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
Applicant’s amendments filed October 28, 2025, amending claims 122-148 and 150, and adding new claim 151 is acknowledged. Claims 122-151 are pending and under examination.
It is noted that claims 133 and 134 still have the mark-up “claim [[1]] 112” from the previous claim set, which should have been removed. Claims 133 and 134 in both the present and previous claim set are dependent from claim 122.
The claim sets and submitted Remarks have a thin line around the border. This border prevents automated text processing used by the USPTO. Please remove the line border from all future claim sets and correspondence.
Richter Declaration
The Declaration by Dr. Hagan Richter filed October 28, 2025 is improper as it does not include 1) an acknowledgment by the declarant that willful false statements and the like are punishable by fine or imprisonment, or both (18 U.S.C. 1001) and may jeopardize the validity of the application or any patent issuing thereon, or 2) an assertion that all statements made of the declarant’s own knowledge are true and that all statements made on information and belief are believed to be true. See MPEP 716. Nevertheless, the substance of the Declaration has been considered. Response to evidence and opinion/arguments made in the Declaration are addressed along with Applicant’s arguments in the Remarks after the § 103 rejections below.
Withdrawn Rejections
The amendments to claims 123 and 131 overcome the §112(b) rejection.
The amendment to claim 122 requiring the invasion factor to be a “heterologous” invasion factor and the guide RNA to hybridize to a sequence in a eukaryotic cell overcomes the §101 rejection. The claims are directed to bacteria that are markedly different than their naturally occurring counterpart because 1) the invasion factor must not be native to the bacterial species and 2) native bacterial guide RNAs naturally target bacteriophage and plasmid sequences, not eukaryotic sequences. See e.g., Schmakov et al., mBio (2017), 8: e01397-17.
The claims in copending US Application 17/660663 that were used to reject the instant claims for nonstatutory double patenting were cancelled. The pending claims in the 17/660663 application are patently distinct from the examined claims. The nonstatutory double patenting rejection over copending US Application 17/660663 is withdrawn.
Any other rejection or objection not reiterated herein has been overcome by amendment. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Priority
As indicated in the previous office action, Provisional Application No. 62/856055 fails to provide support for a nuclease, guide RNA, and/or invasion factor being expressed from the bacterial chromosome (claims 126-127, 138-139) or using eukaryotic promoters to express the nuclease and guide RNA (claims 145-146, 150). The first evidence of support for expression of using eukaryotic promoters or from bacterial chromosomes is the PCT Application PCT/US20/35613 (filed June 1, 2020). As such, the effective filing date for claims 126-127, 138-139, 145-146 and 150 is June 1, 2020. The effective filing date of the remaining claims (122-125, 128-137, 140-144, 147-149 and 151) is June 1, 2019.
Claim Interpretation
As indicated in the previous office action, the preambles of claims 122-146 recite compositions “for the delivery of a gene-editing system to a eukaryotic cell” and are interpreted as intended use for the composition. See MPEP 2111.02.II. In this case, the preamble does not define any component of the bacterium or the gene-editing system. The product claims will be examined based on the structure of the bacterium: a bacterium comprising an invasion factor (heterologous or generic), a gene editing nuclease, and guide RNA (claims 122-142), or a plasmid encoding a gene editing nuclease and guide RNA (claims 143-146). There is no requirement in any of the product or method claims that the gene-editing system, guide RNA, or nuclease have any specific gene-editing activity or efficiency in eukaryotic cells. The method claims only require that the gene-editing system be delivered to a eukaryotic cell.
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.
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 122-124, 129, 132-135, 143, 148-149 and 151 are rejected under 35 U.S.C. 103 as being unpatentable over Xiang (Xiang et al., Nature Biotechnology (2006), 24: 697-702; of record) in view of Falb1 (US20190336544 A1, priority to at least January 5, 2018; of record), Falb2 (WO 2017123675 A1, published July 29, 2017; of record) and Critchley (Critchley et al., Gene Therapy (2004), 11: 1224-1233; of record). This is a maintained rejection that has been unsubstantially modified to address claim amendments and address Applicant’s arguments. This is a new rejection of new claim 151.
Regarding claims 122-124, 134-135 and 151, Xiang teaches E. coli bacteria engineered to deliver short hairpin RNA (shRNA) to eukaryotic cells (i.e., a bacterium for delivery of a gene-modulating system to a eukaryotic cell) (Abstract). Xiang teaches E. coli transformed with a plasmid comprising the Inv locus and HylA gene (i.e., a bacterium engineered to express a heterologous invasion factor) (page 700, ¶7). Xiang teaches Inv codes for invasin, which permits noninvasive E. coli to enter b1-integrin-positive mammalian cells (page 697, ¶2). Xiang teaches the gene product of HylA is listeriolysin O, which permits genetic materials to escape form entry vesicles (page 697, ¶2). Thus, Xiang teaches a bacterium engineered to express an invasion factor to facilitate entry of the bacterium into a eukaryotic cell. Xiang also teaches the plasmid encodes for an shRNA under the control of a T7 promoter (i.e., the bacterium comprises a guide RNA for RNA interference) (page 697, ¶2; Supp Fig 1). Xiang teaches the bacteria-transcribed shRNAs are functional for mediating RNAi in mammalian cells (Figure 1).
Xiang does not teach the engineered bacteria expresses a gene editing nuclease or a guide RNA that is used in gene editing.
Falb1 teaches compositions and uses of microorganisms for targeting cancers (i.e., targeting eukaryotic cells) ([0007]). Falb1 teaches engineered microorganisms include E. coli ([0007]). Falb1 teaches microorganisms can be engineered to display invasin on its surface (Table 16-17). Falb1 teaches invasin allows a bacterium to invade a eukaryotic cell ([1160]). Falb1 teaches anti-cancer molecules that can be delivered by the engineered microorganisms of the invention include CRISPR/Cas9 molecules ([0644]). Falb1 incorporates by reference the entirety of WO 2017123675 (herein referred to as “Falb2”) ([0644])
Falb2 teaches genetically engineered bacteria for delivering a payload, including a CRISPR-regulated genetic circuit ([920]). Falb2 teaches bacteria comprising a Cas9 protein (i.e., a CRISPR nuclease) and a gene encoding a guide RNA ([920]). Falb2 teaches bacteria can be used as delivery vectors, e.g., by bactofection ([154]), which is term of art for the claimed invention delivering transgenic DNA.
Critchley teaches E. coli engineered with an inv gene and the hly locus (i.e., bacterium engineered to express an invasion factor to facilitate entry of the bacterium into a eukaryotic cell) (page 1224, ¶3). Critchley teaches the E. coli also comprising a plasmid encoding GFP (page 1225, ¶3) or plasmid comprising the lacZ gene, which encodes b-galactosidase (page 1227, ¶4). Critchley teaches the engineered E. coli can deliver both the plasmid encoding GFP to mammalian cells for expression in the eukaryotic cells or the GFP protein itself (page 1225, ¶3; Fig 2). Critchley prokaryotic expression of GFP requires a lower dose of bacteria for protein activity when delivered to eukaryotic cells (Fig 2). Critchley teaches bacteria-mediated delivery of b-galactosidase expressed from a prokaryotic promoter (Figs 3 and 6).
Regarding claims 122-124, 134-135 and 151, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used the engineered bacterial-delivery method of Xiang and Critchley for delivering Cas9 and guide RNAs for gene editing. It would have amounted to using a known method that is capable of delivering DNA, RNA and/or proteins to a eukaryotic cell to deliver a known gene editing system. The skilled artisan would have predicted that a guide RNA could be expressed in bacteria and delivered to eukaryotic cells because Xiang teaches such a method for delivering shRNA, which is also a non-coding RNA similar to CRISPR guide RNAs. The skilled artisan would have predicted that Cas9 could also be delivered to eukaryotic cells using Xiang’s engineered E. coli because Critchley demonstrates that a protein of interest encoded by a plasmid and expressed in bacterial cells can be delivered to the mammalian cells via engineered E. coli cells expressing invasin and hylA. The skilled artisan would have been motivated to do so because Falb1 and Falb2 suggest bacteria engineered to express invasin on the cell surface could be used to deliver CRISPR/Cas9 systems to eukaryotic cells.
Regarding claims 129 and 132, Xiang teaches the expression of the shRNA is from a pT7RNAi-Hly-Inv (i.e., a plasmid) (Supp Fig 1). Xiang teaches expression of shRNA was controlled by the T7 promoter (i.e., a prokaryotic promoter) (Supp Fig 1). Xiang teaches E. coli strains comprising the T7 RNA polymerase for expression from a T7 promoter (page 697, ¶2). Critchley also teaches expression of GFP is from a gene located on pGB2Winv-hly/Prok.GFP (i.e., a plasmid) (page 1225, ¶3).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have controlled expression of the guide RNA and Cas9 protein with the T7 prokaryotic promoter from a plasmid because both Xiang and Critchley teach that expression of transgenes encoding non-coding RNAs and proteins from a plasmid is effective for delivery of the payload to eukaryotic cells. The skilled artisan would have been motivated to specifically use the T7 promoter because Xiang teaches commercially available E. coli strains already contain the T7 RNA polymerase needed for expression of transgenes.
Regarding claim 133, the Specification teaches that a promoter can be one that is “synthetically produced” (page 28, lines 27-28). As such a “synthetic promoter” encompasses a promoter that is produced in vitro. Xiang teaches the construction of pT7RNAi-Hly-Inv from oligonucleotides comprising the T7 promoter, enhancer and terminator (i.e., the T7 prokaryotic promoter is a synthetic prokaryotic promoter) (page 700, ¶7; Supp Fig 1).
Regarding claim 143, the teachings of Xiang, Falb1, Falb2 and Critchley and the obviousness of using bactofection to deliver a plasmid-encoded Cas9 nuclease and a guide RNA to eukaryotic cell is recited above for claims 122 and 129.
Regarding claims 148-149, the teachings of Xiang, Falb1, Falb2 and Critchley and the obviousness of using bactofection to deliver a Cas9 nuclease and a guide RNA to eukaryotic cell is recited above for claims 122 and 134.
Claims 125, 128, 130-131, 136-137, 140-142, 144 and 147 are rejected under 35 U.S.C. 103 as being unpatentable over Xiang (Xiang et al., Nature Biotechnology (2006), 24: 697-702), Falb1 (US20190336544 A1, priority to at least January 5, 2018), Falb2 (WO 2017123675 A1, published July 29, 2017) and Critchley (Critchley et al., Gene Therapy (2004), 11: 1224-1233), as applied to claims 122-124, 129, 132-135, 143, 148-149 and 151 above, and further in view of Cong (Cong et al., Science (2013), 339: 819-823 and Supplemental Material; of record). This is a maintained rejection.
The teachings of Xiang, Falb1, Falb2 and Critchley are recited above and applied as for claims 122-124, 129, 132-135, 143, 148-149 and 151.
Xiang, Falb1, Falb2 and Critchley do not teach a single guide RNA or a nuclear localization signal (NLS) attached to the Cas9 nuclease.
Cong teaches gene editing in human cells using CRISPR/Cas systems (Abstract). Cong teaches attaching an NLS to Cas9 “to ensure nuclear compartmentalization in mammalians cells” (page 820, ¶1). Cong teaches the nucleic acid sequence encoding the NLS-Cas9 construct (Supplemental sequences). Cong teaches delivering a plasmid encoding Cas9 nuclease comprising an NLS and a guide RNA targeting the nuclear EMX1 gene (Fig 1; Supp Materials, page 1, ¶2). Cong teaches the Cas9-NLS and guide RNA are capable of editing the EMX1 gene (Fig 1C-E). Cong teaches chimeric guide RNAs (i.e., a single guide RNA) comprised of a direct repeat sequence (black), a guide sequence (i.e., a spacer sequence, blue) and a tracrRNA sequence (red) (Fig 2B). Cong teaches chimeric guide RNAs can direct Cas9-mediated cleavage of nuclear human genes (Fig 2C and 3D).
Regarding claims 125, 128, 130-131, 136-137, 140-142, 144 and 147, the obviousness of using the bactofection system of Xiang and Critchley to deliver the generic Cas9 and guide RNA disclosed in Falb1 and Falb2 is recited above as for claims 122-124, 129, 132-135, 143, 148-149 and 151. It also would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have specifically used a Cas9 fused to an NLS and a single guide RNA as taught in Cong. It would have amounted to the simple combination of known elements by known means to yield predictable results. Regarding the inclusion of an NLS fused to Cas9, the skilled artisan would have predicted that an NLS-Cas9 could be delivered via bactofection because Cong teaches the NLS can be genetically encoded and thus would have recognized that the NLS-encoding sequence could be included on the plasmid encoding Cas9. The skilled artisan would have been motivated to include the NLS so that Cas9 could be used to edit nuclear genes in mammalian cells. Regarding using a single guide RNA, the skilled artisan would have predicted that a single guide RNA could be delivered via bactofection because Cong teaches the single guide RNA is genetically encoded and thus recognized that its coding sequence could be included on the plasmid encoding Cas9. The skilled artisan would have been motivated to use a single guide RNA design to simplify the gene-editing components design.
Claims 126-127 and 138-139 are rejected under 35 U.S.C. 103 as being unpatentable over Xiang (Xiang et al., Nature Biotechnology (2006), 24: 697-702), Falb1 (US20190336544 A1, priority to at least January 5, 2018), Falb2 (WO 2017123675 A1, published July 29, 2017), Critchley (Critchley et al., Gene Therapy (2004), 11: 1224-1233) and Cong (Cong et al., Science (2013), 339: 819-823 and Supplemental Material), as applied to claims 122-125, 128-137, 140-144, and 151 above, and further in view of Linke (WO 2018187381 A3, published October 11, 2018; of record). This is a maintained rejection.
As indicated above in paragraph 10, the effective filing date of claims 126-127 and 138-139 is June 1, 2020. As such the Linke WIPO publication is prior art and outside the one-year grace period for a §102(b)(1)(A) exception.
The teachings of Xiang, Falb1, Falb2, Critchley and Cong are recited above and applied as for claims 122-125, 128-137, 140-144, 148-149 and 151.
Xiang, Falb1, Falb2, Critchley and Cong do not teach methods of using bacteria invasion to deliver transgenes to eukaryotic cells (i.e., bactofection) in which the invasin factor or transgene is expressed from the chromosome of the invading bacteria.
Linke teaches using an invasive bacteria delivery vehicle to deliver therapeutic proteins to eukaryotic cells (i.e., bactofection) (Abstract, Fig 3). Linke teaches expression of invasin and LLO (i.e., the hylA gene product) from the E. coli chromosome (page 16, lines 5-9). Linke also teaches controlling expression of the transgene from the E. coli chromosome (page 16, lines 5-9). Linke teaches that by including the gene encoding the invasion factors and transgenes on the E. coli chromosome the manufacturing process of the invading bacteria can be improved because 1) the bacteria do not need to maintain a plasmid, which can provide a selective disadvantage and cause bacteria stress, and 2) the bacteria do not need to be grown under conditions for maintaining a plasmid (page 16, lines 7, 19-23).
Regarding claims 126-127 and 138-139, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have modified the Cas9/guide RNA bactofection method rendered obvious above by including the inv, hylA, and/or Cas9 genes in the E. coli chromosome instead of providing them on a plasmid. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that the inv, hylA, and/or Cas9 genes could be included on the E. coli chromosome because Linke teaches that invasion factor genes and genes encoding the therapeutic proteins can be included as such in a bactofection gene delivery method. The skilled artisan would have been motivated to do so because Linke teaches that chromosomal-integration of the genes simplifies the manufacturing process.
Claims 145-146 and 150 are rejected under 35 U.S.C. 103 as being unpatentable over Xiang (Xiang et al., Nature Biotechnology (2006), 24: 697-702), Falb1 (US20190336544 A1, priority to at least January 5, 2018), Falb2 (WO 2017123675 A1, published July 29, 2017), Critchley (Critchley et al., Gene Therapy (2004), 11: 1224-1233) and Cong (Cong et al., Science (2013), 339: 819-823 and Supplemental Material), as applied to claims 122-125, 128-137, 140-144, 147-149, and 151 above, and further in view of Cui (Cui and Bikard, Nucleic Acid Research (2016), 44: 4243-4251; of record) and evidenced by Addgene (pECFP-C1, https://www.addgene.org/vector-database/2487/ [retrieved April 23, 2025]; of record). This is a maintained rejection.
The teachings of Xiang, Falb1, Falb2, Critchley and Cong are recited above and applied as for claims 122-125, 128-137, 140-144, 147-149, and 151. Critchley also teaches bactofection of a plasmid encoding GFP under the control of a eukaryotic promoter results in GFP expression in the recipient mammalian cell (page 1225, ¶3; Figure 2). Critchley teaches the plasmid is pEGFP-C1. Addgene teaches that the promoter driving expression of the GFP in the pEGFP-C1 plasmid is CMV.
Although Critchley teaches controlling transgene expression using a eukaryotic promoter, Xiang, Falb1, Falb2, Critchley and Cong do not teach the benefits for limiting nuclease expression in bacteria.
Cui teaches Cas9 cleavage of bacterial chromosomal DNA can kill bacterial cells (Abstract). Cui teaches that most bacteria lack a DNA repair system called nonhomologous end joining (NHEJ), which could explain the lethal effect of Cas9-mediated double strand break formation (page 4243, ¶2). Cui teaches that when there is no additional copy of a gene in the bacterial cell, Cas9-mediated cleavage of the sole chromosomal gene copy is lethal (Fig 1D).
Regarding claims 145-146 and 150, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have modified the Cas9/guide RNA bactofection method rendered obvious above by using a eukaryotic promoter, such as CMV, to drive expression for the Cas9 and guide RNA. It would have amounted to using known promoters for expression by known means to yield predictable results. The skilled artisan would have expected that the CMV promoter could be used for Cas9 expression because Critchley teaches it is sufficient to drive transgene expression in eukaryotic cells delivered via bactofection. The skilled artisan would have been motivated to use the CMV promoter in order to reduce and/or prevent Cas9 expression in bacteria cells, which can be toxic if the guide RNA sequence has off-target targeting of a bacterial gene.
Response to Arguments and Richter Declaration
Applicant summarizes the §103 rejections of the independent claims and presents a preview of the rebuttal arguments (Remarks, pages 14-15).
Applicant argues that not all the words in the claims were addressed in the obviousness rejections (pages 16-23). Applicant argues that Xiang only teaches shRNA delivery by bacteria, which is a 23-33 nt single stranded RNA that forms a hairpin, is processed by a Dicer enzyme in the target host cell, and then binds the RISC complex, which functions in the cytoplasm to degrade a target mRNA (page 17, ¶2). Applicant argues that Falb1 merely suggests using bactofection to deliver DNA-encoding the Cas9, but never actually demonstrates delivering Cas9-encoding DNA and never teaches delivering Cas9 protein that is expressed/produced in the bacteria. Applicant argues that it appears Examiner is using Falb1 to teach bacteria expressing the gene editing enzyme (page 17, ¶3 through page 18). This argument has been fully considered but is not persuasive. First, this argument does not apply to claims 143-146, which encompasses bacteria comprising a plasmid encoding Cas9 and the guide RNA, and does not necessarily require Cas9 or the guide RNA to be expressed in the bacterium. Second, Examiner is using the teachings of Falb1 and Falb2 as a suggestion that bacteria-mediated delivery can generically be used for delivery/expression of Cas9/CRISPR machinery to/in eukaryotic cells. Critchley then provides the teachings of expressing a protein in bacteria using a bacterial promoter for delivery to the host cell. In fact, Critchley teaches that bacterial expression of the protein reduces the MOI needed for appearance of the protein of interest in bacterial cells compared to when the protein of interest was expressed under a eukaryotic promoter and delivered to host cells via a plasmid (Fig 2). Thus, in view of Critchley, the skilled artisan would have been motivated to not only use bacterial-mediated delivery of DNA-encoding the Cas9, but to actually express Cas9 in bacterial cells such that the bacterium comprises the Cas9 nuclease and guide RNA. Third, the standard for obviousness is not whether a prior art reference “demonstrates” protein delivery and functionality, but merely that it was predictable to do so. Because 1) Critchley teaches that proteins produced from transgenes can be expressed in bacteria cells and delivered to a eukaryotic host cell, and 2) Cas9 is a prokaryotic protein, it was entirely predictable that Cas9 would be expressed in bacterial cells and delivered to eukaryotic host cells.
Applicant argues that Examiner is “reading more into” Falb1’s disclosure of using bactofection to delivery CRISPR/Cas9 machinery and is only teaching delivery of DNA or RNA, such that Falb1 really only discloses delivering Cas9-encoding DNA and not the Cas9 nuclease itself (Remarks, page 19, ¶¶3-4). This argument has been fully considered but is not persuasive. As indicated in the previous paragraph, Falb1 is relied up for the general suggestion of using bacteria-mediated delivery of the Cas9 machinery, whether DNA or otherwise. Critchley provides the motivation and teaching of using bacteria-mediated delivery for expressing the transgenic protein in bacteria for delivery to eukaryotic cells.
Applicant argues that Falb1 and Falb2 refer to CRISPR Interference (CRISPRi) which is not gene editing (page 20, ¶2). This argument has been fully considered but is not persuasive. By 2013, at least six years before the effective filing date of the claimed invention, it was well established that CRISPR/Cas technology could be engineered to promote a plethora of genetic manipulations by inactivating Cas9 and or appending sequences onto the guide RNA (See e.g., Mali et al., Nature Methods (2013), 10: 957-963). As such, a skilled artisan would understand that if CRISPR Interference machinery comprising dCas9 and a guide RNA could be delivered using bacteria-mediated delivery methods, a Cas9 and guide RNAs capable of gene editing could also be delivered.
Applicant requests clarification on the Falb2 citation and use of the word “bactofection” (page 20, ¶3 through 21, ¶1). Bactofection is a term of art for the presently claimed invention in claims 143-146. This is evidenced by Pálffy who discusses using bacteria to deliver DNA for gene therapy and uses the term “bactofection” (Pálffy et al., Gene Therapy (2006), 13: 101-105). Pálffy also discusses “alternative gene therapy” to describe the instance where bacteria produce the transgenic protein, but the bacteria remain in the extracellular space (Fig 2). However, “alternative gene therapy” does not appear to be a common term of the art as Examiner could find no other use for it. Nevertheless, because Critchley teaches that bacteria taken up by the target cell can be used to deliver both DNA and proteins produced in bacteria, it would have been obvious to use the bacteria engineered to deliver Cas9-encoding DNA, as taught in Falb1 and Falb2, to deliver the Cas9 protein and guide RNA by expressing Cas9 and guide RNA with prokaryotic promoters.
Applicant argues that the disclosure in Falb2 of Cas9 is regarding using Cas9 as a regulatory circuit in bacteria (page 21, ¶2). This argument has been fully considered but is not persuasive. Falb2 clearly discloses that a heterologous Cas9 protein can be produced in bacteria, as is required for a genetic circuit, and the Cas9/guideRNA cleavage activity is fully functional ([920]). Based on Critchley, it is apparent that proteins expressed in bacteria can be transferred to eukaryotic cells where they are fully functional as well. The combination of Falb1 and Falb2 provides that bacteria can be used to deliver Cas9/gRNA-encoding DNA and that a Cas9/gRNA expressed in bacteria is functional. Critchley then provides that bacterial-expressed proteins are delivered to eukaryotic cells at higher rates than plasmid-encoded proteins, which is a motivation to deliver Cas9/gRNA directly as a protein and RNA.
Applicant and Declarant argue that controlling gene circuits in bacteria, which is the purpose of using Cas9 in Falb2, is fundamentally different than cross-kingdom delivery for genome editing (page 21, ¶3; Declaration, page 31, last ¶ through page 32, ¶7). This argument has been fully considered, but is not persuasive. First, Critchley provides that proteins can be delivered from a prokaryotic cell to a eukaryotic cell (i.e., cross kingdom). Secondly, by the effective filing date of the claimed invention, Cas effectors from at least three different CRISPR types (types II, V and VI) from many different bacterial genera (Staphylococcus, Streptococcus, Francisella, Prevotella, Neisseria) had successfully be used to engineer eukaryotic genomes, including in animal cells, plant cells, fungal cells, and protists. See e.g., Maxhar Adli, Nature Communications (2018), 9:1911. Thus, it was already established in the prior art that 1) a protein and non-coding RNA of bacterial origin could function in eukaryotes, and 2) proteins that are produced in bacteria can be transferred to eukaryotic cells and function as intended.
Applicant reiterates the argument that Falb1 only discloses using bacteria to deliver DNA and RNA encoding Cas9 and not the actual protein (page 21, ¶4 through page 22, ¶5). This argument was fully addressed in a preceding paragraph.
Applicant argues that the reliance on Falb1 an Falb2 was inadvertent hindsight (¶ spanning pages 22-23). This argument has been fully considered, but is not persuasive. In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In this case all the claimed components are taught individually in the prior art (bacteria comprising an invasion factor that can deliver RNA, DNA and proteins to eukaryotic cells, gene editing nucleases, and guide RNAs) and a generic teaching that Cas9 components can be delivered via the invading bacteria.
Applicant argues that Falb1/Falb2 do not demonstrate that CRISPR systems can be delivered to eukaryotic cells either as DNA or as protein/RNA components, and that what Examiner was actually arguing was an ‘obvious to try’ rationale, which is improper (page 23, ¶2). This argument has been fully considered, but is not persuasive. First, if Falb1/Falb2 had demonstrated the claimed invention, the claims would have been rejected under §102 for anticipation. Second, the argument does not address the merits of the rejection, which was based on a combination of elements. The claims were rejected for being obvious based on the well-known methods of using bacteria to deliver DNA, RNA and protein to eukaryotic cells, as evidenced by the 2004 and 2006 publication dates for Xiang and Critchley, and using the methods to deliver a known Cas9/gRNA system. Thus, Applicant’s argument does not address the merits of the rejection.
Applicant and the Richter Declaration argue that there would not have been a reasonable expectation of success of delivering Cas9/gRNA as protein and RNA using the bacteria described in Xiang and Critchley. Applicant provides two lines of argument. First, Applicant points out that gRNAs are longer than shRNAs and require precise folding and interactions with the Cas9 protein (Remarks, page 25, ¶3, Declaration, page 31). Second, Applicant states that Cas9 is a large protein requiring nuclear localization, which is very different than Critchley’s GFP protein (Declaration, page 31). Both lines of argument have been fully considered, but are not persuasive.
First, these arguments only apply to claims 122-142 and 147-151, since claims 143-146 encompass delivering plasmids encoding Cas9 and guide RNAs.
Second, Applicant and Declarant provide no evidence that methods for delivering shRNA have not worked for guide RNAs. Since the dawn of the CRISPR/Cas9 revolution, researchers have used delivery of siRNA and shRNAs as a template for delivering CIRSPR guide RNAs. For instance, both non-coding RNAs have utilized the same RNA III promoters when expressed in a eukaryotic cell (See Cong referenced above), or used a T7 promoter when expressed in bacterial cells (Xiang, page 697; Karvelis et al., RNA Biology (2013), 10: 841-851). Both noncoding RNAs can be also be delivered via liposomes (Zhen et al., Oncotarget (2016), 8: 9375-9387; page 9376, ¶3). Thus, the preponderance of the evidence suggests that means for delivering RNA interference RNAs can be used for delivering CRISPR guide RNA with a reasonable expectation of success.
Third, Critchley demonstrates additional proteins delivered to eukaryotic cells in addition to GFP. Critchley shows animal cell delivery of b-galactosidase that was expressed in bacterial cells (page 1232, ¶5; Figs 3 and 6). A b-galactosidase monomer is 1023 amino acids in length, making it almost as large as Cas9 (Brian Matthews, Comptes Rendus Biologies (2005), 328: 549-556). Additionally, b-galactosidase is tetrameric, underscoring its “complex” quaternary structure. Thus, Critchley demonstrates delivery of a large complex protein that, in its active form, is 3-times the size of Cas9. Additionally, it is important to remember that Cas9 and CRISPR RNAs are bacterial in origin. Cas9’s expression, folding, and complexing with guide RNAs in bacterial cell is not predicted to hinder its activity since Cas9/gRNA complexes are native to bacteria. Cas9 has been shown repeatedly to be able to be expressed, fold correctly, complex with a guide RNA, and have DNA-binding and catalytic activity in vitro, in bacterial cells, and in a multitude of different eukaryotic cells (see references of record and Jinek et al., Science (2012), 337: 816-821). Given 1) Critchley’s demonstration of delivering a large, complex enzyme from bacteria to eukaryotes and 2) the repeated demonstration of Cas9/gRNA stability and activity in a variety of cells types, the preponderance of the evidence suggests that bacteria can be used to express and deliver Cas9/gRNAs to eukaryotic cells with an reasonable expectation of success.
Declarant argues that Falb1 and Falb2 do not provide the necessary guidance to overcome the technical barriers of CRISPR/Cas9 delivery from bacteria to eukaryotic cells including “the inherent instability of sgRNA in the bacterial cytoplasm, degradation of nucleases during the transfer process, inefficient release of bacterial cargo into host nucleic, and the immunogenicity of bacterial delivery systems” (page 32, ¶6). This argument has been fully considered but is not persuasive. First, Applicant provides no evidence that the “technical issues” were inherent or known issues with protein and guide RNA delivery. Xiang and Critchley do not teach such technical issues with DNA/RNA/protein delivery by bacteria. Second, the bacteria do not need to release Cas9/sgRNA directly into host nuclei since means to traffic Cas9/gRNA into the nucleus using nuclear localization signals (NLSs) are well known in the art (See Cong reference of record). Third, it was already known that Cas/sgRNA ribonucleoproteins delivered to eukaryotic cells by other means, such as electroporation, are stable enough for efficient gene editing. For instance, Kim delivered first generation non-engineered Cas9/sgRNA RNPs into human cell lines that resulted in nearly 50% efficiency of genome editing (Kim et al., Genome Research (2014), 24: 1012-1019; Fig 1). Fourth, it is noted that Applicant did not demonstrate the claimed invention for delivery to cells in vivo. Applicant only demonstrates bacteria-mediated delivery of Cas9/gRNA in the A549 cell line (Specification, pages 16-17). As such, it is not known whether Applicant’s own method has overcome the cited technical difficulty of “immunogenicity of bacterial delivery systems”. Finally, in the working examples of Applicant’s Specification, it is not clear what version of pSiCRISP was used to knockout GFP expression (pages 15-17). There are two versions of the pSiCRISP vector illustrated in Fig 2 – one with a prokaryotic promoter driving expression of Cas9 and one with a eukaryotic promoter driving expression of Cas9. The Specification does not disclose which plasmid – the prokaryotic promoter-driven plasmid or eukaryotic-promoter driven plasmid – was used in Example 2. As such, it is not known whether Applicant’s own invention has overcome the supposed technical barriers cited by the Declarant.
Declarant argues that the described invention achieved unexpected results that would not have been anticipated by the skilled artisan given the length and instability of sgRNA and the size of Cas9 (¶ spanning pages 32-33). This argument has been fully considered but is not persuasive. First, the “unexpected result” does not apply to claims 143-146, which also encompass plasmid delivery to cells. Second, as indicated in the preceding paragraph, the size of sgRNA and Cas9 would have not have been considered a hinderance to delivery. Third, it is not clear from the working examples in the Specification which pSiCRISP plasmid was used in Example 2, and as such, Examiner could not evaluate whether the cited results are unexpected.
Declarant argues that there was no precedent in the art for simultaneously delivering both the Cas9 and guide RNA to cell and achieving a functional RNP inside the nucleus (page 33, ¶2). It is not clear if Declarant meant no precedent for delivery of an Cas9/gRNA RNP at all or specifically via invasive bacteria. Either way, the argument has been fully considered but is not persuasive. If there had been a precedent for delivery of Cas9/gRNA to eukaryotic cells via invasive bacteria, then the claims would have been rejected under § 102. However, the claims were rejected under §103, and thus the argument does not address the merits of the rejection. Furthermore, Cas9 and CRISPR guide RNAs have been delivered to cells simultaneously, which showed high editing efficiency. As explained above, Kim discloses RNP electroporation to cells and nuclear genome editing as early as 2014. Additionally, Liu teaches lipofection of Cas9/gRNA using an E-tag system as early as 2015 (US 20150071903 A1).
Declarant argues that “biological barriers at multiple levels” would have discouraged any expectation of success (age 33, ¶3-4). This argument has been fully considered, but is not persuasive. As stated above, Declarant provides no evidence that “biological barriers” for bacteria-mediated protein and RNA delivery exist; whereas both Xiang and Critchley provide examples of successful bacterial-expressed RNA and protein delivery to eukaryotic cells.
Applicant argues that Falb1 and Falb2 are very long and each only mention bactofection-mediated delivery of Cas9 a single time, as such the skilled artisan would need to have had super-human powers. Applicant cites the nonprecedential Genzyme Corp. v. Dr. Reddy’s Laboratories as support for not relying on a prophetic disclosure in a prior art reference (page 25, ¶4 through page 26, ¶2). This argument has been fully considered but is not persuasive because references used in rejections made under § 103 do not need to exemplify all embodiments, only suggest embodiments. "The use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." In re Heck, 699 F.2d 1331, 1332-33, 216 USPQ 1038, 1039 (Fed. Cir. 1983) (quoting In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275, 277 (CCPA 1968)). See MPEP 2123. Because Falb1 and Falb2 disclose delivering DNA-encoding Cas9 via bacteria-mediated means, even just once, it is relevant to what the skilled artisan would have considered.
Applicant argues that Cong, Linke and Addgene do not overcome the shortcomings of Xiang, Falb1, Falb2 and Critchley (pages 27-28). This argument has been fully considered but is not persuasive because a prima facie case of obviousness is established for claims 122-123, 136-137 and 143 for the reasons set for in the §103 rejections of record and the response to Applicant' s arguments above.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 122-144, 147-149 and 151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-14 of U.S. Patent No. 11312954 in view of Falb1 (US20190336544 A1, priority to at least January 5, 2018), Falb2 (WO 2017123675 A1, published July 29, 2017), Critchley (Critchley et al., Gene Therapy (2004), 11: 1224-1233) and Xiang (Xiang et al., Nature Biotechnology (2006), 24: 697-702). Claims 125, 128, 130-131, 136-140, 144 and 147 are rejected further in view of Cong (Cong et al., Science (2013), 339: 819-823 and Supplemental Material). Claims 126 and 139 are rejected in further view of Linke (WO 2018187381 A3, published October 11, 2018).
Patented claim 1 recites A bacterium for nucleic acid delivery to a eukaryotic cell comprising: a nonpathogenic bacterium, wherein the bacterium has been engineered to express at least one invasion factor from a sequence on the chromosome of the bacterium and wherein the bacterium comprises a plasmid having a length less than 4,700 base pairs, the plasmid comprising one or more sequences encoding one or more therapeutic nucleic acids and a prokaryotic promoter to control transcription of the therapeutic nucleic acids. Patented claim 3 recites wherein the plasmid comprises a plurality of therapeutic nucleic acid sequences and a plurality of prokaryotic promoters, wherein a first therapeutic nucleic acid sequence is under the control of a first prokaryotic promoter and a second therapeutic nucleic acid sequence is under the control of a second prokaryotic promoter. Patented claim 5 recites wherein the therapeutic nucleic acid is a small interfering RNA/short hairpin RNA (siRNA/shRNA) (i.e., a guide RNA of an RNAi-silencing system). Patented claims 7 recites wherein the bacterium is E. coli. Patented claim 9 recites wherein the bacterium has been engineered to express at least one of the selection marker elements from a sequence on the chromosome of the bacterium (i.e., the bacterium has at least two transgenes expressed from the chromosome). Patented claim 10 recites wherein the invasion factor is encoded by a gene selected from the group consisting of an inv gene, a hlyA gene, an HA-1 gene or combinations thereof.
The patented claims do not recite the engineered bacterium expressing a gene-editing nuclease or a guide RNA. The patented claims do not recite an NLS (claims 128, 130-131, 137, 144). The patented claims do not recite a specific prokaryotic promoter or a synthetic prokaryotic promoter (claims 132-133, 141).
The teachings of Falb1, Falb2, Critchley and Xiang are recited above in paragraphs 17, 18, 19 and 16, respectively and incorporated here.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used the patented bacterium in a composition for delivering Cas9 and guide RNAs for gene editing. It would have amounted to using the patented method that is predicted to be able to deliver RNA and protein to a eukaryotic cell to deliver a known gene editing system. The skilled artisan would have predicted that a guide RNA could be expressed in bacteria and delivered to eukaryotic cells because CRISPR guide RNA and the patented shRNAs are both non-coding RNAs. Additionally, Xiang demonstrates successful delivery of shRNA via engineered bacteria expressing invasion factors. The skilled artisan would have predicted that Cas9 could also be delivered to eukaryotic cells using the patented bacterium because Critchley demonstrates that a protein of interest encoded by a plasmid can be delivered to the mammalian cells via bactofection, which the patented bacteria would be capable of doing. The skilled artisan would have been motivated use the patented bacterium to delivery Cas9 and a guide RNA because Falb1 and Falb2 suggest that bactofection could be used to deliver CRISPR/Cas9 systems.
Regarding claims 132-133 and 141, Xiang teaches the expression of the shRNA is from a pT7RNAi-Hly-Inv (i.e., a plasmid) (Supp Fig 1). Xiang teaches expression of shRNA was controlled by the T7 promoter that was assembled using oligonucleotides (i.e., a synthetic prokaryotic promoter) (Supp Fig 1). Xiang teaches E. coli strains comprising the T7 RNA polymerase for expression from a T7 promoter (page 697, ¶2). It also would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used a T7 and/or synthetic promoter to drive the expression of the guide RNA in the obvious variation of the patented bacterium because Xiang teaches commercially available E. coli strains already contain the T7 RNA polymerase needed for expression of non-coding RNA transgenes.
Regarding claims 125, 128, 130-131, 136-140, 144 and 147, the teachings of Cong are recited above in paragraph 29 and incorporated here. It also would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have specifically delivered a Cas9 fused to an NLS and a single guide RNA as taught in Cong to eukaryotic cells using the patented bacterium. It would have amounted to the simple combination of known elements by known means to yield predictable results. Regarding the inclusion of an NLS fused to Cas9, the skilled artisan would have predicted that an NLS-Cas9 could be delivered via bactofection because Cong teaches the NLS can be genetically encoded and thus would have recognized that the NLS-encoding sequence could be included on the expression cassette of Cas9. The skilled artisan would have been motivated to include the NLS so that Cas9 could be used to edit nuclear genes in mammalian cells. Regarding using a single guide RNA, the skilled artisan would have predicted that a single guide RNA could be delivered via bactofection because Cong teaches the single guide RNA is genetically encoded and thus would recognize that its coding sequence could be included on the non-coding RNA-encoding plasmid. The skilled artisan would have been motivated to use a single guide RNA design to simplify the gene-editing components design.
Regarding claims 127 and 139, the teachings of Linke are recited above in paragraph 35 and incorporated here. It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have further included the Cas9 expression cassette in the bacterial chromosome in the obvious variation of using the patented bacterium for delivery of Cas9/guide RNA via bactofection to eukaryotic cells. It would have amounted to the simple combination of elemen