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 10/20/2023. Claims 1, 5, 10, 12-14, 17, 19, 24, 30, 32-34, 38, 44, 47, 51, 53-54, and 56 are pending. Claims 33-34, 38, 44, 47, 51, 53-54, and 56 are withdrawn from examination as they are drawn to a non-elected invention. Claims 1, 5, 10, 12-14, 17, 19, 24, 30, and 32 are currently under examination.
Election/Restrictions
Applicant’s elections of Group I in the reply filed on 9/30/2025 and the species elections “bacterial infection,” “siRNA,” “chromosomally,” “Escherichia,” “HEK293,” and “gene product associated with virulence” in the reply filed on 4/13/2026 are acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)). The Applicant’s election reads on claims 1, 5, 10, 12-14, 17, 19, 24, 30, and 32.
Drawings
The drawings are objected to because the figures are not properly labeled.
37 CFR 1.84 (u)(1) states “The different views must be numbered in consecutive Arabic numerals, starting with 1, independent of the numbering of the sheets and, if possible, in the order in which they appear on the drawing sheet(s). Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter. View numbers must be preceded by the abbreviation "FIG." Where only a single view is used in an application to illustrate the claimed invention, it must not be numbered and the abbreviation "FIG." must not appear.”
The drawings are objected to because Figures 6B and 11B are improperly labeled. Figures 6B and 11B contain partial views on separate sheets. For example, Figure 6B spans two separate sheets and is labeled “Figure 6B” and “Figure 6B Continued” but should be labeled “Figure 6B” and “Figure 6C”. The same is true for figure 11B.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
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.
Claims 1, 5, 10, 12-14, 17, 19, 24, 30, and 32 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 “nucleic acid targets (e.g., hybridize to).” Recitation of “e.g., hybridize to” is exemplary language and it is unclear if the claim is meant to be narrowed by the recited parenthetical exemplary language. The metes and bounds are therefor not clearly established in the claim.
Claims 5, 10, 12-14, 17, 19, 24, 30, and 32 depend from claim 1 and do not resolve this 112(b) issue and are therefore also rejected.
Claim Rejections - 35 USC § 112 - Enablement
Claims 1, 5, 10, 12-14, 17, 19, 24, 30, and 32 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of delivering an siRNA inhibitory nucleic acid to an E. coli cell in vitro, where the E. coli cell is contacted with a mammalian extracellular vesicle (EV) derived from an HCT116 cell comprising the siRNA inhibitory nucleic acid in vitro, does not reasonably provide enablement for the delivery of an inhibitory nucleic acid in vivo, or to other prokaryotic cells other than E. coli, or for using mammalian cells other than HCT116 cells to package inhibitory RNAs other than siRNA. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 U.S.C. 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988). Wands states, on page 1404:
Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex parte Forman. They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of these in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
Nature of the Invention/Breadth of the Claims
Regarding claim 1, claim 1 is drawn to a method of delivering one or more inhibitory nucleic acids to a prokaryotic cell, where the method comprises contacting the prokaryotic cell with a mammalian extracellular vesicle (EV) which comprises the inhibitory nucleic acid. Thus, claim 1 recites functional language of the cargo of the mammalian EV because the nucleic acid delivered is recited to be “inhibitory” and targets a gene in the prokaryotic cell. Furthermore, claim 1 broadly encompasses the category “prokaryote,” and therefore includes the kingdom “prokaryote” without limitation, to include prokaryotes found in the environment, in vitro, or in vivo.
Claim 1 is problematic because the specification is not enabling for the scope of the recited method. For instance, a practitioner is not enabled to use the recited method to target specific in vivo cells within a subject such as a human subject because as discussed further below it is known in the art that EVs can not be effectively targeted to specific cells, where furthermore said EVs would be loaded with specific target nucleic acids. The specification does not show that an EV carrying an inhibitory nucleic acid can be effectively targeted to deliver the cargo to a specific target in vivo.
Furthermore, claim 1 is not enabling for the broad genus of “prokaryote” which includes bacteria such as obligate intracellular bacteria and gram-positive bacteria, neither of which were tested in the specification. As discussed further below, it is known in the art that targeting such obligate intracellular bacteria or gram-positive bacteria is highly unpredictable using delivery mechanisms such as EVs.
Additionally, claim 1 is broadly drawn to the genus of “inhibitory nucleic acids” which can target specific genes within a prokaryote. However, such a broad genus of genes to be targeted by the inhibitory nucleic acid is highly unpredictable as taught in the art because large portions of complex microbial ecosystems such as the human microbiome are not characterized genetically, where such complexity and unpredictability would cause undue experimental burden on a practitioner to carry out the method to identify proper target genes so that cargo nucleic acid in claim 1 would act as an “inhibitory” nucleic acid (see below). Gene expression of prokaryotes are largely undefined and uncharacterized, thus selecting a target is highly unpredictable and would task the practitioner with undue experimental burden.
Claim 1 is further broadly drawn to EVs derived from any mammalian cell type. However, it is known in the art that not all mammalian EVs function in the same way, where the practitioner has only reduced to practice EVs from one mammalian cell type (HCT116 cells, see below).
Furthermore, the claims are not enabling for the genus of “inhibitory nucleic acid” because 1) the Applicant has not tested DNA or shown any mechanism that DNA would be delivered to inhibit a prokaryote and 2) it is known in the art that other forms of RNA besides siRNA (e.g., mRNA) are technically challenging to load into an EV (see below).
Additionally, claim 5 recites “bacterial infection” and claim 19 recites “pathogenic bacterium.” The specification is not enabling for these categories of bacteria to be targeted using the method because bacteria such as intracellular pathogens and gram-positive bacteria were not tested, nor is there a predicable expectation of success when using the recited method to target such bacteria (see below). Further, claim 5 and its dependent claim 12 are recited to target prokaryotes in a subject; the specification is not enabling for in vivo use of the method (see below).
Furthermore, claim 17 recites that the genes targeted are located chromosomally. However, the specification has only demonstrated that inhibitory RNA has been used, and does not propose or demonstrate a method where such RNA has targeted the chromosome of a target cell. Rather, the specification only shows that RNA delivered has reduced expression of a target gene, which likely occurs at the level of translation, and not by targeting the chromosome of the target prokaryotic cell (see below).
Guidance Provided in the Specification
With regards to the guidance provided in the specification, the Applicant offers Examples 1-3. In Example 1, the Applicant demonstrates that HCT116 cells can be loaded with siRNA against an antibiotic resistance gene (gentamicin resistance) which can be packaged into extracellular vesicles (EVs). The EVs were harvested and cultured in high saturation (100 EVs per CFU of E coli) with E coli cells which were transformed with a plasmid containing the gentamicin resistance gene. After culturing, E coli cells were plated, where growth was inhibited in E. coli cells which were grown with EVs containing siRNA against the gentamicin resistance gene (see Example 1, pages 33-34). A similar experiment was performed in Example 1 where GFP was targeted and diminished fluorescence was observed in E coli cells that were co-cultured with EVs containing siRNA against the GFP gene.
Example 2 provides data and experiments which focus on human mucosal biofilms and host interactions with said biofilms via altered mRNA and microRNA during cancer (see pages 36-52). While Example 2 provides transcriptomic data relating changes to bacterial gene expression and host gene expression, and its relation to mRNA and microRNA, Example 2 does not reduce to practice a method of delivering inhibitory nucleic acids to prokaryotes via mammalian EVs, and is therefore only a prophetic or speculative example.
Example 3 is similar to Example 1 in that it is also an in vitro experiment where E coli cells are cultured with mammalian EVs loaded with siRNA which targets a flagellar protein endogenous to E coli (see pages 52-53). E coli cells were cultured with a high saturation of EVs loaded with siRNA against the flagellar protein (100 EVs per 1 CFU of E. coli). Example 3 shows that mRNA expression was inhibited in E. coli cells cultured with EVs loaded with siRNA against the flagellar protein but not in the random siRNA control (see Example 3, pages 52-53).
Thus, the Applicant’s specification has reduced to practice examples of HCT116 cells which were loaded with siRNA against exogenous plasmid genes (gentamicin resistance or GFP, Example 1) or an endogenous gene (flagellar protein, Example 3). The Applicant has not reduced to practice the recited method in vivo. The Applicant has not demonstrated that such siRNA-loaded mammalian EVs could target a specific bacterial cell, as Examples 1 and 3 were only performed in a culture of E coli cells. The Applicant has not tested or shown guidance with evaluating other types of prokaryotes/bacteria, including intracellular bacteria or gram-positive bacteria. The Applicant has not offered guidance with the identification of target genes which could be used as inhibitory targets across the broad and complex genus of “prokaryote.”
State of the Art
Regarding the state of the art, it is known that using EVs to target specific cell types for cargo delivery is inherently challenging and unpredictable. For instance, Jiang (Jiang L et al. Gene Ther. 2017 Mar;24(3):157-166) is a review article that focuses on the prospects of using EVs to deliver therapeutic cargo (Title, Abstract, and throughout). Jiang teaches that:
“For mammalian cell derived EVs, target cell specificity seems to be largely lacking, especially after systemic administration, hence improving EV
targeting ability may be advantageous,” (page 162, right column, first paragraph).
Thus, Jiang teaches that there is a shortage/lack of targeting capacity when using mammalian EVs to target specific cells, and in particular when such EVs are administered systemically/generally (page 162, right column, first paragraph). Thus, Jiang teaches inherent unpredictability when attempting the recited methods of the present claims, which include the in vivo administration of the recited EVs. Furthermore, Jiang teaches that there is unpredictability with targeting a specific cell for delivery of EV cargo, which casts doubt on the ability of EVs to target and deliver cargo to the diverse category of prokaryotes presently claimed, including in in vitro methods (page 162, right column, first paragraph), The present specification has only demonstrated in vitro models of delivering such inhibitory nucleic acids to E. coli cells, where target cells (E. coli, Examples 1 and 3) were 1) grown in the absence of any other bacterial cells and 2) were grown at a high saturation of EVs (100 EVs per CFU of E. coli, Examples 1 and 3). The specification does not demonstrate that the siRNA loaded EVs are capable of targeting any specific cell type. This is further problematic because the specific cargo an EV is recited to carry targets a specific gene within a target prokaryote. The Application does not demonstrate that it is possible to deliver an EV to a specific prokaryote so that the EVs cell-specific cargo nucleic acid is properly delivered to specific cell type.
Jiang further teaches that:
“Gene therapy has the potential to be widely used in the treatment of various diseases, including cancer, infection diseases and central nervous disorders as an effective, personalized therapy approach. However, several hurdles exist when translating this potential to the clinic, including the need for delivery of nucleic acids to the diseased target site…Existing challenges include identifying the right donor cells for EV production, finding an efficient way to load therapeutic nucleic acid into EVs, as well as improving the targeting properties,” (Conclusion).
Thus, Jiang teaches that there are several hurdles that are known associated with the actual reduction to practice of using EVs in applied methods such as problems with delivery of the EV to a proper target site, using the correct donor cells, loading the therapeutic nucleic acid into the EV, and targeting the EV to a specific cell (above). The Applicant has not addressed any of these hurdles and has instead only offered an example where EVs are simply directly cultured with E. coli cells in vitro in a culture flask. The specification is therefore not enabling for the scope of what is claimed.
Additionally, the Applicant has only tested one cell type to produce EVs: HCT116 cells (Examples1 and 3). It is known in the art that different mammalian cells are widely variable concerning their ability to produce EVs. For instance, Amiri (Amiri A et al. J Transl Med. 2022 Mar 14;20(1):125.) is a research article focused on the use of exosomes to deliver RNA therapeutics (Title, Abstract, and throughout). Amiri teaches that:
“Despite many benefits, the exosome-based RNA delivery is restricted since generating adequate amounts of RNA-loaded exosomes for in vivo application is technically challenging. First, only a few cell sources have been observed to release an adequate quantity of exosomes that are needed for clinical translation,” (page 5, left column, final paragraph).
Thus, Amiri teaches that it is known that different cell types produce variable amounts of exosomes, and further that only a few cell types are adequate for the delivery of RNA (above). Thus, the Applicant has not characterized the broad genus of mammalian cells which could be used to make exosomes to deliver inhibitory RNA molecules by only testing HCT116 cells.
Furthermore, the Applicant is broadly claiming “inhibitory nucleic acid.” As an initial matter, the Applicant has not demonstrated the delivery of any DNA molecules to a prokaryote, nor identified mechanisms by which DNA would be used as an inhibitory molecule. The specification is therefore a priori not enabling for the use of “DNA” as an inhibitory molecule in the present methods recited. Furthermore, it is known in the art that RNA molecules are difficult to deliver using EVs. For instance, Amiri teaches that:
” Although post-insertion of siRNA and shRNA plasmid into exosomes through electroporation technique has shown more remedial efficacies than synthetic nanocarriers in repressing oncogenic targets in the pancreatic cancer preclinical models, loading of large RNAs like mRNAs into exosomes remains challenging technically,” (page 5, left column final paragraph into right column first paragraph).
Thus, Amiri teaches that there are known technical challenges when attempting to load nucleic acids other than siRNA and shRNA into EVs (above). The specification is therefore not enabling for the general category of “nucleic acid” as recited, as it is known in the art that technical challenges not addressed by the Applicant exist when trying to load EVs with nucleic acids other than siRNA and shRNA (above).
Furthermore, it is known in the art the genus of “prokaryote” is incredibly uncharacterized, broad, and diverse. For instance, even defined microbial niches and environments are incredibly complex. Khalil (Khalil M et al. Microorganisms. 2024 Nov 15;12(11):2333) is a review article that focuses on the gut microbiome and its role in disease (Title, Abstract, and throughout). Khalil teaches that:
“The gut microbiota is the most complicated ecosystem in nature because it supports vast bacterial populations in the intestine and colon, with roughly 1011–1012 microorganisms/gram of intestinal content, and the majority are anaerobes (95% of the total organisms),” (Introduction, second paragraph).
Regarding the complexity of the gut microbiome, Khalil teaches that “At least 2000 species remain largely uncultured candidate bacterial species,” (page 2, sixth paragraph) and further teaches that:
“The microbial composition is highly variable and highly personalized among individuals and can be arranged as the core microbiota, which is constantly associated with a given host genotype or a specific environment, or as the transient microbiota, which can change over time,” (page 2, paragraph 8).
Thus, Khalil teaches that the gut microbiome is incredibly complex, where upwards of 2000 species are largely uncharacterized and have yet to be cultured, where furthermore the gut microbiome is highly dynamic and changes between individuals and also changes over time (page 2, paragraphs 6-8). Taken together, Khalil teaches that there is a high degree of unpredictability within a given bacterial niche such as the microbiome of an individual (above). When coupled with the fact that Jiang teaches that EVs can not be efficiently targeted to specific cells for delivery, the complexity of the present invention is compounded, where it is highly uncertain that a mammalian EV loaded with a bacterial cell-specific target could reach and deliver its cargo to a specific cell, especially in light of the fact that no cell-specific targeting or mechanism was tested in the present application. Given the vast diversity of cells in bacterial niches such as the microbiome, there is a high degree of complexity and lack of certainty known in the art when practicing the recited method.
Furthermore, the present method encompasses prokaryotic cells with known barriers associated with the method steps as claimed. For instance, the genus of “prokaryote” includes organisms such as obligate intracellular pathogens including Coxiella Burnetti. For instance, Clemente (Clemente TM et al. Front Cell Infect Microbiol. 2023 Aug 14;13:1206037), is a review article that focuses on obligate intracellular pathogens (Title, Abstract, and throughout). Clemente teaches that “Pathogens occupying membrane-bound vacuoles are sequestered from the innate immune system and have an extra layer of protection from antimicrobial drugs,” (Abstract). Thus, Clemente teaches that obligate intracellular pathogens can occupy vesicles located within a host cell, where such bacteria are protected from standard immunological defense mechanisms (Abstract). However, the present specification does not offer guidance or support for targeting such intracellular pathogens using EVs, where given the fact that certain infectious diseases such as Q fever caused by Coxiella burnetti are caused by intracellular pathogens, it is unlikely that such an EV vesicle with a nucleic acid targeting C. burnetti could enter into a host cell and then into a vesicle and finally be delivered into a C. burnetti cell (see Figure 1 of Clemente). Thus, the Applicant has not demonstrated functionality for the broad genusof “bacterial infection” as presently recited in claim 5, or “pathogenic bacterium” as recited in claim 19.
In addition, the Applicant has not demonstrated that the recited method is compatible with bacteria such as gram-positive bacteria. For instance, Bose (Bose S et al. Microb Cell. 2020 Oct 5;7(12):312-322) is a research article focused on EVs and their roles in gram-positive bacteria (Title, Abstract, and throughout). Bose teaches that:
“EV biogenesis in gram-positive bacteria is a complex process because of the presence of a thick peptidoglycan barrier. In almost all organisms, the outer layer provides a protective barrier against different stress conditions. Similarly, gram-positive bacteria possess a thick cell wall ranging between 20- 40 nm that helps to withstand the extremities like osmotic pressure, DNA-damaging agents, exposure to antibiotics. Kim et al. reported that peptidoglycan (PGN) is the major component of the cell wall in addition to polysaccharides, proteins, and polymers. The thick PGN layer with a pore size of approximately 2 nm may prevent the release of EVs with a diameter of 20-400 nm. Thus, the intriguing question that remains is “how EVs traverse the thick cell wall?” In S. aureus, it was demonstrated that the action of certain PGN degrading enzymes could remodel the cell wall in a way that may facilitate EVs to transit across the cell wall,” (page 314, right column, first paragraph).
Thus, Bose teaches that gram-positive bacteria possess a thick layer of peptidoglycan which is a known barrier to EV transport which must be traversed using specialized enzymes to remodel the cell wall (above). The Applicant has not demonstrated that such enzymes as would be required to deliver EV cargo to a gram-positive bacteria are present in mammalian EVs, or specifically what mammalian EVs would be capable of delivering cargo to a gram-positive bacteria because they have only tested E. coli, which is a gram-negative bacteria lacking such a peptidoglycan cell wall (Examples 1 and 3 of the specification). Thus, not only is the specification not enabling for the broad genus of “prokaryote” to include gram-positive bacteria, the specification is not enabling for embodiments of the invention as recited in claims 5 and 19, which are drawn to “bacterial infections” or “pathogenic bacteria,” which includes gram-positive bacteria which can cause diseases such as MRSA (page 314, right column, second paragraph).
Furthermore, the art teaches that bacterial/prokaryotic gene expression is incredibly diverse and uncharacterized, where a practitioner faces uncertainty and unpredictability when selecting an “inhibitory nucleic acid” to target a gene, as presently recited. For instance, Joice (Joice R. et al. Cell Metab. 2014 Nov 4;20(5):731-741) is a research article focused on molecular targets in the human microbiome (Title, Abstract, and throughout). Joice teaches that:
“The human microbiome comprises trillions of bacteria, archaea, fungi, protozoa, and viruses…As of 2013, a total of over 20,000 microbial community profiles had been deposited in the sequence read archive (SRA), comprising an estimated 10 million genes…Even prior to this flood, however, it was well-known that functional
characterization of microbial genes lags behind the ability of the field to generate new microbial sequence data (Galperin and Koonin, 2010). Between 30 and 40% (and often as much as 60 to 70%) of the genes from newly-sequenced microbial isolates are functionally uncharacterized despite a growing database of available reference information…The problem of uncharacterized novel microbial gene sequences is further exacerbated in microbial communities, in which a large proportion of genes community-wide remains uncharacterized after annotation. Roughly 50% of genes in the gut microbiomes of HMP participants, for example, could not be characterized using standard annotation methods,” (Introduction, paragraphs 1-3).
Thus, Joice teaches that prokaryotic genes, including in niches such as the human microbiome, are largely uncharacterized and unpredictable. This teaching places uncertainty on a practitioner’s ability to select an inhibitory nucleic acid to target a specific prokaryote, as Joice teaches that the genus of genes within prokaryotes are still largely undefined and uncharacterized.
Additionally, it is known in the art that uncertainty exists in bacterial gene expression even in well-characterized organisms such as E. coli. For instance, Kim (Kim J et al. Sci Rep. 2025 Nov 6;15(1):38870) is a research article that focuses on E. coli gene expression in the gut (Title, Abstract, and throughout). Kim teaches that:
“the complexity of the microbiota poses significant challenges. In this study, we analysed the gene expression of Escherichia coli in the intestines of IBD mouse models in the context of a native gut microbiota. We employed a reporter E. coli expressing reverse transcriptase-Cas1 fusion protein and Cas2 to record transcript data on plasmids as short oligonucleotides. Gene expression profiles differed between IBD models and controls and varied with the type of inflammatory trigger and time point. However, pre-feeding Lactobacillus crispatus prior to IBD induction resulted in E. coli gene expression profiles resembling those of controls despite exacerbation of colitis. In conclusion, altered E. coli gene expression in the inflamed gut may reflect environmental changes driven by interactions between inflammation and the microbiota,” (Abstract).
Thus, Kim teaches that gene expression profiles of a given target prokaryote such as E. coli varies over time in a complex and unpredictable way, as microbiota can interact with environmental changes which shape overall gene expression. Therefore, not only are most genes, and by extension genes which could be targeted for inhibition as recited, not characterized in microbiome niches (per Joice), even well-characterized organisms such as E. coli change target gene expression in uncontrollable ways (per Kim, above).
Given the complexity of the genus “prokaryote” as recited in claim 1, and even the complexity of in vivo microbial niches (claims 5 and 12), the specification is not enabling for the broad genus of inhibitory nucleic acids because many prokaryotic organism are not characterized. Hence, no defined targets of the inhibitory nucleic acids are known for the broad genus of “prokaryote”, let alone targets which could kill the prokaryote, per claim 10.
Regarding claim 14, as discussed above, Amiri teaches that there are inherent technical challenges when loading RNA that is not siRNA or shRNA into EVs (page 5, left and right columns). Thus, the specification is not enabling for what is claimed in claim 14 (e.g., “mRNA”).
Regarding claim 17, claim 17 recites that the genes targeted are located on the chromosome. However, the specification does not offer evidence that genes are targeted chromosomally. Furthermore, proposed mechanisms of action known in the art related to how inter-kingdom cross-talk occurs relate to inhibitory mechanisms at the level of translation, not by targeting chromosomal genes. For instance, Choi (Choi JW et al. Exp Biol Med (Maywood). 2017 Sep;242(15):1475-1481) is a review article that focuses on inter-kingdom communication between bacteria and eukaryotes (Title, Abstract, and throughout). Choi teaches that inhibitory siRNA can target transcribed targets of prokaryotes, but does not propose that the chromosome of the prokaryote itself is targeted by siRNA delivered from the eukaryotic EVs (see Figure 1). Thus, the specification has not demonstrated that chromosomal targeting occurs, where furthermore it is known in the art that the proposed mechanism of action for such EV delivery of RNAs targets prokaryotic transcripts and not the genome/chromosome itself (Figure 1 of Choi). Additionally, Choi’s teachings apply to the lack of enablement of the genus “inhibitory nucleic acid” because Choi teaches that the proposed mechanism of action relies on RNA, and not DNA, which is broadly encompassed by “nucleic acid.” (Choi, Figure 1).
Regarding claims 24 and 32, as discussed above, both Jiang (Conclusion) and Amiri (page 5, left and right columns) teach inherent unpredictability when using different mammalian cells to generate EVs, where only a few mammalian cells are known to be efficient at EV generation. The specification is not enabling for the cell types listed because only HCT116 cells have been tested in the instant Examples.
Undue Experimental Burden
In the present case, undue experimental burden is placed upon the practitioner in order to carry out the recited methods. For instance, the practitioner would tasked with multiple hurdles in order to use the method as recited, including developing their own method to target specific cells with specific inhibiting nucleic acids, selecting the correct mammalian EV, and packaging the EVs with siRNA, each of which are complex and unsolved hurdles as taught by Jiang. Furthermore, the practitioner would be tasked with not only determining a method to deliver EVs to specific cells in complex niches such as the human microbiome, but also identifying inhibitory nucleic acid targets within such complex niches where the art teaches that most genes and genomes of microbial organisms are not well characterized. Thus, the present method encompasses embodiments where EVs are delivered to specific in vivo niches to deliver target-specific cargo. As such, the methods complexity is compounded by the fact that 1) EVs are not efficiently targeted to specific cells and 2) the EVs must be targeted to specific cells because they are carrying cell-specific “inhibitory” cargo, where both the inhibitory nucleic acid cargo and a method of cell-specific delivery would both need to be worked out experimentally by the practitioner.
Furthermore, the practitioner is burdened with tested such methods in prokaryotes which were not reduced to practice, including obligate intracellular pathogens and gram-positive bacteria. The art teaches that there are inherent hurdles when using EVs to target such cells because intracellular pathogens are protected from the extracellular environment and gram-positive bacteria required special enzymes to break down their cell walls for proper EV delivery. Neither of these hurdles are addressed in the specification, and the art teaches that these are significant barriers to practicing the recited method as it relates to specific bacterial types (intracellular bacteria and gram-positive bacteria).
Additionally, owing to the complexity of microbial niches, such as the human microbiome which is encompassed by the claims, there is little predictability that the in vitro examples offered in the specification would function in vivo, as targeted delivery of such EVs is known to be problematic per Jiang.
Conclusion
The practitioner faces undue experimental burden associated with the presently claimed subject matter, where the specification is not enabling for the scope of the recited methods.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DOUGLAS CHARLES RYAN whose telephone number is (571)272-8406. The examiner can normally be reached M-F 8AM - 5PM.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571)-272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/D.C.R./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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