons within each assayed gene and intergenic region were calculated, along with probabilities, including the False Discovery Rates (FDR). Figure 1 presents a volcano plot of the log10 FDR versus the log2 Fold Change for all 5313 known and putative protein-coding genes assayed in the comparison of Tn profiles of infected cells at T0 and T90 (See page 290, left column, paragraph 3; Fig. 1 and below), which can be evidenced by Price’s study. Price et al. teaches that the fitness value of each strain (an individual transposon mutant) is the normalized log2 (strain barcode abundance at end of experiment/strain barcode abundance at start of experiment) (See page 18, paragraph 5). Therefore, Bohm et al. teaches a comparable way as claimed to calculate the log2 between the barcode abundance after the infecting (T90) /barcode abundance prior to the infecting (T0) and acquiring the gene fitness
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as claimed. At the same time, Bohm et al. also teaches the same active steps a-d and that it is well within the purview of one of ordinary skill in the art to analyze the collected data via various methods.
Regarding claim 2, it requires that the barcoded host organism library is an RB-TnSeq library. Price et al. teaches that coupling TnSeq with random DNA barcoding of each mutant (RB-TnSeq) makes it easier to measure phenotypes across many conditions, and they use RB-TnSeq to address the sequence-to-function gap by systematically exploring the mutant phenotypes of thousands of genes from each of 32 bacteria under multiple experimental conditions (See page 2, paragraph 3). It would be obvious for one of ordinary skill in the art to introduce the RB-TnSeq into Bohm’s study to develop a barcoded host organism library as claimed, and the result would be predictable to construct such an RB-TnSeq library.
Regarding claim 3, it is directed to the method of claim 2, wherein the providing one or more host organism libraries comprises inserting a barcoded transposon into a host organism, wherein the host organism is a Yersinia sp., Escherichia sp., Klebsiella sp., Bordetella sp., Neisseria sp.,Aeromonas sp., Franciesella sp., Corynebacterium sp., Citrobacter sp.,Chlamydia sp., Hemophilus sp., Brucella sp., Mycobacterium sp.,Legionella sp., Rhodococcus sp., Pseudomonas sp., Helicobactersp.,Salmonella sp., Vibrio sp., Bacillus sp., Erysipelothrix sp., Salmonella sp.,Streptomyces sp., Bacteroides sp., Prevotella sp., Clostridium sp.,Bifidobacterium sp., or Lactobacillus sp.
Bohm et al. teaches that the host organism library used for inserting the barcoded Tn is Salmonella enterica serovar Typhimurium (See Abstract). Price et al. also teaches 32 types of Bacteria including E. coli. (See Table B and Summary).
Regarding claim 9, it is directed to a method of claim 1, wherein there is one species of host organism and a plurality of bacteriophage species wherein each bacteriophage species is capable of infecting the host organism.
Bohm et al. teaches two other phages of 9NA and Det7 beside P22 (See page 297, left column, paragraph 1), and these phages are used together to infect Salmonella cells with a serial dilution and then spot in 2ul aliquots on top of the cells, and the plates are incubated at 370C overnight. (See page 301, right column, paragraph 1).
Regarding claim 11, it requires wherein the determining step (b) and the determining step (d) are automated and/or high throughout.
Bohm et al. teaches a high throughput assay was developed to screen an SGD S. enterica strain 14028s panel (See page 301, left column, paragraph 2), also teaches the that EZ-Tn5TM <KAN-2> (http://www.lucigen.com) was modified to contain an N18 barcode directly adjacent to an Illumina Read 1 sequence (See e.g. page 300, right column, paragraph 2), where the Illumina's Read 1 sequence is a product of both high-throughput and automated processes.
(Previous rejection- maintained) Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Bohm et al. (Mol Microbiol. 2018 May;108(3):288-305) as evidenced by Price et al. (Nature. 2018 May;557(7706):503-509) as applied to claims 1-3, 9 and 11 above, and further in view of Bielke et al. (Poult Sci. 2007 Dec;86(12):2536-40.).
Regarding claim 10, it requires that there are a plurality of host organism species and one bacteriophage species wherein the bacteriophage species is capable of infecting each host organism species in the plurality of host organism species.
Bohm et al. teaches that they conclude from experiments that LPS and O-antigen production is not affected by the absence of YajC protein. These results also indicate a role for YajC in a narrow range of bacteriophages, as P22 was affected by its absence, but Det7 and 9NA were not (See page 297, right column, paragraph 1), which indicates that the bacteriophage of Det7 and 9NA can infect more than one host. However, Bohm et al. does not explicitly to point out and discuss the broad bacteria host range.
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Bielke et al. describes the Salmonella host range of bacteriophages that infect multiple genera, and teaches that they selected bacteriophages with the ability to infect more than 1 bacterial genus from the wild-type bacteriophages that were selected for ability to form plaques in Salmonella enteritidis agar overlays. Two selected bacteriophages, confirmed to infect Escherichia or Klebsiella, were further infect 10 different Salmonella serovars (Se Abstract). Bielke et al. discloses that of the 35 bacterial isolates screened, bacteriophages originally selected using SE were able to amplify in 4 different isolates (Figure 1, below) and different species of Salmonella (See Figure 3, below).
Bielke et al. also teaches that the selected WHR (wide-host-range) bacteriophages could amplify to a very high titer with just a few passages in the alternative host bacteria. Another potential advantage of WHR bacteriophages amplified in alternative host bacteria, in the case of enteric disease, is the potential to coadminister bacteria capable of colonizing the gastrointestinal tract to replace natural microflora that may have been displaced by the pathogen, WHR bacteriophages, or both (See page 2540, left column, paragraph 3). Also, selection of nonpathogenic host isolates to support replication of Salmonella bacteriophages may allow improved safety for bacteriophage application to poultry because this would reduce the necessity for 100% purification of the bacteriophages(s) from resistant host bacteria (See Abstract).
It would have been obvious to one of skill in the art to combine the teaching from Bohm, Price and Bielke to arrive at an invention as claimed. Because Bielke teaches that the selected WHR bacteriophages can amplify to a very high titer with just a few passages in the alternative host bacteria that provide a tool for selection of broad host-range bacteriophages for the pathogen of interest (See page 2540, left column, paragraph 3, and Abstract), one of ordinary skill in the art would have been motivated to apply the WHR bacteriophages of Bielke into Bohm’s study for screening for gene function, and there would have had a reasonable expectation of success to develop such a method screening for gene function for a WHR bacteriophage.
Responses to Applicant’s Remarks
Applicant’s arguments filed on July 28, 2025 has been received and fully considered as follows:
Applicant’s argument and amendment on the rejection Under §112(b) are considered. The rejection is withdrawn.
Applicant argued that “Bohm et al., Price et al., and BiTeSiZeBio, either alone or combined together, do not teach, suggest, or motivate "determining a gene fitness for each gene, wherein the gene fitness equals log2 (barcode abundance after the infecting/ barcode abundance prior to the infecting)" (See Remark, page 8).
The argument is not persuasive.
Based on the description above, Bohm and Price both teach using the log2 (barcode abundance after the infecting/ barcode abundance prior to the infecting) as claimed to determine a gene fitness. For example, Bohm teaches the aggregated abundances for the input and output libraries were statistically analyzed using DESeq2 and the log2 fold changes and false discovery rates (FDRs) were estimated as described (See page 301, left column, paragraph 1), and Price teaches that they defined gene fitness to be the log2 change in abundance of mutants in that gene during the experiment (Fig. 1a) (See page 3, paragraph 2). Also, Bohm et al. teaches the same steps a-d and that it is well within the purview of one of ordinary skill in the art to analyze the collected data via various methods. These teaching are also extended to address the argument regarding claim 10 (See Remarks, page 8).
Also, the reference of BiTeSiZeBio is not applied in the current office action. The argument for BiTeSiZeBio is moot.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
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/RUIXUE WANG/Examiner, Art Unit 1672
/NICOLE KINSEY WHITE/Primary Examiner, Art Unit 1672