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 .
Applicant’s amendment filed on 01/27/2026 has been entered.
Amended claims 52, 62-63, 65-72 and new claims 73-75 are pending in the present application.
Applicant elected previously the following species: (i) ethanol as a species of an alcohol; (ii) KCl as a species of a salt; and (iii) a gene editing protein as a species of a gene editing composition.
Accordingly, amended claims 52, 62-63 and 65-75 are examined on the merits herein with the above elected species.
Response to Amendment
1. The rejection under 35 U.S.C. 103 as being unpatentable over Held et al (US 7,314,969) in view of Miura et al (JPH03251187; IDS; see attached English translation), Touitou (WO 03/000174; IDS) and Liu et al (US 2015/0071903; IDS) was withdrawn upon further consideration and in light of Applicant’s arguments that were set forth in the Amendment dated 01/27/2026 (pages 8-10). Particularly, the primary Held reference does not teach or fairly suggest any valve between a pneumatic generator and an atomizer other than a pressure regulator in an aerosol beam apparatus, let alone a valve having a switching speed less than 250 milliseconds; and the apparatus relies on the combination of the pressurized gas in the entrainment housing 11 (housing the microflow nebulizer 8) and the partial vacuum in the vacuum chamber (housing the nozzle 12 and a movable stage 14 where target cells are located) to accelerate the aerosol droplets that impact the target cells (Fig. 1).
2. The rejection on the ground of nonstatutory double patenting as being unpatentable over claims 1-38 of U.S. Patent No. 11,981,915 in view of Held et al (US 7,314,969) and Miura et al (JPH03251187; IDS; see attached English translation) was also withdrawn upon further considerations.
3. The rejection on the ground of nonstatutory double patenting as being unpatentable over claims 1-27 of U.S. Patent No. 12,060,570 in view of Held et al (US 7,314,969), Miura et al (JPH03251187; IDS; see attached English translation), Touitou (WO 03/00174; IDS) and Liu et al (US 2015/0071903; IDS) was also withdrawn upon further considerations.
Claim Rejections - 35 USC § 112
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.
Amended claims 52, 62-63 and 65-75 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
An apparatus for delivering a gene editing composition across a plasma membrane of a cell, the apparatus comprising: (i) a pneumatic generator producing a gas (e.g., nitrogen, ambient air or an inert gas) under a pressure; (ii) an atomizer operatively coupled to the pneumatic generator; (iii) a reservoir configured to contain a volume of aqueous solution, the aqueous solution including the gene editing composition (e.g., a Cas9 protein and a gRNA complex) and an alcohol (e.g., ethanol, methanol, isopropyl alcohol) at greater than 2 percent concentration; and (iii) a valve between the pneumatic generator and atomizer, the valve switchable between a closed position for preventing the gas under the pressure from activating the atomizer and an open position for allowing the gas under the pressure to activate the atomizer to produce colloidal droplets from the aqueous solution, the valve having a switchable speed less than 250 milliseconds; wherein the atomizer is oriented to spray an area configured to contain a population of cells with the aqueous solution and wherein the volume is a function of: (i) exposed surface area of the population of cells; or (ii) a number of cells in the population of cells; and wherein the spray includes discrete units of volume ranging in size from 10 nm to 2 um;
does not reasonably provide enablement for an apparatus for delivering a gene editing composition across a plasma membrane of a cell, wherein the spray includes discrete units of volume (or colloidal droplets) with size greater than 2 um as encompassed broadly by the instant claims. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims. This is a modified rejection necessitated by Applicant’s amendment.
The factors to be considered in the determination of an enabling disclosure have been summarized as the quantity of experimentation necessary, the amount of direction or guidance presented, the state of the prior art, the relative skill of those in the art, the predictability or unpredictability of the art and the breadth of the claims. Ex parte Forman, (230 USPQ 546 (Bd Pat. Appl & Unt, 1986); In re Wands, 858 F.2d 731, 8 USPQ 2d 1400 (Fed. Cir. 1988)).
The instant specification is not enabled for the present broadly claimed invention for the reasons discussed below.
1. The breadth of the claims
Amended claims 52, 62-63 and 65-75 encompass an apparatus for delivering a gene editing composition across a plasma membrane of a cell, the apparatus comprising the elements recited in independent claim 52, wherein the spray includes discrete units of volume ranging in size from 10 nm to 100 um or wherein the spray comprises a colloidal or sub-particle comprising a diameter of 10 nm to 100 um (new claim 73).
2. The state and the unpredictability of the prior art
Before the effective filing date of the present application (12/30/2015), little was known about the use of a spray comprising discrete units of volume with size greater than 2 um or colloidal droplets having a diameter of 30-100 um that are generated by an apparatus to deliver a gene editing composition across a plasma membrane of any cell as evidenced at least by the teachings of Mets (US 5,240,842), Held et al (US 7,314,969) and Pui et al (US 6,764,720; IDS). Moreover, the physiological art is also recognized as unpredictable (MPEP 2164.03).
3. The amount of direction or guidance provided
Apart from generically stating that that the spray can include discrete units of volume ranging in size from 10 nm to 100 um (e.g., 30-100 um in diameter) (see at least first full paragraph on page 7; paragraph bridging pages 24-25); the instant specification fails to provide sufficient guidance for an ordinary skilled artisan on how to use the claimed apparatus to generate spray comprising discrete units of volume with size greater than 2 um or colloidal/subparticle droplets having a diameter of 30-100 um for delivering a gene editing composition across a plasma membrane of a cell as encompassed by the instant claims. Particularly, Mets already stated “Preferably, the projectiles (droplets of solvent and suspended particulate matter) have a mass mean diameter of from about 0.1 to about 2 microns….Projectiles having a diameter of greater than about 2 microns were found to cause an unacceptable level of cell death following impact, while projectiles having a diameter of below about 0.1 microns were unable to efficiently penetrate into the target cell” (col. 5, lines 20-30). Held et al stated “The size of the droplet is of particular importance when molecules are to be introduced into small cells or cellular organelles, for example, cells less than about 20 microns. The importance of droplet size has been demonstrated with particle bombardment technology where particles of greater than 1 micron in diameter were shown to be unsuitable for introducing DNA into cells of 10 to 20 microns in diameter” (col. 5, lines 6-12); and “In part, because aerosol droplets generated by the present invention are so much smaller than the particles produced by the prior art, the present invention is superior in delivering molecules to small cells, for example cells less than 20 microns in size. Many animal, plant and microbial cells are in this size range. Entry of micron-size particles into cells of this size can be expected to cause significant damage.” (col. 5, line 66 continues to line 6 on col. 6). There is no evidence of record indicating and/or suggesting that the apparatus of the present invention successfully delivered a gene editing composition across a plasma membrane of a cell using a spray comprising discrete units of volume with size 2 um or greater, or colloidal/sub-particle droplets have a diameter of 30-100 micrometers, and the cell is still viable/undamaged. There is no use for delivering a gene editing composition across a plasma membrane of a dead cell.
Since the prior art before the effective filing date of the present application failed to provide sufficient guidance regarding to the aforementioned issues, it is incumbent upon the present application to do so. Given the state of the prior art discussed above, coupled with the lack of sufficient guidance provided by the present application, it would have required undue experimentation for a skilled artisan to make and/or use an apparatus for delivering a gene editing composition across a plasma membrane of a cell as claimed broadly.
Accordingly, due to the lack of sufficient guidance provided by the specification regarding to the issues set forth above, the state and unpredictability of the relevant art, and the breadth of the instant claims, it would have required undue experimentation for one skilled in the art to make and use the instant broadly claimed invention.
Response to Argument
Applicant’s arguments related to the above 35 U.S.C. 112(a) rejection in the Amendment filed on 01/27/2026 (pages 5-6) have been fully considered, but they are respectfully not found persuasive for the reason discussed below.
Applicant argued basically that the specification discloses specific operating conditions of the claimed spray apparatus demonstrating that the apparatus is capable of generating and dispensing the discrete units of volume (e.g., the specification on page 7 disclosing “spray delivery volumes adjusted for microtiter plates with more wells and/or smaller approximate cell growth/cell-occupied area per single well of the multi-well plate”). Additionally, claim 64 is cancelled, and this ground of rejection is moot.
First, it is noted that Applicant incorporated the limitation of claim 64 (now cancelled) into the currently amended independent claim 52. Please refer to the above modified 112(a) rejection for details. Additionally, the above 112(a) rejection is an Enablement rejection, and not a Lack of Written Description rejection.
Second, simple statements that the spray can include discrete units of volume ranging in size from 10 nm to 100 um (e.g., 30-100 um in diameter) (see at least first full paragraph on page 7; paragraph bridging pages 24-25) do not automatically make the claimed invention to be fully enabled. Please refer to the Wands Factor analysis set forth in the above 112(a) rejection. Particularly, Mets already stated “Preferably, the projectiles (droplets of solvent and suspended particulate matter) have a mass mean diameter of from about 0.1 to about 2 microns….Projectiles having a diameter of greater than about 2 microns were found to cause an unacceptable level of cell death following impact, while projectiles having a diameter of below about 0.1 microns were unable to efficiently penetrate into the target cell” (col. 5, lines 20-30). Moreover, Held et al stated “The size of the droplet is of particular importance when molecules are to be introduced into small cells or cellular organelles, for example, cells less than about 20 microns. The importance of droplet size has been demonstrated with particle bombardment technology where particles of greater than 1 micron in diameter were shown to be unsuitable for introducing DNA into cells of 10 to 20 microns in diameter” (col. 5, lines 6-12); and “In part, because aerosol droplets generated by the present invention are so much smaller than the particles produced by the prior art, the present invention is superior in delivering molecules to small cells, for example cells less than 20 microns in size. Many animal, plant and microbial cells are in this size range. Entry of micron-size particles into cells of this size can be expected to cause significant damage.” (col. 5, line 66 continues to line 6 on col. 6). Please note that the spray includes discrete units of volume ranging in size from 10nm to 100um of the instant claims contain produced colloidal droplets, and the spray comprises a colloidal or sub-particle comprising a diameter of 10 nm to 100 um (see new claim 73). Furthermore, there is no evidence of record indicating and/or suggesting that the apparatus of the present invention successfully delivered a gene editing composition across a plasma membrane of a cell using a spray comprising discrete units of volume with size 2 um or greater, or colloidal/sub-particle droplets have a diameter of 30-100 micrometers, and the cell is still viable/undamaged. There is no use for delivering a gene editing composition across a plasma membrane of a dead cell.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Amended claims 52, 62-63 and 65-73 are still rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-30 of U.S. Patent No. 12,195,748 (IDS) in view of Touitou (WO 03/00174; IDS) and Liu et al (US 2015/0071903; IDS).
The claims of the present application differ from claims 1-30 of U.S. Patent No. 12,195,748 in reciting specifically that an apparatus comprising a reservoir configured to contain a volume of aqueous solution comprising a gene editing composition and an alcohol at greater than 2 percent concentration, preferable the gene editing composition includes a Cas9 protein and a gRNA complex; and an atomizer is oriented to spray an area configured to contain a population of stem cells.
Before the effective filing date of the present application (12/30/2015), Touitou already disclosed an intracellular delivery alcoholic lipid composition for various applications (e.g., pharmaceutical, medical, research), the composition comprises at least a phospholipid, ethanol (or other C2-C4 volatile alcohol), water and at least one active molecule for the penetration through biological membranes and for the facilitation of the delivery of entrapped or complexed molecules through biological and cellular membranes, into cells, wherein ethanol is in an amount of 10%-50% and the active molecule includes proteins, peptides, glycoproteins as well as nucleic acids such as antisense oligonucleotides, DNA, RNA, plasmids (see at least Abstract; particularly pages 3-7; Examples 4-5). Touitou also taught that the composition may be in the form of a liquid spray, and it can be administered via microporation, electroporation or jet (page 6, lines 8-10). Touitou stated clearly “The presence of ethanol in an amount of 10 to 50% provides a negative charge to the vesicle” (page 4, lines 11-12), “The composition is added to a culture in a quantity of 10-200ul/well, wherein the well volume is 1-2 ml. The same ratio is maintained also in other sizes of wells” (page 6, lines 11-13), and “The advantages of the method and the composition of the invention are as follows: Improved cellular uptake and trafficking. The composition is easy to prepare. Delivery to cells, tissues, glands, follicles and organs. Delivery to nucleus (or other cellular organelles)” (page 4, lines 1-6). Example 1 showed penetration of the amphiphilic fluorescent dye D-289 into mouse 3T3 fibroblasts within 10 minutes and as early as within 3 minutes; Example 4 illustrated cultured Osteosarcoma South cells were efficiently transfected with p53 plasmid after 24 hours; and Example 5 demonstrated intracellular delivery of CMV-GFP cDNA to the dorsal skin surface of 5-week, female, CD-1 nude mice.
Additionally, Liu et al already taught successful delivering functional effector proteins, including RNA-programmable nucleases such as Cas9 proteins, Cas9 variants and Cas9 fusion thereof associated with gRNAs in the absence of a supercharged protein using cationic lipids into cells for therapeutic and research purposes, including for targeted manipulation of a gene associated with a disease and the programming of cell fate (Abstract; Summary of the Invention; particularly paragraphs [0004]-[0005], [0011]-[0012], [0014], [129]-[134], [0137], [0140]-[0143], [0256]-[0276]; Example 7; Figs. 30-31 and 36- 43). Liu et al stated explicitly “It was surprisingly found that when a Cas9 protein is associated with a gRNA, the complex can be encapsulated by cationic lipids and effectively delivered to cells. This may be accomplished with or without a supercharged protein….In some embodiments, the composition exhibits low toxicity when delivered to a population of cells, for example, wherein at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells are viable following administration of the composition. In some embodiments, the Cas9 protein is a wild type Cas 9 protein, a Cas9 nickase, or comprises a nuclease inactivated (dCas9) protein” (paragraph [0011]); and “[i]t is believed that the gRNA acts as a polyanionic mediator between the otherwise cationic Cas9 protein and the cationic lipids. Delivery of Cas9:gRNA complexes is not only highly efficient (e.g., up to 80% modification from a single treatment) but also results in markedly higher genome modification specificity compared with plasmid transfection, typically resulting in >10 fold higher on-target:off-target modification ratios, presumably due to the transient nature of the delivered Cas9:gRNA activity” (paragraph [0131]). Example 7 demonstrated efficient delivery of genome editing proteins, particularly Cas9:sgRNA nuclease complexes with a cationic lipid agent, in vitro (e.g., in cultured human cells such as U2OS-EGFP cells, HEK293T cells, mouse ES cell line Tau-GFP; and assayed for gene inactivation either 4 or 16 hours after delivery, or assayed for gene modification 72 hours after delivery) and in vivo (e.g., inner ear of a mouse) (paragraphs [0230],[0237]-[0240], [0256]-[0276]; Figs. 30-31 and 36- 43). Liu et al also demonstrated that lipid-mediated Cas9:sgRNA delivery can support efficient multiplexed genome editing in U2OS EGFP reporter cells treated with a single lipid-mediated delivery treatment of Cas9 complexed with a mixture of four gRNAs targeting EGFP, CLTA, EMX and VEGF (paragraph [0263] and Fig. 30C). Gene disruption efficiency in U2OS EGFP reporter cells were also obtained from delivery of Cas9 D10A nickase with appropriate EGFP-targeting sgRNA pairs targeting opposite strands (paragraph [0264] and Fig. 30D).
Accordingly, it would have been obvious for an ordinary skilled artisan to modify an apparatus for delivering a payload across a plasma membrane of a cell in claims 1-30 of US Patent No. 12,195,748 by also using an aqueous solution comprising a gene editing composition containing a Cas9 protein and a gRNA complex for delivering across a plasma membrane of a cell, including a stem cell; in light of the teachings of Touitou and Liu et al as presented above with a reasonable expectation of success.
An ordinary skilled artisan would have been motivated to carry out the modifications because: (i) Touitou already successfully disclosed an intracellular delivery alcoholic lipid composition for the delivery of entrapped or complexed molecules through biological and cellular membranes of a cell; and wherein the composition may be in the form of a liquid spray; and (ii) Liu et al also taught successful delivering functional effector proteins, including RNA-programmable nucleases such as Cas9 proteins, Cas9 variants and Cas9 fusion thereof associated with gRNAs in the absence of a supercharged protein using cationic lipids into cells (e.g., U2OS-EGFP cells, HEK293T cells, mouse ES cell line Tau-GFP) for therapeutic and research purposes.
The modified apparatus resulting from claims 1-30 of U.S. Patent No. 12,195,748 along with the combined teachings of Touitou and Liu et al is indistinguishable from the apparatus as claimed.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
New claims 74-75 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-30 of U.S. Patent No. 12,195,748 (IDS) in view of Touitou (WO 03/00174; IDS) and Liu et al (US 2015/0071903; IDS) as applied to claims 52, 62-63 and 65-73 above, and further in view of Jinek et al (Science 337:816-821, 2012 with Supplementary Materials; IDS). This is a new ground of rejection necessitated by Applicant’s amendment.
New claims 74-75 differ from claims 1-30 of U.S. Patent No. 12,195,748 (IDS) in view of Touitou and Liu et al in reciting the limitations “wherein the aqueous solution further comprises 0.1 to 10 mM of a magnesium salt” and “wherein the magnesium salt is MgCl2”, respectively.
Before the effective filing date of the present application (12/30/2015), Jinek et al already characterized and demonstrated that Cas9 endonuclease can be programmed with guide RNA engineered as a single transcript to target and cleave any dsDNA of interest (Abstract). Jinek et al also taught that the cleavage reaction of Cas9 endonuclease required both magnesium and the presence of a crRNA sequence complementary to the DNA (section titled “Cas9 is a DNA endonuclease guided by two RNAs” on pages 816-817). A cleavage buffer supplemented with 1, 5 or 10 mM MgCl2 as well as cleavage buffers supplemented with other metals were used to investigate metal-dependent Cas9 cleavage; and linear cleavage product was observed and increased with increased concentrations of MgCl2 in the cleavage buffer (Section “Metal-dependent cleavage assay” on page 4 of the Supplementary Materials; and Figure S4A on page 11).
Accordingly, it would have been obvious for an ordinary skilled artisan to further modify claims 1-30 of U.S. Patent No. 12,195,748 along with the combined teachings of Touitou and Liu et al by also further incorporating into the aqueous solution at least MgCl2 at a concentration of between 1 mM to 10 mM, in light of the teachings of Jinek et al as presented above.
An ordinary skilled artisan would have been motivated to further carry out the above modification because Jinek et al already demonstrated that magnesium chloride at a concentration of 1, 5 or 10 mM MgCl2 is required or essential for Cas9 endonuclease cleavage activity in a gene editing composition.
An ordinary skilled artisan would have a reasonable expectation of success in light of claims 1-30 of U.S. Patent No. 12,195,748 along with the combined teachings of Touitou, Liu et al and Jinek et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified apparatus for delivering a gene editing composition across a plasma membrane of a cell resulting from claims 1-30 of U.S. Patent No. 12,195,748 along with the combined teachings of Touitou, Liu et al and Jinek et al as set forth is indistinguishable from and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Amended claims 52, 62-63 and 65-73 are still provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 in view of Touitou (WO 03/00174; IDS) and Liu et al (US 2015/0071903; IDS).
Claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 are drawn to an apparatus for delivering a payload across a plasma membrane of a cell (e.g., primary or immortalized mesenchymal stem cells; dependent claim 124), the apparatus comprising: (i) a pneumatic generator configured to produce gas under a pressure; (ii) an atomizer operatively coupled to the pneumatic generator and capable of forming a spray; (iii) a reservoir configured to contain an aqueous solution (e.g., the aqueous solution comprising one or more of 75 to 98% H2O, 2 to 45% ethanol, 6 to 91 mM sucrose, 2 to 35 mM KCl, 2 to 35 mM ammonium acetate, and 1 to 14 mM HEPES; see dependent claim 132), the aqueous solution including the payload; and (iii) a valve between the pneumatic generator and atomizer, the valve switchable between a closed position for preventing a gas under the pressure from activating the atomizer and an open position for allowing the gas under the pressure to activate the atomizer to produce the spray, the spray including colloidal droplets, the valve having a switching speed less than 250 milliseconds; wherein the atomizer is oriented towards a support capable of supporting a population of cells for contacting the population of cells with a volume of the aqueous solution.
The claims of the present application differ from claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 in reciting specifically that an apparatus comprising a reservoir configured to contain a volume of aqueous solution comprising a gene editing composition and an alcohol at greater than 2 percent concentration, and preferable the gene editing composition includes a Cas9 protein and a gRNA complex.
Before the effective filing date of the present application (12/30/2015), Touitou already disclosed an intracellular delivery alcoholic lipid composition for various applications (e.g., pharmaceutical, medical, research), the composition comprises at least a phospholipid, ethanol (or other C2-C4 volatile alcohol), water and at least one active molecule for the penetration through biological membranes and for the facilitation of the delivery of entrapped or complexed molecules through biological and cellular membranes, into cells, wherein ethanol is in an amount of 10%-50% and the active molecule includes proteins, peptides, glycoproteins as well as nucleic acids such as antisense oligonucleotides, DNA, RNA, plasmids (see at least Abstract; particularly pages 3-7; Examples 4-5). Touitou also taught that the composition may be in the form of a liquid spray, and it can be administered via microporation, electroporation or jet (page 6, lines 8-10). Touitou stated clearly “The presence of ethanol in an amount of 10 to 50% provides a negative charge to the vesicle” (page 4, lines 11-12), “The composition is added to a culture in a quantity of 10-200ul/well, wherein the well volume is 1-2 ml. The same ratio is maintained also in other sizes of wells” (page 6, lines 11-13), and “The advantages of the method and the composition of the invention are as follows: Improved cellular uptake and trafficking. The composition is easy to prepare. Delivery to cells, tissues, glands, follicles and organs. Delivery to nucleus (or other cellular organelles)” (page 4, lines 1-6). Example 1 showed penetration of the amphiphilic fluorescent dye D-289 into mouse 3T3 fibroblasts within 10 minutes and as early as within 3 minutes; Example 4 illustrated cultured Osteosarcoma South cells were efficiently transfected with p53 plasmid after 24 hours; and Example 5 demonstrated intracellular delivery of CMV-GFP cDNA to the dorsal skin surface of 5-week, female, CD-1 nude mice.
Additionally, Liu et al already taught successful delivering functional effector proteins, including RNA-programmable nucleases such as Cas9 proteins, Cas9 variants and Cas9 fusion thereof associated with gRNAs in the absence of a supercharged protein using cationic lipids into cells for therapeutic and research purposes, including for targeted manipulation of a gene associated with a disease and the programming of cell fate (Abstract; Summary of the Invention; particularly paragraphs [0004]-[0005], [0011]-[0012], [0014], [129]-[134], [0137], [0140]-[0143], [0256]-[0276]; Example 7; Figs. 30-31 and 36- 43). Liu et al stated explicitly “It was surprisingly found that when a Cas9 protein is associated with a gRNA, the complex can be encapsulated by cationic lipids and effectively delivered to cells. This may be accomplished with or without a supercharged protein….In some embodiments, the composition exhibits low toxicity when delivered to a population of cells, for example, wherein at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells are viable following administration of the composition. In some embodiments, the Cas9 protein is a wild type Cas 9 protein, a Cas9 nickase, or comprises a nuclease inactivated (dCas9) protein” (paragraph [0011]); and “[i]t is believed that the gRNA acts as a polyanionic mediator between the otherwise cationic Cas9 protein and the cationic lipids. Delivery of Cas9:gRNA complexes is not only highly efficient (e.g., up to 80% modification from a single treatment) but also results in markedly higher genome modification specificity compared with plasmid transfection, typically resulting in >10 fold higher on-target:off-target modification ratios, presumably due to the transient nature of the delivered Cas9:gRNA activity” (paragraph [0131]). Example 7 demonstrated efficient delivery of genome editing proteins, particularly Cas9:sgRNA nuclease complexes with a cationic lipid agent, in vitro (e.g., in cultured human cells such as U2OS-EGFP cells, HEK293T cells, mouse ES cell line Tau-GFP; and assayed for gene inactivation either 4 or 16 hours after delivery, or assayed for gene modification 72 hours after delivery) and in vivo (e.g., inner ear of a mouse) (paragraphs [0230],[0237]-[0240], [0256]-[0276]; Figs. 30-31 and 36- 43). Liu et al also demonstrated that lipid-mediated Cas9:sgRNA delivery can support efficient multiplexed genome editing in U2OS EGFP reporter cells treated with a single lipid-mediated delivery treatment of Cas9 complexed with a mixture of four gRNAs targeting EGFP, CLTA, EMX and VEGF (paragraph [0263] and Fig. 30C). Gene disruption efficiency in U2OS EGFP reporter cells were also obtained from delivery of Cas9 D10A nickase with appropriate EGFP-targeting sgRNA pairs targeting opposite strands (paragraph [0264] and Fig. 30D).
Accordingly, it would have been obvious for an ordinary skilled artisan to modify an apparatus for delivering a payload across a plasma membrane of a cell in claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 by also using an aqueous solution comprising a gene editing composition containing a Cas9 protein and a gRNA complex for delivering across a plasma membrane of a cell; in light of the teachings of Touitou and Liu et al as presented above with a reasonable expectation of success.
An ordinary skilled artisan would have been motivated to carry out the modifications because: (i) Touitou already successfully disclosed an intracellular delivery alcoholic lipid composition for the delivery of entrapped or complexed molecules through biological and cellular membranes of a cell; and wherein the composition may be in the form of a liquid spray; (ii) Liu et al also taught successful delivering functional effector proteins, including RNA-programmable nucleases such as Cas9 proteins, Cas9 variants and Cas9 fusion thereof associated with gRNAs in the absence of a supercharged protein using cationic lipids into cells (e.g., U2OS-EGFP cells, HEK293T cells, mouse ES cell line Tau-GFP) for therapeutic and research purposes.
The modified apparatus resulting from claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 along with the combined teachings of Touitou and Liu et al is indistinguishable from the apparatus as claimed.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
This is a provisional nonstatutory double patenting rejection.
New claims 74-75 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 in view of Touitou (WO 03/00174; IDS) and Liu et al (US 2015/0071903; IDS) as applied to claims 52, 62-63 and 65-73 above, and further in view of Jinek et al (Science 337:816-821, 2012 with Supplementary Materials; IDS). This is a new ground of rejection necessitated by Applicant’s amendment.
New claims 74-75 differ from claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 in view of Touitou and Liu et al in reciting the limitations “wherein the aqueous solution further comprises 0.1 to 10 mM of a magnesium salt” and “wherein the magnesium salt is MgCl2”, respectively.
Before the effective filing date of the present application (12/30/2015), Jinek et al already characterized and demonstrated that Cas9 endonuclease can be programmed with guide RNA engineered as a single transcript to target and cleave any dsDNA of interest (Abstract). Jinek et al also taught that the cleavage reaction of Cas9 endonuclease required both magnesium and the presence of a crRNA sequence complementary to the DNA (section titled “Cas9 is a DNA endonuclease guided by two RNAs” on pages 816-817). A cleavage buffer supplemented with 1, 5 or 10 mM MgCl2 as well as cleavage buffers supplemented with other metals were used to investigate metal-dependent Cas9 cleavage; and linear cleavage product was observed and increased with increased concentrations of MgCl2 in the cleavage buffer (Section “Metal-dependent cleavage assay” on page 4 of the Supplementary Materials; and Figure S4A on page 11).
Accordingly, it would have been obvious for an ordinary skilled artisan to further modify claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 along with the combined teachings of Touitou and Liu et al by also further incorporating into the aqueous solution at least MgCl2 at a concentration of between 1 mM to 10 mM, in light of the teachings of Jinek et al as presented above.
An ordinary skilled artisan would have been motivated to further carry out the above modification because Jinek et al already demonstrated that magnesium chloride at a concentration of 1, 5 or 10 mM MgCl2 is required or essential for Cas9 endonuclease cleavage activity in a gene editing composition.
An ordinary skilled artisan would have a reasonable expectation of success in light of claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 along with the combined teachings of Touitou, Liu et al and Jinek et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified apparatus for delivering a gene editing composition across a plasma membrane of a cell resulting from claims 100-133, 139, 143 and 150-177 of copending Application No. 16/841,093 along with the combined teachings of Touitou, Liu et al and Jinek et al as set forth is indistinguishable from and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
This is a provisional nonstatutory double patenting rejection.
It is noted that in the Amendment filed on 01/27/2026 (pages 10-11), Applicant simply requested that the above non-statutory double patenting rejections be held in abeyance until there is an indication of allowability.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Quang Nguyen, Ph.D., at (571) 272-0776.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s SPE, James Douglas (Doug) Schultz, Ph.D., may be reached at (571) 272-0763.
To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Group Art Unit 1631; Central Fax No. (571) 273-8300.
Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to (571) 272-0547.
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/QUANG NGUYEN/Primary Examiner, Art Unit 1631
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