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
The Amendments and Remarks filed 04 December 2025 in response to the Office Action 04 June 2025 are acknowledged and have been entered. Claim 45 is newly added. Claims 2-3, 6-14, 19-25, and 27-30 are cancelled. Claims 1, 4, 5, 15-18, 26, and 31-45 are under examination on the merits.
Any objection or rejection not reiterated in this office action has been overcome by applicant’s claim amendments and or arguments.
This office action contains rejections that are not necessitated by applicant’s amendments and is therefore a non-final office action.
Priority
The instant application claims priority to 63/118,593 filed 11/25/2020 and 62/959,976 filed 1/11/2020.
Information Disclosure Statement
The information disclosure statement filed 12/04/2025 have been considered.
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.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
Claim 1, 15-17, and 38-40 is rejected under 35 U.S.C. 103 as being unpatentable over Hone (US 6,500,419 B1, 12/31/2002) in view Brodel (Brodel et al. 2013 PLoS ONE 8(12): e82234. doi:10.1371/journal.pone.0082234) and Ko (Ko et al. J. Microbiol. Biotechnol. (2019), 29(1), 127–140). This is a new rejection.
Regarding claims 1 and 40, Hone teaches a method for introducing RNA molecules into eukaryotic cells, wherein the RNA molecules are capable of being translated in the eukaryotic cells and nucleic acids which can be introduced into bacteria for practicing the method of the invention [abstract]. Hone teaches an isolated bacterium comprising a DNA which is transcribed into a messenger RNA molecule in the bacterium, wherein the RNA is capable of being translated in a eukaryotic cell [col. 4, lines 56-60]. Hone teaches that the bacteria can be naturally non-pathogenic and non-invasive, where any bacterial strain can be modified
to modulate, in particular to increase, its invasive characteristics [col.9, lines 48-59]. Hone teaches that the DNA can be heterologous with respect to the bacterium and can be operably linked to a prokaryotic promoter [col. 1, lines 61-62]. Hone teaches that the RNA preferably comprises a 5’ Cap Independent Translation Enhancer (CITE) sequence for allowing efficient translation in a eukaryotic cell [col. 5, lines 11-13; Fig. 2]. Hone teaches that "CITE sequences", also referred to herein as "Internal Ribosome Entry Site" and "IRES sequence", arr any nucleotide sequences, which, when present in an RNA molecule, increase the translation efficiency of the RNA molecule in eukaryotic cells [col. 20, lines 23-27]. Hone teaches a CITE sequence from the encephalomyocarditis virus (ECMV) 5' non-coding region [Fig. 1]. Hone teaches that another sequence which may be included in a eukaryotic RNA expression cassette is a 3’ polyadenylate sequence, and that the polyadenylate tail on an RNA may improve its stability both in the invasive bacterium and in the target eukaryotic cells which is a desirable element [col. 21, lines 15-20; Fig. 2].
Hone does not teach where the IRES is Cricket paralysis virus (CrPV) IRES.
Brodel teaches that internal ribosome entry site (IRES) elements found in the untranslated region of mRNAs enable translation initiation [abstract]. Brodel evaluates the ability of a number of viral IRESs to initiate protein synthesis and direct efficient protein expression in different eukaryotic cell-free expression systems [abstract; pg. 2, col.1, para 2]. Brodel teaches that the IGR IRES from the CrPV genome, versus the IRESs from the Encephalomyocarditis virus (EMCV) and Rhopalosiphum padi virus (RhPV) genomes, was the most efficient across all cell-free systems [pg. 2, col.1, para 2; Fig. 1]. Brodel teaches that this result may facilitate the development of novel eukaryotic cell-free protein expression platforms as well as the high-yield synthesis of target proteins, in particular glycoproteins and membrane proteins, in already established in vitro transcription-translation systems [pg. 2, col.1, para 2].
Ko teaches the development of cap-independent RNA expression platforms using viral IRES with better expression efficiency that will be useful in useful in developing potential RNA-based prophylactic or therapeutic vaccines [abstract, pg. 128, col. 1, para 3]. Ko teaches that EMCV is a Class II IRES which require most eukaryotic initiation factors (eIFs) while Class IV
IRESs, such as CrPV, have a simple translational mode that does not require any eIFs [pg. 128, col. 2, para 1]. Ko investigates cellular GOI expression efficiency in five viral IRESs to include EMCV and CrPV [pg. 128, col. 2, para 2]. Ko teaches that EMCV IRES and CrPV IRES had the highest cellular expression efficiency in comparison to cap-dependent expression platform [pg. 128, col. 2, para 2; Fig. 1B and 1C], and teaches that EMCV and CrPV IRES showed comparable levels of expression in single-RNA expression platforms [Fig. 4]. Ko also teaches that the RNA expression platforms can be injected into mice for in vivo expression [pg. 136, col. 2; Fig. 6].
Taken together, it would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to substitute the ECMV IRES in the bacterium of Hone for the CrPV IRES of Brodel or Ko. One of ordinary skill would be motivated to make this modification since Ko demonstrated comparable cellular GOI expression levels with RNA expression platforms comprising ECMV or CrPV, and CrPV IRES has a simple translational mode by requiring extra initiation factors. Furthermore, one of ordinary skill would be motivated to try this modification given Brodel disclosure that the IRES derived from CrPV is a suitable IRES known to initiate translation and the IRES derived from CrPV was most efficient at protein initiation and synthesis compared to other IRESs with the hope that the combination of the cell type/CrPV RNA platform will yield higher GOI expression. One of ordinary skill would have a reasonable expectation of success because Hone, Brodel, and Ko all teach the use of viral IRES for protein initiation.
Regarding claims 15-16, Hone teaching that the DNA can be heterologous with respect to the bacterium and can be operably linked to a prokaryotic promoter [col. 1, lines 61-62] is a teaching where the transcription of the eukaryote-translatable mRNA is under the control of a promoter that is inactive in a eukaryotic cell.
Regarding claim 17, Hone teaches the bacterium can be a naturally non-pathogenic invasive bacterium [col. 6, lines 10-13 and 31-36].
Regarding claim 38, Hone teaches the DNA will allow transcription in bacteria and in eukaryotic cells. In these embodiments, the DNA will contain transcriptional regulatory elements
directing transcription in prokaryotic cells and in eukaryotic cells (i.e., a eukaryotic promoter) [pg. 30, lines 43-47].
Regarding claims 39, the teachings of claims 1 and 38 are applied as discussed above.
Claim 4, 18 and 26 is rejected under 35 U.S.C. 103 as being unpatentable over Hone (US 6,500,419 B1, 12/31/2002) in view of Brodel (Brodel et al. 2013 PLoS ONE 8(12): e82234. doi:10.1371/journal.pone.0082234) and Ko (Ko et al. J. Microbiol. Biotechnol. (2019), 29(1), 127–140) as applied to claim 1 and 15, and further in view of Lage (Lage et al. J. Vis. Exp. (42), e2099, doi:10.3791/2099. 2010). This is a new rejection.
Regarding claims 4, 18 and 26, the teachings of Hone, Brodel and Ko are discussed above as applied to claim 1 and 15, and similarly apply to claim 4 and 18.
While Hone teaches the bacterium can be a naturally non-pathogenic bacterium [col. 6, lines 10-13], Hone, Brodel nor Ko teach where the bacterium is engineered to have at least one invasion factor to facilitate entry into a eukaryotic cell or release from a eukaryotic cell endosome.
Lage teaches the use of non-pathogenic bacteria, e.g., Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells [abstract]. Lage teaches that the vector that contains the RNA of interest contains the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter β1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes , which produces listeriolysin O and allows the therapeutic RNA to escape from entry vesicles within the cytoplasm of the target cell [abstract].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the bacterium as taught and suggested by Hone, Brodel and Ko to additionally contain the Inv locus or HlyA gene of Lage. One of ordinary skill in the art would have been motivated to have made the modification for the advantage of permitting natural noninvasive bacteria to enter β1-integrin-positive mammalian cells and/or allowing the therapeutic RNA to escape from entry vesicles within the cytoplasm of the target cell. One of ordinary skill would be motivated to make the modification with a reasonable expectation of success because Hone teaches that whether traditionally described as naturally invasive or non-invasive, any bacterial strain can be modified to modulate, in particular to increase, its invasive characteristics [col. 9, lines 48-59]; and both Hone and Lage teach the possibility of increasing the invasiveness of nonpathogenic bacterium.
Regarding claim 26, Hone teaches the administration of invasive bacteria to a subject for the treatment of a disease or condition [col. 32, lines 62-67].
Claims 5, 31-37, and 41-44 is rejected under 35 U.S.C. 103 as being unpatentable over Hone (US 6,500,419 B1, 12/31/2002) in view of Brodel (Brodel et al. 2013 PLoS ONE 8(12): e82234. doi:10.1371/journal.pone.0082234) and Ko (Ko et al. J. Microbiol. Biotechnol. (2019), 29(1), 127–140) as applied to claim 1 and 15, and further in view of Wesselhoeft (Wesselhoeft et al 1 Nature Communications (2018) 9:2629, page 1-10), Petkovic (Petkovic et al. Nucleic Acids Research, 2015, Vol. 43, No. 4) and Ford (Ford et al. Proc. Natl. Acad. Sci. USA Vol. 91, pp. 3117-3121, April 1994). This is a new rejection.
The teachings of Hone, Brodel and Ko are discussed above as applied to claim 1 and similarly apply to claim 5, 31-37, and 41-44.
Regarding claims 5, 31-37, and 41-44, Hone, Brodel and Ko do not teach where the expression cassette encodes a PIE sequence that promotes circularization and covalent closure of the eukaryote-translatable mRNA following its transcription. Hone, Brodel and Ko do not teach where the PIE sequence comprises bacteriophage T4 phage intron and exon elements.
Petkovic teaches that a spontaneous group I intron self-splicing system, designated as the PIE method, allows circular RNA production in vitro and in vivo [pg. 2461, col.2, para 1; Fig. 6]. Petkovic teaches that Ford (and Ares Jr) demonstrated that foreign sequences can be placed in the exon of a permuted group I intron self-splicing system (here termed ‘cyclase ribozyme’) derived from the phage T4 td gene and made circular in vitro [pg. 2461, col.2, para 1]. Ford teaches that synthesis of circular RNA by such transcripts (called RNA cyclase ribozymes) after transcription in vitro [pg. 3117, col.1, para 2]. Petkovic teaches the formation of covalently closed circRNAs using the PIE method [pg. 2454, col. 2, para 2; Fig. 6, pg. 2461, col.2, para 1-3]. Petkovic teaches the generation of circular RNAs in the range of 71–1130 nt and that for circularization of largeRNAs, protocols using the PIE strategy may be the better alternative [pg. 2459, col. 1, para 1, pg. 2461, col. 2, para 2]. Petkovic teaches that the advantage of RNA being in a circular form rather than in the traditional linear one is stability and being protected from degradation [pg. 2454, col. 2, para 2].
Wesselhoeft teaches an engineering approach to generating exogenous circRNAs for potent and durable protein expression in eukaryotic cells [pg. 2, col. 1, para 2]. Wesselhoeft teaches that circularization may allow for the stabilization of mRNAs that generally suffer from short half-lives, extend the duration of protein expression from full-length RNA messages, and may therefore improve the overall efficacy of exogenous mRNA in a variety of applications [pg. 2, col. 1, para 1; abstract]. Wesselhoeft teaches that one of the approaches is a ribozymatic methods using self-splicing introns [pg. 2, col.1, para 3]. Wesselhoeft teaches the ribozymatic method utilized a permuted intron-exon (PIE) splicing strategy that consists of fused partial exons flanked by half-intron sequences [pg. 2, col.1, para 3]. Wesselhoeft teaches that the PIE construct comprises bacteriophageT4 phage intron and exon elements [pg. 2, col.1, para 3]. Wesselhoeft also teaches the design of a pair of spacer sequences to adapt the circRNA construct for efficient circularization of a variety of long intervening RNA sequences [pg. 3] and homnology arms [pg. 2, col. 1, para 3-col. 2, para 1].
Hone provides for additional regulatory elements, which can, e.g., affect the stability of the RNA in the eukaryotic cell [col. 7, lines 13-16], or reduce degradation [col. 21, lines 50-60].
While Ko teaches the development of cap-independent RNA expression platforms, Ko discloses that the capping in cap-dependent RNA expression platforms is necessary to protect against RNA degradation and increase RNA translation [pg. 128, col. 1, para 2].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to place the expression cassette of the bacterium as taught and suggested by Hone, Brodel and Ko in the exon of a permuted group I intron self-splicing system derived from the phage T4 td gene as taught by Wesselhoeft, Petkovic and Ford along with any other factors needed to ensure RNA circularization. One of ordinary skill in the art would have been motivated to attempt the modification for the advantage of stabilizing and protecting the mRNA from degradation, given that the RNA expression construct doesn’t comprise a cap and teachings of circularizing large RNAs.
Response to Arguments
Applicant's arguments filed 04 December 2025 have been fully considered.
Applicant’s arguments regarding the rejection of claims 1, 15-17, 38 and 39 under 35 U.S.C. 103 as being unpatentable over Hone in view of Santamaria and the rejection of claims 4, 18, and 26 under 35 U.S.C. 103 as being unpatentable over Hone in view of Santamaria, and further in view of Lage in that one of ordinary skill in the art would not deem the various IRESes mentioned in Kieft and Santamaria as interchangeable because Kieft teaches that they are mechanistically different was found persuasive and are hereby withdrawn.
Applicants argue that the claimed invention yields unexpectedly improved properties/results relative to the prior art because Applicant's system resulted in 2 orders of magnitude higher translation efficiency using the CRPV IRES compared to Hone's use of the Encephalomyocarditis virus IRES. First, other than claim 40 of the current application, the claimed are not limited to the use of only a CRPV IRES. Second, the teachings of Brodel and Ko, as described in the new rejection discussed above, provides a rationale to combine and suggests that the use of CRPV IRES could results in more than 2 orders of magnitude higher translation efficiency compared to ECMV IRES in a eukaryotic cell or animal, not just a cell-free expression system as applicants argue. Thereby applicant’s arguments regarding claims 1, 15-17, and 38-40 under 35 U.S.C. 103 as being unpatentable over Hone in view Brodel has been modified above to include the additional teachings of Ko.
Applicant’s arguments with respect to the rejection of claims 5, 31-37, and 41-44 under 35 U.S.C. 103 as being unpatentable over Hone in view of Santamaria and further in view of Petkovic and Ford have been fully considered and is unpersuasive. As discussed in the modified rejection above, Hone provides for additional elements in the RNA molecule that stabilizes and prevents degradation of the RNA. Hone also teaches that the delivery of RNA by use of a bacterium provides the advantage that the RNA is “less” likely to be degraded. The term “less likely” does not provide security that the RNA will not be degraded. Furthermore, the claimed expression cassette is not limited to comprising only the IRES, polypeptide, poly A-tail and a promoter. Therefore, based on the teachings of Hone, KO Wesselhoeft, Petkovic and Ford, one would be motivated to modify Hone to achieve stabilization and would not conclude that the PIE system is not scalable.
Conclusion
No claims allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIFFANY N GROOMS whose telephone number is (571)272-3771. The examiner can normally be reached M-F 830-530.
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/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
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