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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 15, 2026 has been entered.
Application Status
Applicant’s amendments filed January 15, 2026 amending claims 180, 183-184, 186, 190-191, 193, 196, 201-203 and 209, canceling claim 200, and adding new claim 211 is acknowledged. Claims 180-199, 201-203 and 205-211 are pending and under examination.
Claim 180 was 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 arrangement of canceled claim 200 and includes limitations from amended claim 202. 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 200 and amended claim 202 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 of step (b) to the protein that comprise a gag and a pro within the extracellular lipid containing particle 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, 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. Although the claims are directed to methods of making using the VLPs, claim 211 does not refer to the exogenous polynucleotides of step (a). 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 specifically 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 (i.e., exogenous polynucleotide) 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 provide 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 180-199, 201-203, 205-210 are rejected under 35 U.S.C. 103 as being unpatentable over Doudna (US 20220403379 A1, priority to May 28, 2021) in view of 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). Claims 181-182, 194-195 and 205 are evidenced by Addgene1 (psPAX2, Plasmid #12260, https://www.addgene.org/12260/ [retrieved January 14, 2025]). Claims 198 and 205-206 are evidenced by Schneider (Schneider et al., Virology (2012), 188-196).
Regarding claim 180, Doudna teaches virus like particles comprising Cas9 (VLPs, i.e., an extracellular lipid-containing particle) (Fig 1B; [0006]-[0007]). Doudna teaches producing the Cas9-VLPs by (a) providing Lenti-X cells (i.e., producer cells) with the Gag-Cas9 plasmid (i.e., an exogenous polynucleotide comprising a nucleic acid sequence encoding a fusion protein) ([0328]-[0329]). Doudna teaches the Cas9-Gag fusion protein comprises (i) the MA, CA, NC and P6 proteins of Gag (i.e., a plasma membrane localization protein) (Fig 1B). Doudna teaches the fusion protein comprises (ii) SpCas9-NLS (i.e., a therapeutic cargo comprising at least one NLS) (Fig 1B). Doudna teaches the fusion protein comprises (iv) 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 teaches (b) expressing the Gag-Cas9 fusion protein in the producer cells ([0329]). Doudna teaches (c) producing the VLP from the producer cells ([0329]). Doudna teaches the VLPs comprise the Gag-Cas9 fusion protein (FIG 1C).
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]-[at least 2xNES]-[cleavable linker]-[cargo]-[NLS].
Robert teaches producing VLPs from packaging (i.e., producer) cells by transfecting plasmids encoding Gag-cargo fusion proteins into the packaging cells, for the purpose of delivering nuclear proteins to cells (Title, Abstract). Robert teaches VLPs comprise a fusion comprising the Gag polyproteins (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).
Wu also teaches producing VLPs from producer cells that comprise nuclear transcriptions factors fused to Gag proteins 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 Cas9 fragment in the cytoplasm, 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 fusion protein to form the fusion protein [Gag]-[2xNES]-[cleavable linker]-[Cas9]-[NLS] in the method of producing Cas9-VLPs. 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 one 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 two 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 NESs 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 claims 181-182, Doudna teaches also transfecting the Lenti-X cells with psPax2 ([0329]). Doudna teaches psPax2 encodes at least a Gag protein ([0328]). Doudna teaches the Cas9 was cleaved from the Gag in the mature VLPs, indicating that there was also a protease in the VLP (Fig 1e). Doudna teaches the cells transfected with a plasmid labeled Gag-Pol (i.e., a plasmid that encodings Gag and Pol, which encodes a protease) (Fig 1A). Doudna teaches that the Gag coding sequence for the Gag-Cas9 fusion protein was derived from psPax2 (Addgene #12260) ([0328]). Doudna is silent whether psPax2 in [0328] also encodes a protease. Addgene teaches psPax2 encodes the Pol gene (i.e., the gene that encodes the protease) (pages 1-2). Therefore, Doudna’s method inherently included a producer cell that comprises an exogenous polynucleotide comprising a nucleic acid sequence encoding gag and pro.
Regarding claim 183-185 and 209, Doudna teaches that VLPs produced from cells transfected with 1:1 (w/w) ratio of the Gag-Cas9 plasmid and the Gag-pol plasmid comprised both the Gag-Cas9 fusion protein and Cas9 cleaved from the fusion protein (Fig 1C, VLP formation C). Based on the size and darkness of the Cas9 band compared to the Gag-Cas9 in FIG 1C with VLP formulation C, Doudna teaches the amount of cleaved Cas9 in the VLP is higher than the amount of Gag-Cas9 fusion protein in the VLP (Fig 1C).
Regarding claim 186, Doudna teaches that the VLPs comprise both Gag-Cas9 and the cleaved Cas9 (Fig 1C). The obviousness of including two NESs 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-2xNES without the Cas9.
Regarding claim 187-188 and 207, Doudna teaches transfecting the Lenti-X cells with a plasmid encoding the HIV Env glycoprotein (i.e., a polynucleotide encoding a membrane fusion protein that is a viral glycoprotein ([0329]; FIG 1A). Doudna teaches expressing the HIV Env protein in the Lenti-X producer cells and producing VLPs comprising the HIV Env glycoprotein (Fig 1A-B, Fig 4; [0359]).
Regarding claims 189-190, the obviousness of including two NESs between the Gag protein and the protease cleavage site in the [Gag]-[linker]-[Cas9]-[NLS] fusion protein of Doudna is recited above for claim 180. Doudna does not teach that the Gag-Cas9 fusion protein localizes at the outer cytoplasmic membrane of the producer cell when expressed.
However, Robert teaches that when the NES was added to the Gag-VP16KLF4 fusion protein, the NES-containing fusion protein was localized to the plasma membrane (i.e., the outer cytoplasmic membrane) of the HEK293A producer cells to a greater extent than the Gag-VP16KLEF4 fusion protein that lacks the NES (Fig 7b, compare panel v and ix). Wu also teaches that the 2xNES-containing Gag-Oct4-2NES fusion protein localized to the plasma membrane in the producer cells to a greater extent than the Gag-Oct4 fusion protein that lacked the two NESs (Fig 2, compare panels b and c).
The skilled artisan would have predicted that the obvious [Gag]-[2xNES]-[linker]-[Cas9]-[NLS] fusion protein would have localized to the plasma membrane to a great extent that the [Gag]-[linker]-[Cas9]-[NLS] lacking the 2xNES because both Wu and Robert teach that adding NESs to a Gag-cargo fusion protein increases its localization to the plasma membrane in the producer cells.
Regarding claim 191, the obviousness of including two NESs between the Gag protein and the protease cleavage site in the [Gag]-[linker]-[Cas9]-[NLS] fusion protein of Doudna is recited above for claim 180. Additionally, Wu and Robert teach that adding an NES 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]-[2xNES]-[linker]-[Cas9]-[NLS] fusion protein in the VLPs would be higher than the packaging of Doudna's [Gag]-[linker]-[Cas9]-[NLS].
Regarding claim 192, Doudna teaches harvesting and purifying the VLPs produced from the Lenti-X cells ([0329]).
Regarding claims 193, the obviousness of including two NESs between the Gag protein and the protease cleavage site in the [Gag]-[linker]-[Cas9]-[NLS] fusion protein of Doudna is recited above for claim 180. Given the domain structure disclosed in FIG 1A and 1B in Doudna, the cleaved Cas9 product detected in FIG 1C, inherently must have comprised an NLS.
Regarding claim 194-195, Doudna teaches the Gag protein comprises the MA, CA, NC and P6 proteins that forms the nucleocapsid of the VLP (i.e., the plasma membrane protein comprises a gag nucleocapsid 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 comprises a retroviral gag protein and a retroviral gag nucleocapsid polyprotein.
Regarding claim 196, 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 197, as indicated above for claim 180 Doudna teaches the Gag-Cas9 fusion protein comprises an HIV-1 protease cleavage site (Fig 1A).
Regarding claims 198, 205-206 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 MLV proviral vectors encoding Gag-protein fusions with MLV and 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 additional WT gag and pro proteins) were based on the pNCA-C (IN- D184N/K376A) proviral vector as a backbone (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., a site with at least 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 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 MMLV viral vectors, MMLV gag-pro structural components, and MMLV cleavage site that is RSSLYPALTP (i.e., 90% identical to SEQ ID NO 2) instead of the HIV-1-derived components in Doudna’s method of producing VLPs comprising the [Gag]-[NES]-[linker]-[Cas9] protein rendered obvious above. It would have amounted to the simple substitution of one retroviral Gag structural components and protease for another by known means to yield predictable results. The skilled artisan would have predicted that an [MMLV Gag]-[NES]-[MMLV protease site: RSSLYPALTP]-[Cas9] fusion protein could be produced from Lenti-X producer cells 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 199, 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 a third NES in the fusion protein rendered obvious for claim 180, is also obvious.
Regarding claims 201-202, 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 for claim 180 comprises the structure N-terminus to C-terminus: [Gag]-[2xNES]-[cleavable linker]-[Cas9]-[NLS] (i.e., wherein n=2, m1=0, and m2=1).
Regarding claim 203, as indicated above for claims 200 and 201, Doudna teaches a Gag-Cas9-NLS fusion protein (FIG 1A-B). The obviousness of adding 3x NESs to the [Gag]-[cleavable linker]-[Cas9]-[NLS] of Doudna to produce a [Gag]-[3xNES]-[cleavable linker]-[Cas9]-[NLS] fusion protein is recited above for claims 180 and 199. Doudna also 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 included an additional NLS at the N-terminus of Cas9 in the fusion protein rendered obvious for claim 199 to produce a fusion protein with the structure [Gag]-[3xNES]-[cleavable linker]-[NLS]-[Cas9]-[NLS]. It would have amounted to the simple addition of a known element 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 208, 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 method to produce VLPs 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 claim 211, for the purposes of compact prosecution and applying prior art, claim 211 is interpreted as “wherein the exogenous polynucleotide encoding the fusion protein and the exogenous polynucleotide encoding the protein that comprises a gag and a pro are provided to the producer cells in a mass ratio of approximately 1:3”.
Doudna teaches varying the ratio of plasmid encoding the gag-pol protein relative to the plasmid encoding the [Gag]-[linker]-[Cas9]-[NLS] fusion 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). Doudna teaches plasmid mass ratios for the gag-Cas9 fusion protein and gag-pol protein of 1:4.8 (formulation A), 2:1 (formulation B) and 1:1 (formulation C) (FIG. 1C). Doudna teaches formulations A and B had the VLP production (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 plasmids-encoding Gag-pro to Gag-Cas9 fusion protein in the method of producing VLPs rendered obvious for claim 180 to arrive at a mass ratio of 3:1. It would have amounted to optimizing the plasmid ratios by known means to yield predictable results. The skilled artisan would have predicted that a 3:1 Gag-Pro:Gag-Cas9 plasmid mass ratio could be used to produce VLPs because Doudna teaches optimization with 1:4.8 and 1:2 plasmid mass ratios are both useful for producing high VLP titers. One would have been motivated to optimize in order to maximize Cas9 loaded into VLPs, as taught in Doudna.
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 already recognizes 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.
Non-Statutory Double Patenting - 18957216
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 180-199, 201-203 and 205-211 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 180-204 of copending Application No. 18957216 in view of Doudna (US 20220403379 A1, priority to May 28, 2021). Claims 189-190 are rejected further in view of Wu (Wu et al., Biomaterials (2014) 35: 8416-8426).
Copending claim 180 recites a lipid containing particle that comprises a protein core and a fusion protein encapsulated by a lipid membrane, wherein the fusion protein comprises:(a) a plasma membrane localization protein, (b) a therapeutic cargo comprising at least one nuclear localization sequence (NLS), (c) a cleavable linker, and (d) at least two nuclear export sequences (NESs), and wherein the fusion protein comprises from N-terminus to C-terminus: the plasma membrane localization protein, the NES, the cleavable linker, and the therapeutic cargo and wherein the fusion protein comprises a greater number of NESs than NLSs. Copending claims 181 and 194 recite the lipid containing particle of claim 180, wherein the protein core comprises a protein that comprises a group-specific antigen (gag) and a protease (pro), including an MMLV gag pro polyprotein. Copending claims 182-183 recite the lipid containing particle of claim 180, wherein the plasma membrane localization protein comprises a retroviral gag protein, including a retroviral gag protein comprises a gag nucleocapsid polyprotein. Copending claim 184 recites the lipid containing particle of claim 180, wherein the therapeutic cargo comprises a nuclease. Copending claim 185 and 187 recites the lipid containing particle of claim 180, wherein the cleavable linker comprises a protease cleavage site, including an MMLV protease cleavage site. Copending claim 186 recites the lipid containing particle of claim 180, wherein the protein core comprises a cleavage product comprising the plasma membrane localization protein and the NESs, and lacking the therapeutic cargo. Copending claim 192 recites the fusion protein comprises the structure: [plasma membrane localization protein]-[3X NES]-[cleavable linker]-[NLS]-[therapeutic cargo]-[NLS]. Copending claim 195 recites the lipid containing particle of claim 180, wherein the plasma membrane localization protein comprises an MMLV gag nucleocapsid protein or an FMLV gag nucleocapsid protein. Copending claims 196-198 recites the lipid containing particle further comprises a viral envelope glycoprotein including a retroviral envelope glycoprotein and a baboon retroviral glycoprotein. Copending claims 202-203 recite the lipid containing particle of claim 180, wherein the lipid containing particle further comprises the therapeutic cargo cleaved from the fusion protein and the amount of the therapeutic cargo cleaved from the fusion protein is higher than the amount of the fusion protein in the lipid containing particle. Copending claim 210 recites the lipid containing particle of claim 180, wherein the cleavable linker comprises an amino acid sequence at least 90% identical to TSTLLMENSS (SEQ ID NO: 1), PRSSLYPALTP (SEQ ID NO: 2), VQALVLTQ (SEQ ID NO: 3), or PLQVLTLNIERR (SEQ ID NO: 4). Copending claim 211 recites wherein the ratio of the fusion protein to the protein that comprises a gag and a pro within the protein core is approximately 1:3.
The copending claims do not recite methods of making the lipid-containing particles. The copending claims do not recite the localization of the fusion proteins in a cell (claims 189-190).
The teachings of Doudna are recited above in paragraphs 15, 21-24, 29-34, 38-39, 41 and 44, and incorporated here. Briefly, Doudna teaches that producing VLPs comprising Gag-Cas9 fusion proteins produced from Lenti-X producer cells by (b) contacting the Lenti-X cells with polynucleotides encoding comprising the Gag-Cas9 fusion protein. Doudna teaches further harvesting and purifying the VLPs. Doudna teaches the resulting VLPs comprise 1) the fusion protein, 2) the cleaved Cas9, and 3) therefore the cleaved Gag protein. Doudna also teaches that by titrating the ratio of plasmid encoding Gag-Cas9 and plasmid encoding Gag-Pol transfected into the producer cells, it is possible to optimize the ratio of fusion protein and cleaved cargo in the VLPs (FIG 1C).
Regarding claims 180-203, 205-210, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have produced the copending VLPs comprising the copending fusion proteins by contacting producer cells with polynucleotides encoding the copending fusion proteins. It would have amounted to producing known lipid-containing particles by known methods to yield predictable results. The skilled artisan would have predicted that the copending lipid containing particles could be produced merely by contacting a producer cell with polynucleotides encoding the copending fusion proteins because Doudna teaches those are the steps needed to produce VLPs, a type of lipid-containing particle.
Regarding claims 189-190, the teachings of Wu are recited above in paragraphs 18, 26, 28, 34, and 36 and incorporated here. Briefly, Wu teaches that adding a cleavable NES to a Gag-cargo fusion protein that contains an NLS 1) traffics the Gag-NES-cargo protein to the plasma membrane in the producer cells, 2) increases the level of Gag fusion protein at the plasma membrane compared to a Gag-fusion protein without an NES, and 3) increases the amount of Gag fusion protein in the VLPs compared to a Gag-fusion protein without an NES. It would have been obvious to one skilled in the art that the copending method would include the copending fusion protein localized to the plasma membrane because Wu teaches that NES-containing Gag fusion proteins traffic to the plasma membrane. It also would have bene obvious that the copending method would include more Gag-NES-cargo at the plasma membrane with subsequent packaging in VLPs than a Gag-cargo fusion without the NES because Wu teaches that including an NES causes such effects.
This is a provisional nonstatutory double patenting rejection.
Non-Statutory Double Patenting - 18715578
Claims 180-199, 201-203 and 205-210 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 180-204 of copending Application No. 18715578 in view of Doudna (US 20220403379 A1, priority to May 28, 2021). Claims 189-190 are rejected further in view of Wu (Wu et al., Biomaterials (2014) 35: 8416-8426).
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 203 recites A vector comprising the polynucleotide of claim 180.
The copending claims do not recite a method of using the polynucleotide to produce an extracellular lipid containing particle.
The teachings of Doudna are recited above in paragraphs 15, 21-24, 29-34, 38-39, 41 and 44, and incorporated here. Briefly, Doudna teaches that producing VLPs comprising Gag-Cas9 fusion proteins produced from Lenti-X producer cells by (b) contacting the Lenti-X cells with polynucleotides encoding comprising the Gag-Cas9 fusion protein. Doudna teaches further harvesting and purifying the VLPs. Doudna teaches the resulting VLPs comprise 1) the fusion protein, 2) the cleaved Cas9, and 3) therefore the cleaved Gag protein. Doudna also teaches that by titrating the ratio of plasmid encoding Gag-Cas9 and plasmid encoding Gag-Pol transfected into the producer cells, it is possible to optimize the ratio of fusion protein and cleaved cargo in the VLPs (FIG 1C).
Regarding claims 180-199, 201-203, and 205-211, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used the copending polynucleotides to produce a VLP in producer cells with an additional polypeptide encoding a gag-pro protein using the method of Doudna. It would have amounted to producing known lipid-containing particles by known methods to yield predictable results. The skilled artisan would have predicted that the copending polynucleotides could be used to produce VLPs merely by contacting a producer cell with the polynucleotides because Doudna teaches those are the steps needed to produce VLPs, a type of lipid-containing particle.
Regarding claims 189-190, the teachings of Wu are recited above in paragraphs 18, 26, 28, 34, and 36 and incorporated here. Briefly, Wu teaches that adding a cleavable NES to a Gag-cargo fusion protein that contains an NLS 1) traffics the Gag-NES-cargo protein to the plasma membrane in the producer cells, 2) increases the level of Gag fusion protein at the plasma membrane compared to a Gag-fusion protein without an NES, and 3) increases the amount of Gag fusion protein in the VLPs compared to a Gag-fusion protein without an NES. It would have been obvious to one skilled in the art that the copending method would include the copending fusion protein localized to the plasma membrane because Wu teaches that NES-containing Gag fusion proteins traffic to the plasma membrane. It also would have bene obvious that the copending method would include more Gag-NES-cargo at the plasma membrane with subsequent packaging in VLPs than a Gag-cargo fusion without the NES because Wu teaches that including an NES causes such effects.
This is a provisional nonstatutory double patenting rejection.
Non-Statutory Double Patenting - 18715587
Claims 180-199, 201-203 and 205-210 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 257-271 and 273-286 of copending Application No. 18715587 in view of Doudna (US 20220403379 A1, priority to May 28, 2021). Claims 189-190 are rejected further in view of Wu (Wu et al., Biomaterials (2014) 35: 8416-8426).
Copending claim 257 recites A virus-like particle (VLP) comprising:(a) a protein that comprises a viral structural protein linked to a viral protease, wherein the protein is fused to a first coiled-coil peptide;(b) 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 claims 262-263 recite wherein the fusion protein further comprises a cleavable linker and a nuclear export sequence (NES), including at least three NESs. Copending claims 265 recites wherein the protease cleavage site comprises a Moloney murine leukemia virus (MMLV) protease cleavage site or a Friend murine leukemia virus (FMLV) protease cleavage site. Copending claims 266-267 recite wherein the fusion protein comprises a gag nucleocapsid protein, including MMLV gag or FMLV gag nucleocapsid protein (i.e., a PMLP). Copending claims 270 recites wherein the fusion protein comprises a napDNAbp, and wherein the cleavable linker is located between (i) the napDNAbp and (ii) the NES. Copending claims 273 recites wherein the fusion protein comprises an NLS. Copending claim 276 recites 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. Copending claims 277-279 recite wherein the lipid membrane comprises a viral envelope glycoprotein, including a baboon retroviral envelope glycoprotein.
The copending claims do not recite methods of making the lipid-containing particles. The copending claims do not recite the localization of the fusion proteins in a cell (claims 189-190).
The teachings of Doudna are recited above in paragraphs 15, 21-24, 29-34, 38-39, 41 and 44, and incorporated here. Briefly, Doudna teaches that producing VLPs comprising Gag-Cas9 fusion proteins produced from Lenti-X producer cells by (b) contacting the Lenti-X cells with polynucleotides encoding comprising the Gag-Cas9 fusion protein. Doudna teaches further harvesting and purifying the VLPs. Doudna teaches the resulting VLPs comprise 1) the fusion protein, 2) the cleaved Cas9, and 3) therefore the cleaved Gag protein. Doudna also teaches that by titrating the ratio of plasmid encoding Gag-Cas9 and plasmid encoding Gag-Pol transfected into the producer cells, it is possible to optimize the ratio of fusion protein and cleaved cargo in the VLPs (FIG 1C).
Regarding claims 180-203, 205-210, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have produced the copending VLPs comprising the copending fusion proteins by contacting producer cells with polynucleotides encoding the copending fusion proteins. It would have amounted to producing known lipid-containing particles by known methods to yield predictable results. The skilled artisan would have predicted that the copending lipid containing particles could be produced merely by contacting a producer cell with polynucleotides encoding the copending fusion proteins because Doudna teaches those are the steps needed to produce VLPs, a type of lipid-containing particle.
Regarding claims 189-190, the teachings of Wu are recited above in paragraphs 18, 26, 28, 34, and 36 and incorporated here. Briefly, Wu teaches that adding a cleavable NES to a Gag-cargo fusion protein that contains an NLS 1) traffics the Gag-NES-cargo protein to the plasma membrane in the producer cells, 2) increases the level of Gag fusion protein at the plasma membrane compared to a Gag-fusion protein without an NES, and 3) increases the amount of Gag fusion protein in the VLPs compared to a Gag-fusion protein without an NES. It would have been obvious to one skilled in the art that the copending method would include the copending fusion protein localized to the plasma membrane because Wu teaches that NES-containing Gag fusion proteins traffic to the plasma membrane. It also would have bene obvious that the copending method would include more Gag-NES-cargo at the plasma membrane with subsequent packaging in VLPs than a Gag-cargo fusion without the NES because Wu teaches that including an NES causes such effects.
This is a provisional nonstatutory double patenting rejection.
Non-Statutory Double Patenting - 18945691
Claims 180-199, 201-203, 205-207 and 209 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 90-102 of copending Application No. 18945691 in view of Doudna (US 20220403379 A1, priority to May 28, 2021) and Wu (Wu et al., Biomaterials (2014) 35: 8416-8426). Claims 198, 205-206 are evidenced by Schneider (Schneider et al., Virology (2012), 188-196).
Copending claims 90-91 recite a lipid containing particle comprising: a virally derived glycoprotein selected from the group consisting of: RD 114, Fug-E, FuG-E (P440E), and MLV 10A1; 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 coupled to a freight (i.e., a cargo). Copending claims 91-92 recite the lipid containing particle of claim 90, wherein the combinatorial protein comprises the NES, 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 recite the lipid containing particle of claim 93, wherein the combinatorial protein further comprises the cleavable linker and wherein the cleavable linker is positioned between the plasma membrane localization protein and the freight or between the NES and the freight. Copending claim 96 recites the lipid containing particle of claim 94 wherein the combinatorial protein comprises a nuclear localization sequence (NLS) C-terminal of the cleavable linker. Copending claim 101 recites the freight comprises a therapeutic freight or a binding partner for the therapeutic freight. Copending claim 102 recites wherein the lipid containing particle comprises a lipid containing membrane encapsulating a protein core, wherein the protein core comprises the combinatorial protein.
The copending claims do not recite methods of making the lipid-containing particles. The copending claims do not recite the fusion protein comprising 2 or 3 NESs. The copending claims do not recite an additional protein comprised of gag and pro (claims 181-182). The copending claims do not recite the freight cleaved from the fusion protein (claims 183-186). The copending claims do not recite the localization of the combinatorial protein when produced in a producer cell (claims 189-190). The copending claims do not recite comparisons between fusion proteins that do and do not contain NESs (claim 191). The copending claims do not recite harvesting and purifying the lipid containing particles (claim 192). The copending claims do not recite the plasma membrane proteins or cleavable linkers are derived from retroviruses in general or MMLVs specifically (claims 194-195, 197-198, 205-206). The copending claims do not recite a specific therapeutic freight (claim 196).
The teachings of Doudna are recited above in paragraphs 15, 21-24, 29-34, 38-39 and 41, and incorporated here. Briefly, Doudna teaches that producing VLPs comprising Gag-Cas9 (i.e., a nuclease) fusion proteins produced from Lenti-X producer cells by (b) contacting the Lenti-X cells with polynucleotides encoding comprising the Gag-Cas9 fusion protein. Doudna teaches further harvesting and purifying the VLPs. Doudna teaches the resulting VLPs comprise 1) the fusion protein, 2) the cleaved Cas9, and 3) therefore the cleaved Gag protein. Doudna also teaches that by titrating the ratio of plasmid encoding Gag-Cas9 and plasmid encoding Gag-Pol transfected into the producer cells, it is possible to optimize the ratio of fusion protein and cleaved cargo in the VLPs (FIG 1C). Doudna also teaches that VLPs can be produced from MMLV-derived machinery.
The teachings of Wu are recited above in paragraphs 18, 26, 28, 34, and 36 and incorporated here. Briefly Wu, as evidenced by Schneider, teaches producing MMLV-derived lipid containing particles for delivery of transcription factors to recipient cells and the beneficial effect of adding an NES to the Gag-cargo fusion proteins. Wu teaches an MMLV protease cleavage site added between the gag protein and the cargo that comprises RSSLYPALTP (i.e., at least 90% identical to SEQ ID NO 2).
Regarding claims 180-182, 187-192, 198, 201-202, 205-207 and 210, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included an addition 1 or 2 NESs in the copending fusion protein and produced the copending lipid-containing particles by contacting a producer cell with a polynucleotide encoding the copending combinatorial protein and a Gag-pol proteins. It would have amounted to a simple duplication of an element in the copending protein and producing known lipid-containing particles by known methods to yield predictable results. The skilled artisan would have predicted that two or three NESs could be included in the combinatorial 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 or 1 NES, the triplication of the NES in the copending combinatorial protein in the obvious method of producing the copending lipid containing particle is also obvious. The skilled artisan would have predicted that the copending lipid containing particles could be produced merely by contacting a producer cell with polynucleotides encoding the copending fusion proteins and additionally a Gag-Pol plasmid taught in Doudna because Doudna teaches those are the steps needed to produce VLPs, a type of lipid-containing particle. The skilled artisan would have been motivated to do so because Doudna teaches the claimed method is how the copending VLPs can be produced.
Regarding claims 183-186, 193 and 209, it also would have been obvious to one skilled in the art before the effective filing date of the claimed invention that resulting VLPs would have both the combinatorial fusion protein and the cleaved therapeutic freight because Doudna teaches when making VLPs comprising a fusion protein comprising a plasma membrane localization protein and a cargo separated by a cleavable linker, the VLP will have both the combinatorial protein and the cleavage products. It also would have been obvious to vary the ratios of the polynucleotide encoding the copending combinatorial protein and the Gag-Pol plasmid taught in Doudna such that there would be higher levels of the cleaved therapeutic freight than the combinatorial protein. Doudna teaches that having equal weights of plasmids encoding polynucleotide encoding the copending combinatorial protein and the Gag-Pol plasmid results in the VLPs having both fusion protein and cleaved cargo, wherein the cleaved cargo is present at a higher level than the fusion protein. The skilled artisan would have been motivated to do so in order to produce VLPs that produce the cleaved product which are more active in the recipient cells.
Regarding claims 194-195, and 197, 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 retroviral nucleocapsid gag polyprotein as the plasma membrane protein and a retroviral protease site as the cleavable linker in the copending combinatorial protein. It would have amounted to using a specific membrane localization protein and cleavable linker instead of the copending generic elements by known means to yield predictable results. The skilled artisan would have predicted that the gag proteins and HIV-1 cleavage sites taught in Doudna could be used in the copending combinatorial protein, and been motivated to do so, because Doudna demonstrates that such elements are functional in VLPs to deliver freight to recipient cells.
Regarding claim 196, 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 the Cas9 nuclease in the copending combinatorial protein. It would have amounted to using a specific known therapeutic cargo instead of the copending generic elements by known means to yield predictable results. The skilled artisan would have predicted that the Cas9 taught in Doudna could be used in the copending combinatorial protein, and been motivated to do so, because Doudna demonstrates successful VLP-mediated delivery of Cas9.
Regarding claim 203, as indicated above for claims 180, 200 and 201, the structure of the copending combinatorial protein in copending claim 96 is [PM protein]-[NES]-[cleavable linker]-[freight] with an [NLS] either between the linker and the freight or at the C-terminal end of the freight. The obviousness of adding 3x NESs to the copending combinatorial protein is recited above for claim 199. Doudna also 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 included an additional NLS at the N-terminus or C-terminus of the freight in the copending combinatorial protein rendered obvious for claim 199 to produce a fusion protein with the structure [PM protein]-[NES]-[cleavable linker]-[NLS]-[freight]-[NLS]. It would have amounted to the simple addition of a known element 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 freight because Doudna teaches adding NLSs to both the N- and C-terminus of nuclear cargoes delivered by lipid containing particles. The skilled artisan would have been motivated to do so because Doudna suggests it.
Regarding claims 189-191, the obviousness of having increased levels of the copending NES-containing fusion protein localized to the plasma membrane and subsequent packaging in VLPs than a combinatorial protein without the NES is recited above in paragraph 54.
Regarding claims 198 and 205-206, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used MMLV viral vectors, MMLV gag-pro structural components, and MMLV cleavage site taught in Wu in the obvious method of producing the copending lipid containing particles comprising the [membrane protein]-[NES]-[cleavable linker]-[freight] protein. It would have amounted to using retroviral Gag structural components and protease known to be useful for producing VLPs for the generic components in the copending claims. The skilled artisan would have predicted that an [MMLV Gag]-[NES]-[MMLV protease site: RSSLYPALTP]-[Cas9] fusion protein could be produced from producer cells and loaded into the copending lipid containing particles and been motivated to use the MMLV components 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.
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.
Conclusion
No claims are allowable.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635