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 .
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 1,3-4 are rejected under 35 U.S.C. 103 as being unpatentable over Han M. et al 2006 and further in view of Leenhouts M. et al 1998.
Han M. et al 2006 discloses mapping the origin of replication in the ColE2-P9 plasmid by expressing wild type and variants of the initiator protein (Rep) in bacteria. The mutational analysis of the ColE2-P9 Rep protein in bacteria revealed an origin of replication of 40bp (Figure 1A-C). “We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches…” (abstract).
Han M. et al 2006 does not disclose having the Rep protein integrated into the bacteria genome/chromosome.
Leenhouts M. et al 1998 discloses a system where plasmids containing the pWV01 replicon are mutated to a RepA null phenotype. Therefore, plasmid replication can only occur when RepA is provided in-trans by various bacterial strains carrying RepA integrated into their genome. (Figure 1). Specifically, “The system consists of plasmids that are all based on the broad-host-range lactococcal replicon pWV01 which has been deprived of its gene encoding the replication initiation protein RepA. These so-called pORI plasmids can only replicate if RepA is provided in trans. Special Escherichia coli, Bacillus subtilis and L. lactis helper strains, producing RepA in trans (RepA+), are used as intermediate hosts for the construction of pORI integration plasmids.” (abstract).
Therefore, it would be obvious to a person of ordinary skill in the art, before the effective filing date, to use the ColE2-P9 plasmid with its uniquely minimal 40bp origin of replication as disclosed in Han M. et al 2006, and adapt it into a system analogous to the one disclosed by Leenhouts M. et al 1998, wherein the essential ColE2-P9 replication protein (RepA) is provided in-trans via integration into the host chromosome. In-trans complementation of an essential replication element for ColE2-P9 effectively prevents unwanted plasmid dissemination via bacterial conjugation between probiotic and consensual strains in-vivo.
Claims 22,24-25,27-30 are rejected under 35 U.S.C. 103 as being unpatentable over Han M. et al 2006 and Leenhouts M. et al 1998 as applied to claims 1,3-4 above, and further in view of Trieu-Cuot P et al 1991, and Tabor S et al 1985.
Han M. et al 2006 and Leenhouts M. et al 1998 disclose a recombinant bacteria containing a ColE2-P9-based expression plasmid wherein plasmid replication is controlled through in-trans complementation of the RepA protein.
They do not disclose controlling expression of the genomically integrated replication protein, RepA, via a T7 RNA polymerase-dependent promoter. They also do not disclose a circular DNA vector with two segments wherein one segment contains the replication origin and coding sequence but lacks a selection marker, and is flanked by different restriction enzyme sites.
Tabor S et al 1985 discloses a recombinant bacterial system wherein transgene expression is controlled by the T7 sequence (and therefore T7 RNA polymerase). Specifically, “The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the X PL promoter… The specificity of T7 RNA polymerase for its own promoters…permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.” (abstract).
Cuot P et al 1991 discloses an expression plasmid containing two segments. 1) the first segment comprising the pUC origin of replication and LacZ alpha expression sequence (also home to the Multiple Cloning Site or MCS) is flanked by the exogenous and non-homologous Ndel-AvaII restriction sites, and the second/remaining segment comprising the orthogonal origin of replication pAMB1 and the selection protein erm (aka methyltransferase-encoding gene) (Figure 1 and associated legend).
It would be obvious to a person of ordinary skill in the art, before the effective filing date, to employ the T7 RNA polymerase-dependent promoter system disclosed by Tabor S et al 1985 to control the expression of the genomically integrated RepA protein of the ColE2-P9 origin-based expression system disclosed above. This type of controlled expression allows high resolution titration of RepA expression, helping dial in plasmid copy number for appropriate trans-gene dosage and allowing appropriate tuning of overall metabolic/toxic burdens associated with transgene expression.
It would also be obvious to a person of ordinary skill in the art, before the effective filing date, to replace the pUC origin of replication in the two-segment, orthogonal replication origin containing plasmid disclosed by Cuot P et al 1991 with the ColE2-P9 origin and use the resulting plasmid in the T7-RNA polymerase controlled RepA in-trans complementation/ColE2-P9 origin-based plasmid system disclosed above. This type of dual and orthogonal replication origin plasmid system containing a removable selection cassette facilitates plasmid engineering wherein various therapeutic transgenes can be swapped out from a standard vector backbone by allowing easy maintenance and selection of recombinant plasmids in alternative bacterial species (eg; E. coli.).
Claims 23, 101-106 are rejected under 35 U.S.C. 103 as being unpatentable over Han M. et al 2006, Leenhouts M. et al 1998, Trieu-Cuot P et al 1991, and Tabor S et al 1985 as applied to claim 22 above, and further in view of US 2004142452 A1.
Han M. et al 2006, Leenhouts M. et al 1998, Trieu-Cuot P et al 1991, and Tabor S et al 1985 disclose an easily manipulable vector backbone which contains features that make it an ideal component of engineered probiotic treatments (eg; tightly controlled in-trans complementation of plasmid replication for preventing unwanted dissemination and allowing tunable control over transgene expression/dosages).
They do not disclose the aforementioned plasmid-system wherein segment 1 is flanked by identical recombination sequences, or a method of producing a circular DNA vector containing only the first segment from a parental DNA vector containing both segments.
US 2004142452 A1 discloses a method for inducing recombination of a parental plasmid using exogenous recombinases and their associated recognition sequences. The result is two independent circular DNA vectors, wherein one vector contains the selection marker and the other contains the coding sequence. Figure 30 details the structure of the parental plasmid/vector backbone (pXL4256, approx. 8kb) wherein the selection marker (AmpR) containing segment is separated from the coding sequence segment by flanking exogeneous recombinase sequences (AttB and the segment containing P’, O, P). Additionally, figure 30 details the method of plasmid creation via transformation of the parental plasmid/vector backbone into G6264 E. coli and subsequent recombinase expression induction via arabinose. The result is the 4256 mini-plasmid vector and 4256 minicercle vector. Importantly, they also disclose that, “Sequences permitting site-specific recombination may also be derived from the loxP region of phage P1…” (paragraph 0102).
It would be obvious to a person of ordinary skill in the art, before the effective filing date, to modify the vector backbone plasmid system outlined above by exchanging Ndel-AvaII restriction sites with identical loxP sequences as detailed in US 2004142452 A1, and furthermore, providing an arabinose-controlled Cre-recombinase expression system as disclosed in US 2004142452 A1. This modular design, wherein orthogonal replication origins are floxed, and recombination methodology, allows for easy excision of genetic elements that are; 1) essential solely for therapeutic plasmid construction, from, 2) the minimal plasmid elements required to transform an innocuous bacterium into a probiotic.
Claims 112-113 are rejected under 35 U.S.C. 103 as being unpatentable over Han M. et al 2006, Leenhouts M. et al 1998, Trieu-Cuot P et al 1991, and Tabor S et al 1985, and US 2004142452 A1 as applied to claim 101 above, and further in view of Mullick A et al 2006 and Jusiak B et al 2019.
Han M. et al 2006, Leenhouts M. et al 1998, Trieu-Cuot P et al 1991, and Tabor S et al 1985, and US 2004142452 A1 disclose the vector backbone, therapeutic sub-plasmid creation methodology, and its associated recombinant bacterial-probiotic replication system.
They do not disclose that the recombinase used for non-essential element excision be the Bxb1-GA recombinase, and they do not disclose that it is under control of the cumate-inducible promoter.
Mullick et al 2006 discloses the regulation of gene expression using the bacterial cym operon. Specifically, “In the repressor configuration, regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a strong constitutive promoter. Addition of cumate, a small molecule, relieves the repression.” (results paragraph in the abstract section).
Jusiak B et al 2019 discloses the use of the Bxb1-GA site mutant. Specifically, “Here, we show that a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency…” (abstract).
Therefore, it would be obvious to a person of ordinary skill in the art, before the effective filing date, to use the cuminic acid-inducible promoter system of Mullick et al 2006 to control the expression of the Bxb1-GA site mutant integrase as disclosed by Jusiak B et al 2019, and to substitute that recombinase system for the arabinose-controlled Cre-recombinase expression system as disclosed above (aka the therapeutic sub-plasmid creation methodology). Cumate and its metabolism are alien to most eukaryotic and bacterial pathways and thus the cumate-inducible system won’t inadvertently trigger other cellular cascades, or result in unwanted stress, toxicity, or growth inhibition like other systems (including arabinose). This helps maintain plasmid and sub-plasmid integrity, lowers the probability of random point mutations and the probability of aberrant recombination events. Additionally, the Bxb1-GA site mutant integrase has significant integration efficiency gains over other recombinases, making non-essential therapeutic element segment excision more efficient.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Adam M Smith whose telephone number is (571)272-7517. The examiner can normally be reached Monday- Friday 10:30AM-5PM.
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/Tracy Vivlemore/ Supervisory Primary Examiner, Art Unit 1638
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