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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on January 14, 2026 has been entered.
Application Status and Withdrawn Rejections
Applicant’s amendments filed January 14, 2026 amending claims 180, 184, 186, 190-192, 205, and 209-210, canceling claim 189, and adding new claim 211 is acknowledged. Claims 180-188, 190-192 and 194-211 are pending and under examination.
The amendment to claim 205 overcomes the §112(b) rejections.
Claims 180 and 205 were amended to require the fusion protein to comprise at least one NLS and at least two NESs, with the total number of NESs greater than the total number of NLSs while still requiring the arrangement of the fusion protein to comprise [PMLP]-[at least 2x NES]-[cleavable linker]-[Cargo/NLS]. Claim 180 encompasses the composition and arrangement of canceled claim 189 and includes limitations from amended claim 191. The obviousness rejections of the claims over prior art references Doudna, Robert, Wu and Zetsche are modified to reflect the limitations from claims canceled claim 189 and amended claim 191 incorporated into claim 180.
The NSDP rejections of the instant claims over copending claims are modified to reflect the amendments to the instant and copending claims.
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.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 211 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a NEW MATTER rejection.
MPEP 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims” and “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112a, as lacking adequate written description". According to MPEP § 2163.I.B, "While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure" and "The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117".
In the instantly rejected claims, the new limitation of “wherein the ratio of the fusion protein to the protein that comprise a gag and a pro within the protein core is approximately 1:3” in claim 211 appears to represent new matter. No specific basis for this limitation was identified in the specification, nor did a review of the specification by the examiner find any basis for the limitation. Paragraphs [0018], [0147] and [0193] discuses ratios of proteins in the VLP and VLP core, but the ratio is regarding the amount of cleaved cargo protein to the cargo protein remaining in the fusion protein. Paragraphs [0537]-[0539] disclose varying the stoichiometry of the plasmids encoding the gag-pro and the fusion protein, but don’t disclose what the final protein concentrations of gag-pro and gag-BE are in the VLPs. The Specification does not provide evidence, such as an immunoblot or a correlation between the amount of plasmid delivery and the level of protein expression, to provide support for final protein ratios in the VLPs. It is noted that the claims are directed to the VLPs themselves and methods of using the VLPs. The claims do recite methods of making the VLPs and do not recite nucleic acids encoding the gag-pro and fusion proteins. A thorough review of the Specification found no disclosure of ratios of gag-pro and gag-cargo fusion protein in the final VLPs. Since no basis has been identified, the claims are rejected as incorporating new matter.
Response to Arguments – New Matter
Applicant argues that support for new claim 211 is found in the Specification in paragraph [0539] (Remarks, page 8, ¶1). This argument has been fully considered but is not persuasive because paragraph [0539] does not disclose final protein concentrations in the VLPs. [0539] only discloses the plasmid ratios transfected into producer cells. Fig 2F illustrates a continuum between gag-pro-pol and gag-cargo ratios while Fig 2G is a readout of editing efficiency based on plasmid ratios provided to producer cells. As indicated in the rejection above, the Specification does not show immunoblots for final protein ratios or provides a correlation between plasmid transfection levels and protein concentration in produced VLPs. As such, the claims are rejected as incorporating new matter.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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 180-188, 190-192, 194-198, 200-206 and 208-211 are rejected under 35 U.S.C. 103 as being unpatentable over Doudna (US 20220403379 A1, priority to May 28, 2021; of record) in view of Robert (Robert et al., Molecular biotechnology (2017), 59: 9-23; of record), Wu (Wu et al., Biomaterials (2014) 35: 8416-8426; of record), and Zetsche (Zetsche et al., Nature Biotechnology (2015), 33: 139-142 and Supplemental Material; of record). Claims 182-183 are evidenced by Addgene (psPAX2, Plasmid #12260, https://www.addgene.org/12260/ [retrieved January 14, 2025]; of record). Claims 187, 194-195 are evidenced by Schneider (Schneider et al., Virology (2012), 188-196; of record).
Regarding claim 180, Doudna teaches virus like particles (VLPs, i.e., lipid-containing particles) (Fig 1B; [0006]-[0007]). Doudna teaches the VLPs comprise a lipid envelope encapsulating proteins in the core and fusion proteins (Fig 1B). Doudna teaches the fusion protein comprises (a) the MA, CA, NC and P6 proteins of Gag located at the N-terminus based on the direction of transcription (i.e., a plasma membrane localization protein) (Fig 1A-B). Doudna teaches the fusion protein comprises (b) SpCas9-NLS at the C-terminus (i.e., a therapeutic cargo comprising at least one NLS) (Fig 1B). Doudna teaches the fusion protein comprises (c) a protease cleavage site (i.e., a cleavable linker) (Fig 1A-B). Doudna teaches the cleavable linker is located between Cas9 and Gag protein (Fig 1A-B). Thus Doudna teaches the fusion protein comprises N-terminus to C-terminus [plasma membrane localization protein]-[cleavable linker]-[cargo]-[NLS]. Doudna also teaches using the Gag-Cas9 VLPs for delivering Cas9 to cells for genome editing of nuclear genes ([0343]; Fig 1E-F).
Doudna does not teach the fusion protein comprises an NES that is between the plasma membrane localization protein and the cleavable linker such that the structure of the fusion protein is [plasma membrane localization protein]-[2xNES]-[cleavable linker]-[cargo]-[NLS].
Robert teaches VLPs for delivering nuclear proteins to cells (Title). Robert teaches VLPs comprise a fusion comprising the Gag polyprotein (i.e., a plasma membrane localization protein), protease cleavage sites between most of the Gag proteins (i.e., cleavable linkers), and the KLF4 transcription factor (i.e., a therapeutic cargo) (Fig 1b, Table 1). Robert teaches adding a VP16 domain to the Gag-KLF4 fusion to form the fusion protein Gag-VP16KLF4 (Fig 5a). Robert teaches that it was previously shown that insertion of an NES could improve the packaging effectiveness of transcription factors within VLPs (page 16, ¶5). Robert teaches that adding an NES to the Gag-VLP16KLF4 fusion protein to form Gag-NES-VP16KLF4 (Fig 6a), in which the NES was inserted at the 5’ end (N-terminal end) of the VP16 sequence and at the C-terminal end of the Gag protein (page 10, ¶5; Fig 6a). Robert teaches that inclusion of the NES increased fusion protein packaging in the VLPs (Fig 7a-b). Robert teaches the activation of transcription of a reporter protein in recipient cells was higher when an NES was included in the fusion protein (Fig 6a). Robert teaches the presence of the NES stimulates incorporation of Gag into VLPs and/or the production of VLPs (page 17, ¶2).
Wu also teaches delivering nuclear transcriptions factors fused to Gag proteins and encapsulated in VLPs for the purpose of delivering the transcription factors (TFs) to recipient cells (Fig 1a-b). Wu teaches that cargos that don’t have NLSs, such as GFP, do not need an NES to be efficiently packaged in VLPs (Fig 2a-b). Wu teaches including 2 NESs in the fusion protein increases the packaging of the Gag-TF fusion protein in VLPs (Fig 2c, 3a). Wu teaches including a protease cleavage site between the transcription factor, the Gag protein, and the NES releases the protein of interest from the viral proteins and allows it to traffic independently in the recipient cell (Section 3.1, ¶1; Fig 1a-b; Fig 3a-b).
Zetsche teaches engineering Cas9 proteins to develop an inducible Cas9 system (Figure 1). Zetsche teaches that appending an NES to the N-terminal half of Cas9 sequesters the N-terminal fragment in the cytoplasmic, thereby inhibiting its nuclear localization and subsequent editing upon auto-dimerization with the C-terminal half that comprises two NLS (page 140, ¶2; Fig 1C).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included two NESs between the Gag protein and protease cleavage site in Doudna’s Gag-Cas9-NLS fusion protein to form the fusion protein [Gag]-[2xNES]-[cleavable linker]-[Cas9]-[NLS]. It would have amounted to the simple combination of known protein domain elements by known means to yield predictable results. The skilled artisan would have been motivated to include two NESs because both Robert and Wu teach that including an single or two NESs in Gag-nuclear protein fusions increases the packaging of the fusion proteins in VLPs. Based on the teachings of Robert and Wu, the skilled artisan would have considered placing the NESs either 1) between the protease cleavage site and the cargo, or 2) between the Gag protein and the protease cleavage site. The skilled artisan specifically would have been motivated to place the two NESs of Wu between Gag and the protease cleavage site for two reasons. First, Wu teaches cleaving the NESs away from the PT cargo allows the cargo to traffic independently in the cell. Second, Zetsche teaches that adding an NES to a Cas9 fragment sequesters the Cas9 fragment in the cytoplasm. The skilled artisan would not have been motivated to retain an NES on Cas9 because both Doudna and Zetsche teaches that Cas9 functions to edit genes in the nucleus of eukaryotic cells. The skilled artisan would have predicted that two NESs could be added between the Gag protein and cleavable linker because Wu and Robert teach that the Gag polyprotein is amenable to NES placement at various positions, including at the C-terminus of the Gag polyprotein.
Regarding claim 181, Doudna teaches including structural Gag proteins in the VLP (Fig 1B). Doudna teaches the Cas9 was cleaved from the Gag in the mature VLPs, indicating that there was also a protease in the VLP (Fig 1C).
Regarding claim 182-183, Doudna teaches the Gag protein comprises MA, CA, NC and P6 (i.e., is a gag polyprotein) (Fig 1B). Doudna teaches that the Gag coding sequence for the Gag-Cas9 fusion protein was derived from psPax2 (Addgene #12260) ([0328]). Although Doudna teaches that the VLPs are derived from HIV-1 vectors, Doudna is silent on whether the Gag protein is a retroviral gag protein. Addgene teaches that the Gag coding sequence is the HIV gag sequence (pages 1-2). Thus, Doudna’s Gag-Cas9 fusion protein inherently comprised a retroviral gag protein and a retroviral gag nucleocapsid polyprotein.
Regarding claim 184, Doudna teaches the Gag-Cas9 protein comprises Cas9 (Fig 1B). Doudna teaches that Cas9 has nucleic acid cleavage activity (i.e., Cas9 is a nuclease) ([0057]).
Regarding claim 185, as indicated above for claim 180 Doudna teaches the Gag-Cas9 fusion protein comprises an HIV-1 protease cleavage site (Fig 1A).
Regarding claim 186, Doudna teaches that the VLPs comprise both Gag-Cas9 and the cleaved Cas9 (Fig 1C). The obviousness of including 2x NES between the cleavable linker and Gag to form [Gag]-[2xNES]-[linker]-[Cas9]-[NLS] is recited above for claim 180. The VLP taught in Doudna having the obvious fusion protein would result in a cleavage product comprising Gag-NES without the Cas9.
Regarding claims 187, 194-195 and 210, Doudna teaches that Cas9 VLPs have also be made using MLV VLPs ([0343]). Wu also teaches VLPs derived from Murine Leukemia Viruses (MLVs) (Abstract). Wu teaches Gag-protein fusions (Fig 1b). Wu teaches VLPs derived from MMLV proviral vectors encoding Gag-protein fusions with MMLV protease cleavage sites between the NES and cargo (Fig 1b, black and pink arrows). Wu teaches providing WT gag-pro to increase VLP production (Fig 3a). Wu teaches the protein transduction vectors (i.e., the vectors encoding the fusion protein and the protein core) were based on the pNCA-C (IN- D184N/K376A) proviral vector as a backbone and the gag-PT-Pol (page 8417, last ¶ through page 8418, ¶2). Wu teaches that the pNCA-C (IN- D184N/K376A) proviral vector was described in Reference 8 – Schneider et al (page 8416, last ¶). There is no indication in Wu that the protease cleavage sites normally found in the proviral vector or the gag protein sequences were altered (page 8416, last ¶ through page 8417, ¶3), thus the gag polyprotein in the fusion protein, the gag nucleocapsid protein in the WT gag-pro vector, and the cleavage site between the NES and the cargo, are derived from MLV (Fig 1b). Additionally, Wu teaches adding an additional RSSLY/PALTP site (i.e., a MMLV protease site; i.e., the linker comprising an amino acid at last 90% identical to SEQ ID NO: 2) (Fig 1b). Wu is silent on whether the MLV gag polyprotein and protease cleavage site are derived from an Moloney MLV. Schneider teaches the pNCA-C vector is an Moloney murine leukemia virus (i.e., M-MuLV or MMLV) proviral vector (Abstract; page 193, ¶5). Thus, the gag polyprotein and protease cleavage sites in Wu’s Gag-protein fusion and in the WT gag-pro protein core were inherently from an MMLV.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used the MMLV viral vectors, MMLV gag-pro structural components, and MMLV cleavage site with the RSSLY/PALTP sequence to make VLPs comprising the [Gag]-[2xNES]-[linker]-[Cas9]-[NLS] protein rendered obvious above. It would have amounted to the simple substitution of one retroviral Gag structural components and protease cleavage site for another by known means to yield predictable results. The skilled artisan would have predicted that an [MMLV Gag]-[2xNES]-[MMLV protease site: RSSLYPALTP]-[Cas9]-[NLS] fusion protein could be produced and loaded into VLPs because 1) Doudna teaches that an MLV-based Cas9 VLP has been produced previously and 2) Wu teaches that MMLV-derived elements can be used to make VLPs that deliver nuclear proteins. Because the prior art recognizes the equivalence of HIV-1 derived gag and protease sites and MMLV derived gag and protease sites for the purpose of producing VLPs that can deliver nuclear cargo, an express suggestion to substitute one equivalent component or process for another is not necessary to render such substitution obvious. MPEP 2144.06.II.
Regarding claim 188, as indicated above for claim 180, Wu teaches adding 2x NESs to the Gag-protein fusion (Fig 1b). Doudna, Robert, Wu and Zetsche do not teach at least three NESs in the Gag-Cas9 fusions. However, it would have been obvious to have included an additional NES in the [Gag]-[2xNES]-[linker]-[Cas9]-[NLS] fusion protein rendered obvious above to have produced a fusion protein with structure [Gag]-[3xNES]-[linker]-[Cas9]-[NLS]. It would have amounted to the simple duplication of known elements by known means to yield predictable results. The skilled artisan would have predicted that three NESs could be included in the fusion protein since Wu teaches that multiple NES can be included in Gag-fusion proteins. “[M]ere duplication of parts has no patentable significance unless a new and unexpected result is produced” and “require[s] only ordinary skill in the art and hence [is] considered routine expedients.” See MPEP 2144.04 and 2144.04.VI.B. Thus, in the absence of any showing of the criticality of 3 NESs versus 2 NESs, the addition of another NES in the fusion protein rendered obvious for claim 180, is also obvious.
Regarding claims 190-191, Doudna teaches that Gag-Cas9 fusion protein comprises an NLS at the C-terminus of Cas9 (Figure 1A). Thus, the fusion protein rendered obvious above comprises the structure N-terminus to C-terminus: [Gag]-[2xNES]-[linker]-[Cas9]-[NLS] (i.e., wherein n=2, m1=0, and m2=1).
Regarding claim 192, Doudna teaches the Cas9 can include two or more NLSs ([0065], [0074]). Doudna teaches the Cas9 can have an NLS at the N-terminus and an NLS at the C-terminus ([0074]).
It would have been obvious to have include an additional NLS at the N-terminus of Cas9 in the fusion protein rendered obvious for claim 188 to produce a fusion protein with the structure [Gag]-[3xNES]-[linker]-[NLS]-[Cas9]-[NLS]. It would have amounted to the simple addition of known elements by known means to yield predictable results. The skilled artisan would have predicted that an NLS could be added to both the N- and C-terminus of the Cas9 because Doudna teaches such an NLS-Cas9-NLS protein. The skilled artisan would have been motivated to do so because Doudna suggests it.
Regarding claim 196, Doudna teaches the VLPs comprise a viral glycoprotein in the envelope (Fig 1A-B).
Regarding claim 197, Doudna teaches the viral envelope glycoprotein is the CD-4 tropic HIV-1 envelope glycoprotein (i.e., a retroviral envelope glycoprotein) ([0325]).
Regarding claim 198, Doudna teaches that VLPs can be engineered with different envelope proteins for binding to different target cells ([0120]). Doudna teaches one such envelope glycoprotein that can be used is a Baboon Endogenous retrovirus (BAEV) envelope protein ([0120]). Doudna provides the sequences for the Baboon retrovirus envelope glycoproteins (pages 60-61; SEQ ID NO 49).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have replaced Doudna’s HIV-1 glycoprotein in the VLP rendered obvious in claim 180 for a Baboon retroviral envelope glycoprotein. It would have amounted to the simple substitution of one known retroviral envelope glycoprotein for another by known means to yield predictable results. The skilled artisan would have predicted that the substitution could be made and be motivated to do it because Doudna teaches that the Baboon retroviral envelope glycoprotein can be used in VLPs delivering Cas9. The skilled artisan would have also been motivated to make the substitution in order to use VLPs in Baboon animal model studies.
Regarding claims 200-201, Doudna teaches VLPs can include a polypeptide that provides for binding to a target cell or target cell type (i.e., a targeting moiety) ([0208]). Doudna teaches that such targeting polypeptides include antibodies, scFV (antibody fragments), and nanobodies ([0208]).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included a targeting antibody as taught in Doudna in the VLP rendered obvious in claim 180. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that targeting antibodies could be added to the obvious VLP because Doudna teaches that targeting antibodies can be used in VLPs delivering Cas9. The skilled artisan would have been motivated to include targeting antibodies for the purpose of targeting the obvious VLP to a specific cell type.
Regarding claim 202-204, Doudna teaches the VLPs comprise Cas9 cleaved from the fusion protein (Fig 1C). Based on the size and darkness of the Cas9 band compared to the Gag-Cas9 in the lane with 6.7 mg Gag-pol plasmid and 3.3 mg Gag-Cas9 plasmid, Doudna teaches the amount of cleaved Cas9 is at least 1.5 times higher than the amount of Gag-Cas9 fusion protein (Fig 1C).
Regarding claim 205, the teachings of Doudna regarding Cas9-NLS (i.e., a gene editing agent comprising at least one NLS) encased in a VLP (i.e., a lipid containing particles) comprising (1) a [Gag]-[protease cleavage linker]-[Cas9]-[NLS] fusion protein, and the obviousness of including two NESs between the Gag and protease cleavage site to form [Gag]-[2xNES]-[linker]-[Cas9] is recited above as for claim 180. Doudna also teaches the VLPs comprise (2) Cas9 (i.e., the gene editing agent) cleaved from the fusion protein (Fig 1C). Doudna teaches both the Gag-Cas9 fusion protein and cleaved Cas9 protein are detected in VLPs harvested from producer cell supernatants (i.e., the fusion protein and cleaved agent are encapsulated by the lipid membrane) (Figs 1C; [0329]-[0330]). Doudna also teaches contacting 293T, Jurkat and A549 cells with the VLPs comprising the Gag-Cas9 fusion protein for the purpose of gene editing ([0332]-[0333]; Figs 1-2). Doudna teaches the targeted B2M gene was edited (Figs 1F and 2B).
Regarding claims 206 and 208, Doudna teaches the Cas9 can have reduced or undetectable nuclease activity (i.e., is a nickase or a dead Cas protein) ([0057]-[0058]). Doudna teaches using Cas effectors that are dead or have reduced activity for the purpose of fusing a heterologous polypeptide that provides additional function to the Cas effector including transcriptional activation, transcriptional repression, protein modification, or nucleic acid modification like deamination ([0057]).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used a dead or nicking Cas9 as taught in Doudna in the VLP rendered obvious in claim 180 and the method of claim 205. It would have amounted to the simple substitution of one known Cas9 for another by known means to yield predictable results. The skilled artisan would have predicted that dead or nicking Cas9 proteins could be used in the obvious VLP because Doudna teaches that such Cas9 variants can be delivered by VLPs. The skilled artisan would have been motivated to have made the substitution for the purpose of delivering Cas9-effector domain fusions to cells for base editing, increasing expression, or decreasing expression of a target gene.
Regarding claim 209, as indicated above for claim 180, Wu and Robert teach that adding a single and multiple NESs to Gag-Cargo fusions increases cargo packaging in VLPs (Robert Fig 7a-b; Wu Fig 2C). Additionally, Robert teaches that the activity of the cargo protein in the cells increases, which is due to the increased packaging of the cargo in the VLPs (Fig 6a). Based on the findings in Wu and Robert, the skilled artisan would have predicted that the efficiency of packaging the obvious [Gag]-[NES]-[linker]-[Cas9]-[NLS] fusion protein in the VLPs would be higher than the packaging of Doudna’s [Gag]-[linker]-[Cas9]. The skilled artisan would have then predicted that the increased efficiency in VLP packaging would result in higher rates of delivery and subsequent activity of Cas9 (i.e., gene editing) in cells compared to Doudna’s Gag-Cas9 without the NES.
Regarding claim 211, Doudna teaches varying the ratio of plasmid encoding the core protein Gag-pol relative to the plasmid encoding [Gag]-[linker]-[Cas9]-[NLS] to optimize Cas9 incorporation into budded VLPs (Fig 1C; [0340]). Doudna teaches that by varying the ratios of the Gag-pol coding sequence and the [Gag]-[linker]-[Cas9]-[NLS]-coding sequence introduced into the producer cells, the skilled artisan can vary the final amount of the intact fusion protein in the VLP (Fig 1C), which can alter the VLP yield (FIG. 1D-E).
It would have been obvious to one skilled in the art before the effective filing date to have optimized the ratio of Gag-pro to Gag-Cas9 fusion protein in the VLP rendered obvious for claim 180 to arrive at a ratio of 3:1. It would have amounted to optimizing the protein ratios by known means to yield predictable results. The skilled artisan would have predicted that a 3:1 Gag-Pro:Gag-Cas9 ratio could be achieved because Doudna teaches such optimization. One would have been motivated to optimize in order to maximize Cas9 loaded into VLPs, as taught in Doudna.
Claim 199 is rejected under 35 U.S.C. 103 as being unpatentable over Doudna (US 20220403379 A1, priority to May 28, 2021), Robert (Robert et al., Molecular biotechnology (2017), 59: 9-23), Wu (Wu et al., Biomaterials (2014) 35: 8416-8426), and Zetsche (Zetsche et al., Nature Biotechnology (2015), 33: 139-142 and Supplemental Material) as applied to claims 180-188, 190-192, 194-198, 200-206 and 208-211 above, and further in view of Urano (Urano et al., Journal of General Virology (2008), 89: 3144-3149; of record).
The teachings of Doudna, Robert, Wu and Zetsche are recited above and applied as for claims 180-188, 190-192, 194-198, 200-206 and 208-211.
Doudna, Robert, Wu and Zetsche do not teach protein transduction VLPs in with the Gag-fusion protein comprises a pleckstrin homology (PH) domain.
Urano teaches VLPs derived from HIV-1 (Fig 2). Urano teaches replacing the myristylation signal sequence on the N-terminal Gag polyprotein with a PH domain from phospholipase C-d1 (¶ spanning pages 3144-3145; Fig 1). Urano teaches VLP production with the PH-Gag protein variant increased VLP production from 293T producer cells compared to the normal myristylated gag protein (Table 1, compare pgag-pol rows to pPH-gag-pol rows). Urano teaches the added PH domain did not affect VLPs overall structure (¶ spanning pages 3146-3147) or the ability to deliver transgenes to recipient cells (page 3175, ¶1).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have further modified the [Gag]-[2xNES]-[linker]-[Cas9]-[NLS] fusion protein in the VLP rendered obvious for claim 180, by replacing the myristylation signal in the Gag protein with the PH domain taught in Urano. It would have amounted to the simple combination of elements and engineering Gag proteins in known ways to yield predictable results. The skilled artisan would have predicted that the obvious [Gag]-[2xNES]-[linker]-[Cas9]-[NLS] protein could be modified to include a PH domain because the prior art demonstrates that retroviral Gag proteins can be easily engineered. It also would have been predictable that VLPs with a [PH-Gag]-[2xNES]-[linker]-[Cas9]-[NLS] could be produced since Urano teaches that inclusion of the PH domain does not affect VLP morphology or the ability to infect cells. One would have been motivated to include the PH domain in the Gag-Cas9 protein for the purpose of increasing the VLP production as indicated in Urano.
Claim 207 is rejected under 35 U.S.C. 103 as being unpatentable over Doudna (US 20220403379 A1, priority to May 28, 2021), Robert (Robert et al., Molecular biotechnology (2017), 59: 9-23), Wu (Wu et al., Biomaterials (2014) 35: 8416-8426), and Zetsche (Zetsche et al., Nature Biotechnology (2015), 33: 139-142 and Supplemental Material) as applied to claims 180-188, 190-192, 194-198, 200-206 and 208-211 above, and further in view of Thakore (Thakore et al., Nature Methods (2015), 12: 1143-1149 and online methods; of record).
The teachings of Doudna, Robert, Wu and Zetsche are recited above and applied as for claims 180-188, 190-192, 194-198, 200-206 and 208-211.
Although Doudna teaches that Cas9 delivered in VLPs can be fused to heterologous proteins, including transcriptional repressor domains, Doudna, Robert, Wu and Zetsche do not teach that the Cas9 editing agent comprises an epigenetic editor or a component of an epigenetic editing complex.
Thakore teaches that Cas9 can be fused to a KRAB repressor domain to form dCas9-KRAB in order to silence target gene expression (Abstract; Fig 1). Thakore teaches dCas9-KRAB induces highly specific H3K9 trimethylation and decreases chromatin accessibility (i.e., Cas9-KRAB is an epigenetic editor) (Abstract; Fig 4). Thakore teaches that dCas9-KRAB can be used for gene therapy, cellular reprogramming, and modulate cell phenotypes (page 1148, ¶6)
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted a dCas9-KRAB domain for the active Cas9 proteins in the [Gag]-[2xNES]-[linker]-[Cas9]-[NLS]-containing VLP rendered obvious above in claim 180 and the method of claim 205. It would have amounted to the simple substitution of one known Cas9 effector for another by known means to yield predictable results. The skilled artisan would have predicted that dCas9-KRAB could be used in the obvious VLP because Doudna teaches that such dead Cas9 variants fused to transcriptional repressors can be delivered by VLPs. The skilled artisan would have been motivated to have made the substitution for the purpose of delivering dCas9-KRAB to recipient cells for cellular reprogramming as taught in Thakore.
Response to Arguments - §103
Applicant argues that the cited references in combination do not teach or suggest a fusion protein comprising a greater number of NESs than NLSs (Remarks, page 9, ¶2). In the same paragraph Applicant argues that although Doudna teaches a Gag-Cas9-NLS fusion protein and Robert and Wu teaches Gag fusion proteins comprising NESs, it would not have been predictable what the combination of the NLS and NES with competing activities would have on the therapeutic cargo and its cellular localization (page 9, ¶2). This argument has been fully considered but is not persuasive. Robert teaches a fusion protein with both an NLS and an NES and teaches that an NES is essential for efficient packaging of an NLS-containing protein into VLPs. Because Wu teaches that cleavage of the NES away from the cargo after packaging allows independent cellular trafficking in recipient cells, the skilled artisan would predict that whatever localization signals are retained on the cargo after cleavage would dictate localization in the recipient cell.
Applicant argues that the claimed architecture resulted in a substantial increase in cytosolic localization of the fusion protein in producer cells and a substantial increase in Cas9-based gene editing when the VLP was delivered to target cells, which the skilled artisan would not have had a reasonable expectation of achieving from the teachings of Doudna, Robert and Wu (page 9, ¶3). This argument has been fully considered but is not persuasive. First, Applicant provides no evidence of why the skilled artisan would not have expected increased cytosolic localization in producer cells and increased Cas9-editing in recipient cells. MPEP 716.0l(c) makes clear that arguments of counsel cannot take the place of evidence in the record. Second, Wu teaches including an NES between the viral membrane protein and protease cleavage site increased packaging of the cargo protein in VLPs (i.e., increased cytosolic localization) and subsequent activity (i.e., localization) of the cargo in the recipient cells. From this teaching, the skilled artisan would have expected an increase in Cas9 packaging in VLPs and a concomitant increase in Cas9 delivery and activity in the VLP-recipient cells when an NES is included to form N-[gag]-[2xNES]-[cleavable linker]-[Cas9]-[NLS]-C in the VLP packaging cells. As such, the proffered evidence of increased cytosolic localization in producer cells and increased Cas9 activity in recipient cells when an NES is included between the gag protein and the cleavable linker is not unexpected.
Applicant argues that Urano and Thakore to do not cure the deficiencies of Doudna, Robert Wu and Zetsche (pages 10, ¶2-3). This argument has been fully considered but is not persuasive because a prima facie case of obviousness is established for the independent claims for the reasons set for in the §103 rejections of record and the response to Applicant’s arguments above.
Non-Statutory Double Patenting - 18715587
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.
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Claims 180, 184-192, 194-204 and 211 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 257-286 of copending Application No. 18715587. Claims 186, 200-204 and 211 are rejected in view of Doudna (US 20220403379 A1, priority to May 28, 2021). Claim 199 is rejected in view of Urano (Urano et al., Journal of General Virology (2008), 89: 3144-3149).
Copending claim 257 recites a virus-like particle (VLP) comprising… a fusion protein that comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp) or a domain comprising DNA polymerase activity fused to (ii) a second coiled-coil peptide; and (c) a lipid membrane encapsulating the protein and the fusion protein. Copending claim 276 recites the VLP of claim 257, wherein the fusion protein comprises the structure: NH2-[a gag nucleocapsid protein]-[an 1X-3X NES]-[a cleavable linker]-[an NLS]-[the napDNAbp]-[the domain comprising DNA polymerase activity]-[the NLS]-COOH, wherein each instance of “]-[“ independently comprises an optional linker, which was recited in canceled claim 39. Copending claims 264-265 recites wherein the cleavable linker comprises a protease cleavage that is an MMLV protease cleavage site or a FMLV protease cleavage site, which was recited in canceled claims 28-29. Copending claims 266-267 recite wherein the fusion protein comprises an MMLV or FMLV gag nucleocapsid protein, which was recited in canceled claim 36. Copending claim 277-279 recites, wherein the viral envelope glycoprotein a retroviral envelope glycoprotein, including a baboon retroviral envelope glycoprotein, which was recited in canceled claims 45-47. Thus, the copending claims anticipate instant claims 180, 184-185, 187-188, 190-192 and 194-198.
The copending claims do not recite the VLPs comprising both the Gag-Cas9 fusion protein and the cleaved products or ratios of proteins in the VLPs (claims 186, 202-204, 211). The copending claims do not recite the fusion protein comprising a pleckstrin homology domain (claim 199). The copending claims do not recite an antibody targeting moiety (claims 200-201).
The teachings of Doudna are recited above in paragraphs 16, 22-27, 30-31, 33-35, 37, 39-41 and 44 and incorporated here. Briefly, Doudna teaches VLPs that contain both the Gag-Cas9 fusion protein and the cleaved products. Doudna teaches including targeting moieties including antibody and antibody fragments to the surface of VLPs to target them to specific cells. Doudna teaches optimizing ratios of delivering plasmids encoding the Gag-pro and Gag-Cas9 fusion proteins to producer cells.
Regarding claims 186 and 202-204, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used VLPs that contain both the copending fusion proteins and the cleaved products at least in amounts where the cleaved products are in the VLP 1.5 the amount of the fusion protein because Doudna teaches in VLPs with Gag-Cas9 fusion proteins, the protease cleavage site is cleaved between 50-100%, thereby resulting in both the cleaved and un-cleaved products.
Regarding claims 200-201, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included a targeting antibody as taught in Doudna in the lipid containing particle of the copending claims. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that targeting antibodies could be added to the copending VLP because Doudna teaches that targeting antibodies can be used in VLPs delivering Cas9. The skilled artisan would have been motivated to include targeting antibodies for the purpose of targeting the obvious VLP to a specific cell type.
Regarding claim 211, it would have been obvious to one skilled in the art before the effective filing date to have optimized the ratio of Gag-pro to Gag-Cas9 fusion protein in the copending VLPs to arrive at a ratio of 3:1. It would have amounted to optimizing the protein ratios by known means to yield predictable results. The skilled artisan would have predicted that a 3:1 Gag-Pro:Gag-Cas9 ratio could be achieved because Doudna teaches such optimization. One would have been motivated to optimize in order to maximize Cas9 loaded into VLPs, as taught in Doudna.
Regarding claim 199, the teachings of Urano are recited above in paragraph 49 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 modified the fusion protein in the copending VLP by replacing the myristylation signal in the Gag protein with the PH domain taught in Urano. It would have amounted to the simple combination of elements and engineering Gag proteins in known ways to yield predictable results. The skilled artisan would have predicted that the copending fusion protein could be modified to include a PH domain because the prior art demonstrates that retroviral Gag proteins can be easily engineered. It also would have been predictable that VLPs with a [PH-Gag] could be produced since Urano teaches that inclusion of the PH domain does not affect VLP morphology or the ability to infect cells. One would have been motivated to include the PH domain in the copending fusion protein for the purpose of increasing the VLP production as indicated in Urano.
This is a provisional nonstatutory double patenting rejection.
Non-Statutory Double Patenting - 18957270
Claims 180-188, 190-192, 194-204 and 210-211 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 180-203 and 205-211 of copending Application No. 18957270. Claims 200-201 and 204 are rejected in view of Doudna (US 20220403379 A1, priority to May 28, 2021). Claim 199 is rejected in view of Urano (Urano et al., Journal of General Virology (2008), 89: 3144-3149).
Copending claim 180 recites a method of making an extracellular lipid containing particle comprising:(a) providing a producer cell that comprises: an exogenous polynucleotide comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises: (i) a plasma membrane localization protein, (ii) a therapeutic cargo comprising at least one NLS, (iii) at least two a nuclear export sequence (NES), and (iv) a cleavable linker, wherein the fusion protein comprises from N-terminus to C-terminus: the plasma membrane protein, the NES, the cleavable linker and the therapeutic cargo, and wherein the fusion protein comprises a greater number of NESs than NLSs (b) expressing the fusion protein from the exogenous polynucleotide in the producer cell; and (c) producing the extracellular lipid containing particle, wherein the extracellular lipid containing particle comprises the (1) the fusion protein from step (b) or (2) the therapeutic cargo cleaved from the fusion protein of step (b), or (3) both. Copending claim 181 recites the method of claim 180, wherein the producer cell further comprises an exogenous polynucleotide comprising a nucleic acid sequence encoding a protein that comprises a group- specific antigen (gag) and a protease (pro), and wherein (b) further comprises expressing the protein that comprises the gag and the pro in the producer cell (i.e., becomes the protein core). Copending claim 182 recites the method of claim 181, wherein the extracellular lipid containing particle further comprises the protein that comprises the gag and the pro. The product of the method recited in copending claim 182 is the product of instant claim 180. Thus, copending claim 182 anticipates instant claim 180. Copending claim 183-184 recites wherein the extracellular lipid containing particle comprises the therapeutic cargo cleaved from the fusion protein of (b) and the fusion protein of (b). Copending claim 185 recites wherein the amount of the therapeutic cargo that is cleaved from the fusion protein in the extracellular lipid containing particle is higher than the amount of the fusion protein in the extracellular lipid containing particle. Copending claim 186 recites wherein the extracellular lipid containing particle further comprises a cleavage product that comprises the plasma membrane localization protein and the NES from the fusion protein of (b) and lacks the therapeutic cargo. Copending claim 188 recites wherein the extracellular lipid containing particle further comprises a membrane fusion protein (i.e., an envelope glycoprotein. Copending claims 194-195 recite wherein the plasma membrane localization protein comprises a retroviral gag nucleocapsid polyprotein. Copending claim 196 recites wherein the therapeutic cargo comprises a nuclease or a transcription factor. Copending claims 197-198 recite wherein the cleavable linker comprises a protease cleavage site, including an MMLV protease cleavage site. Copending claim 199 recites wherein the fusion protein comprises at least three NESs. Copending claim 203 recites wherein the fusion protein comprises [plasma membrane localization protein]-[3X NES]-[cleavable linker]-[NLS]- [therapeutic cargo]-[NLS]. Copending claim 205 recites wherein the protein comprises an MMLV gag pro polyprotein or an FMLV gag pro polyprotein. Copending claim 206 recites wherein the plasma membrane localization protein comprises an MMLV gag nucleocapsid protein or an FMLV gag nucleocapsid protein. Copending claims 207-208 recite wherein the membrane fusion protein comprises a viral envelope glycoprotein, including a baboon retroviral envelope glycoprotein. Copending claim 210 recites wherein the cleavable linker comprises an amino acid sequence at least 90% identical to TSTLLMENSS. Copending claim 211 recites wherein teh ratio of the fusion protein of step (b) to the protein that comprise a gag and a pro within the extracellular lipid containing particle is approximately 1:3. Thus, the copending claims anticipate instant claims 180-188, 190-192, 194-198, 202-203 and 210-211.
The copending claims do not recite the fusion protein comprising a pleckstrin homology domain (claim 199). The copending claims do not recite an antibody targeting moiety (claims 200-201). The copending claims do not recite producing a VLP in which the amount of cleaved product is at least 1.5 times the amount of fusion protein in the VLP (claim 204).
The teachings of Doudna are recited above in paragraphs 16, 22-27, 30-31, 33-35, 37, 39-41 and 44 and incorporated here. Briefly, Doudna teaches VLPs that contain both the Gag-Cas9 fusion protein and the cleaved products, wherein the cleaved product is president at least 1.5 times as much as the fusion protein. Doudna teaches including targeting moieties including antibody and antibody fragments to the surface of VLPs to target them to specific cells.
Regarding claims 200-201, the obviousness including a targeting antibody in VLPs is recited above in paragraph 65.
Regarding claim 204, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used VLPs that contain both the copending fusion proteins and the cleaved products at least in amounts where the cleaved products are in the VLP 1.5 the amount of the fusion protein because Doudna teaches VLPs with Gag-Cas9 fusion proteins, the protease cleavage site is cleaved less than 100% resulting in ratios of cleaved and un-cleaved products to be 1.5 or higher.
Regarding claim 199, the teachings of Urano are recited above in paragraph 49 and incorporated here. The obviousness of modifying the fusion protein in the copending method of producing VLPs by replacing the myristylation signal in the Gag protein with the PH domain taught in Urano is recited above in paragraph 67.
This is a provisional nonstatutory double patenting rejection.
Non-Statutory Double Patenting - 18715569
Claims 180-188, 190-192, 194-211 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 134 of copending Application No. 18715569. Claims 185, 187 and 210 are rejected in view of Wu (Wu et al., Biomaterials (2014) 35: 8416-8426; of record). Claims 186 and 200-209 are rejected in view of Doudna (US 20220403379 A1, priority to May 28, 2021). Claim 199 is rejected in view of Urano (Urano et al., Journal of General Virology (2008), 89: 3144-3149).
Copending claim 134 recites a method of delivering a gene editing agent to (i.e., editing a nucleic acid molecule in) a target cell comprising contacting the target cell with a lipid containing particle that comprises that comprises:(1) a fusion protein that comprises:(i) the gene editing agent, (ii) a cleavable linker, and (iii) a nuclear export sequence (NES), and (2) the gene editing agent cleaved from the fusion protein, wherein the gene editing agent comprises a napDNAbp, and wherein the fusion protein and the gene editing agent cleaved from the fusion protein are encapsulated by a lipid membrane, thereby delivering the gene editing agent cleaved from the fusion protein to the target cell. Copending claim 171 recites wherein the cleavable linker is between the gene editing agent and the NES. Claims 172-173 recite wherein the fusion protein further comprises a gag protein, and wherein the NES is located between the gag protein and the gene editing agent. Copending claims 174 recites wherein the fusion protein comprises at least three NESs. Copending claim 181 recites NH2-[a gag protein]-[1X-3X NES]-[the cleavable linker]-[NLS]-[the gene editing agent]-[NLS]- COOH, wherein each instance of ]-[ independently comprises an optional linker. Copending claim 177 recites wherein the lipid containing particle further comprises a cleavage product that comprises the gag protein and the NES and lacks the gene editing agent. Copending claim 176 recites wherein the gag protein comprises an MMLV gag protein or an FMLV gag protein. Copending claim 179 recites wherein the lipid containing particle further comprises a viral envelope glycoprotein. Thus, the copending claims anticipates instant claims 180-184, 187-188, 190-192, 194-196.
The copending claims do not recite the VLPs comprising both the Gag-Cas9 fusion protein and the cleaved products or a ratio between the Gag-Cas9 fusion and the gag-pro protein (claims 186, 202-204, 211). The copending claims do not recite the cleavable linker is a protease cleavage site with a specific sequence (claims 185, 187, 210). The copending claims do not recite the fusion protein comprising a pleckstrin homology domain (claim 199). The copending claims do not recite an antibody targeting moiety (claims 200-201). The copending claims do not recite editing a nucleic acid molecule.
Regarding claims 185, 187 and 210, the teachings of Wu are recited above in paragraph 19 and 27 and incorporated here. Briefly, Wu teaches VLPs derived from MMLV proviral vectors encoding Gag-protein fusions with MMLV protease cleavage sites between the NES and cargo (Fig 1b, black and pink arrows). Wu teaches adding an additional RSSLY/PALTP site (i.e., a MMLV protease site; i.e., the linker comprising an amino acid at last 90% identical to SEQ ID NO: 2) (Fig 1b). It would have been obvious to specifically use the MMLV protease sequence as the cleavable linker in the copending fusion protein because Wu teaches the protease site can be used with the copending MMLV based system and is functional for producing cargo protein cleaved from the gag/NES portion of the fusion protein.
Regarding claims 186 and 202-209, the teachings of Doudna are recited above in paragraphs 16, 22-27, 30-31, 33-35, 37, 39-41 and 44 and incorporated here. Briefly, Doudna teaches VLPs that contain both the Gag-Cas9 fusion protein and the cleaved products. Doudna teaches including targeting moieties including antibody and antibody fragments to the surface of VLPs to target them to specific cells. Doudna teaches optimizing ratios of delivering plasmids encoding the Gag-pro and Gag-Cas9 fusion proteins to producer cells. Doudna teaches delivering the VLP comprising the Gag-Cas9 fusion protein and free Cas9 to cells for the purpose of editing a target locus. Regarding claims 186 and 202-204, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used VLPs that contain both the copending fusion proteins and the cleaved products at least in amounts where the cleaved products are in the VLP 1.5 the amount of the fusion protein because Doudna teaches VLPs with Gag-Cas9 fusion proteins, the protease cleavage site is cleaved less than 100%, thereby resulting in ratios of the cleaved and un-cleaved products to be 1.5 or higher. Regarding claims 205-209, it would have been obvious to one skilled in the art before the effect filing date of the claimed invention to have further included a step wherein the gene editing agent actually edits the targeted gene because Doudna teaches that is the end purpose of delivering gene editing agents by VLPs to cells.
Regarding claims 200-201, the obviousness including a targeting antibody in VLPs is recited above in paragraph 72.
Regarding claim 211, the obviousness of optimizing the ratio between the gag-pro protein and the gag-Cas9 fusion protein in the copending lipid containing particles is recited above in paragraph 66.
Regarding claim 199, the teachings of Urano are recited above in paragraph 49 and incorporated here. The obviousness of modifying the fusion protein in the copending method of producing VLPs by replacing the myristylation signal in the Gag protein with the PH domain taught in Urano is recited above in paragraph 67.
This is a provisional nonstatutory double patenting rejection.
Non-Statutory Double Patenting - 18715578
Claims 180, 184-188, 190-192, 195-196, 199 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 180-203 of copending Application No. 18715578 in view of Doudna (US 20220403379 A1, priority to May 28, 2021).
Copending claim 180 recites A polynucleotide encoding a fusion protein, wherein the fusion protein comprises:(i) a plasma membrane localization protein, (ii) a therapeutic cargo, (iii) a nuclear export sequence (NES); wherein a) the NES is located between the plasma membrane localization protein and the therapeutic cargo, or b) the NES is N-terminal to the therapeutic cargo; and(iv) a cleavable linker located between the therapeutic cargo and the NES. Copending claim 184-185 recites wherein the plasma membrane localization protein comprises a retroviral gag protein, wherein the retroviral gag protein comprises a gag nucleocapsid protein. Copending claim 187 recites wherein the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease, a recombinase, a transcription factor. Copending claims 192-193 recite wherein the cleavable linker comprises a protease cleavage site including a Moloney murine leukemia virus (MMLV) protease cleavage site or a Friend murine leukemia virus (FMLV) protease cleavage site. Copending claim 198 recites wherein the fusion protein comprises from N- terminus to C-terminus: [plasma membrane localization protein]-[3X NES]-[cleavable linker]- [NLS]-[therapeutic cargo]-[NLS]. Copending claim 200 recites wherein the plasma membrane localization protein comprises an MMLV gag nucleocapsid protein or an FMLV gag nucleocapsid protein. Copending claim 201 recites wherein the plasma membrane localization protein comprises a human endogenous retroviral (HERV) structural protein, a humanized viral structural protein, a pleckstrin homology (PH) domain, or a non-immunogenic plasma membrane recruitment protein. Copending claim 202 recites A fusion protein encoded by the polynucleotide of claim 180. Copending claim 203 recites A vector comprising the polynucleotide of claim 180.
The copending claims do not recite the fusion protein within the context of a lipid containing particle.
The teachings of Doudna are recited above in paragraphs 16, 22-27, 30-31, 33-35, 37, 39-41 and 44 and incorporated here. Briefly, Doudna teaches VLPs containing a Gag-Cas9 fusion protein with the copending claimed structure.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used included the copending fusion protein in a lipid containing particle because Doudna teaches including the gag domains fused to Cas9 is for the purpose of creating a VLP.
This is a provisional nonstatutory double patenting rejection.
Non-Statutory Double Patenting - 18945691
Claims 180, 182, 189-191, 196-197 and 199 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 90-109 of copending Application No. 18945691.
Copending claim 90 recites A lipid containing particle comprising:(a) a virally derived glycoprotein, (b) a combinatorial protein (i.e., a fusion protein) comprising a plasma membrane localization protein coupled to a nuclear export sequence (NES) or a cleavable linker; and (c) a freight (i.e., a cargo). Copending claim 91 recites the lipid containing particle of claim 90, wherein the combinatorial protein further comprises the freight. Copending claim 93 recites the lipid containing particle of claim 92, wherein the combinatorial protein comprises the plasma membrane localization protein, the NES, and the freight arranged in order from an N- terminus of the combinatorial protein to a C-terminus of the combinatorial protein. Copending claims 94-95 recites the lipid containing particle of claim 93, wherein the combinatorial protein further comprises a cleavable linker; wherein the cleavable linker is positioned between the NES and the freight. Copending claim 96 recites wherein the combinatorial protein comprises a nuclear localization sequence (NLS) C-terminal of the cleavable linker. Copending claim 103 recites a lipid containing particle comprising (a) a human endogenous retroviral (HERV) structural protein, optionally HERV gag (i.e., a retroviral gag that comprises the nucleocapsid polyprotein) and (b) a combinatorial protein comprising a plasma membrane localization protein that is a PH domain. Copending claim 102 recites the lipid containing particle of claim 90, wherein the lipid containing particle comprises a lipid containing membrane encapsulating a protein core. Thus, the copending claims anticipate the lipid-containing particle of instant claims 180, 182, 189-191, 193, 196-197, and 199.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments - NSDP
Applicant requested reconsideration of the nonstatutory double patenting (NSDP) rejections in view of the amendments (Remarks, page 11). Examiner has considered the NSDP rejections in view of the amendments to the instant claims and the amendments to the copending claims since the mailing of the previous office action. The rejections recited above are updated to reflect claim amendments.
Conclusion
No claims are allowable.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635