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 .
This is the First Office Action on the Merits of US18/587,120 filed on 02/26/2024 which is a CON of 16/903,971 filed on 06/17/2020 (now US Patent 11,939,585) which is a CON of 15/727,150 filed on 10/06/2017 (now US Patent 10,724,043) which claims US priority benefit of US Provisional 62/404,993 filed on 10/06/2016.
Election/Restrictions
Applicant’s election without traverse of the Species: A: transfection/electroporation parameters of claim 1; B: NK cells; and C: isotonic medium in the reply filed on 10/27/2025 is acknowledged.
However, upon further consideration the Species election requirement mailed on 09/24/2025 is withdrawn and species are rejoined in this office action.
Claims 4-5, 7, 12, and 24 are cancelled.
Claims 1-3, 6, 8-11, 13-20, 22-23, and 25-26 are pending and under examination.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-3, 6, 8-11, 13-18, 19-20, 22-23, and 25-26 is/are rejected under 35 U.S.C. 103 as being unpatentable over US2003/0073238 to Dzekunov et al in (published April 17, 2003, now US Patent 7,141,425 B2, issued November 28, 2006), in view of Heiser et al in “Optimizing Electroporation Conditions for the Transformation of Mammalian Cells” (Springer Nature, Transcription Factor Protocols, Methods in Molecular Biology”, 2000 Vol 130: 117-134; IDS ref), in view of Takahashi et al in “Cytoplasmic expression of EGFP in dendritic cells transfected with in vitro transcribed mRNA or cellular total RNA extracted from EGFP expressing leukemia cells” (Medical Oncology 2003 Vol 20, pages 335-348), in view of Kubiniec et al in “Effects of pulse length and pulse strength on transfection by electroporation” Biotechniques 1990 January Vol 8, No. 1. Pages 16-20 (Abstract only; IDS ref).
Regarding base claims 1, 16, and 25-26, Dzekunov et al teach electroporation of mammalian cells with DNA and/or RNA (e.g., para 0181, line 1). Dzekunov et al teach electroporation of mammalian cells using multiple pulses, specifically two or four pulses, specifically with various type of mammalian cells including lymphocytes cells (para 0350-0354). Dzekunov et al point to preferred field strength range of 1 kV/cm to 3.5 kV/cm (para 0191; which is the same as 1000 V/cm to 3500 V/cm) and meets the limitation of instant claims 1 and 26 of 600-1,400 V/cm and of claim 16 of 800-1200 V/cm.
Regarding claims 2-3, Dzekunov et al discloses that the mammalian cells are immune competent cells, and specifically lymphocytes and macrophages (para 0060, line 6).
Regarding claim 6, Dzekunov et al discloses that the transfection medium can be an isotonic medium and a growth medium or a high conductance medium. (See Example 2 & 3, which disclose using B& K buffer (125 mM KCl, 15 mM NaCl, 1.2 mM MgCl.sub.2, 3 mM glucose, 25 mM Hepes, pH 7.4) which meets the limitation of an isotonic medium and a growth medium or a high conductance medium.
Regarding claim 8, Dzekunov et al teach electroporation of mammalian cells using multiple pulses, specifically two or four pulses, specifically with various type of mammalian cells including lymphocytes cells (para 0350-0354). (See para 0299; 0323, line 2; para 0311, line 3).
Regarding claims 9-10, Dzekunov et al teach electroporation of mammalian cells that a “preferred set of parameters is a field strength of 1-2 kV/cm with a pulse width of 1-2 ms (i.e., 1000-2000 µs) and 1-2 seconds between pulses” which meets the limitation of between 1-15 seconds (claim 9) and between 2-10 seconds (claim 10).
Regarding claims 11 and 25, Dzekunov et al teach mini-flow cell with an electrode gap of 4 mm which is 0.4 cm. (See para 0355).
Regarding claim 19, Dzekunov et al teach a step of culturing the transfected mammalian cells after pulsing. (See para 0308, lines 4-5; stating: “Cells were cultured in culture medium for various periods (up to 72 hours) and the transfection efficiency was analyzed.”).
Regarding claim 20, Dzekunov et al discloses that the transfected mammalian cells have a viability of at least 70% (See para 0183 and especially Table 7 for example, showing 85% viability for CHO cells, and 90% for Huh-7 cells).
Regarding claim 22, Dzekunov et al discloses that the pulses provide a transfection efficiency of at least 80% (See Table 7 for example showing 80% for CHO cells and 80% for Huh-7 cells).
However, regarding claims 1, 13-14, and 25-26, Dzekunov et al differ from the present claims because while they disclose that a “preferred set of parameters is a field strength of 1-2 kV/cm with a pulse width of 1-2 ms (i.e., 1000-2000 µs) and 1-2 seconds between pulses” they do not appear to disclose a particular time constant in the range of from about 0.5 msec to about 10 msec (claims 1, and 25-26), or about 0.5/1 msec to about 5/3 msec (claims 13/14). Note that pulse width and time constant both describe the duration of the electric pulse. Pulse width refers specifically to the duration of a square wave pulse while the time constant is the decay rate of an exponential decay pulse. For pulse width (square wave) the voltage is held constant for a specific, set duration (e.g., milliseconds) and then drops immediately. It is a direct input parameter. For Time Constant (Exponential Decay) the voltage starts at a maximum and decays to a percentage (%) of that value. It is dependent on the system resistance (R) and capacitance (C). Time constant is [tau equals R x C]. Pulse width is pre-set by the user and the time constant is measured after the pulse, determined by the resistance of the buffer/cell mixture and the capacitator machine settings. Thus time constant is a function of buffer/cell mixture and capacitance.
Dzekunov et al suggests optimizing a time constant for their electroporation methods in Table 3. Further, each of Heiser et al and Kubiniec et al, and Takahashi et al disclose a preferred embodiment of a time constant in the range of from about 0.1 msec to about 10 msec for transfection/electroporation of mammalian cells.
Heiser et al disclose methods of transfecting mammalian cells with a nucleic acid cargo by disposing the mammalian cells with DNA in a transfection medium and applying a pulse to the mammalian cells at a suggested field strength of “500-2000 V/cm and a time constant of 10-30 ms. Heiser suggest starting points for mammalian cells are “low-resistance media”, “lower field strengths” (i.e., “500-2000 V/cm) and “longer time constants (10-30 ms)”. In page 5, Heiser disclose for HuT 78 (T-cell), a time constant of 4 ms and a Field strength of 750 V/cm and a voltage of 150 V (See page 5, second row).
Takahashi et al disclose optimization of electroporation parameters for transfection of mRNA into mammalian dendritic cells. Dendritic cells meet the limitation of immune cells. Takahashi et al disclose optimization of the standard electroporation parameters. Takahashi et al disclose using pulse widths up to 1000 µs (1 ms). Further, Kubiniec et al teach that pulses with 2.2 to 4.6 ms time constant showed an optimal result for stable transfection of mammalian cells with plasmid DNA. For example, Kubiniec et al reports the following in the Abstract:
The relative importance of pulse field strength E and pulse length tau 1/2 (half decay time of an exponential decay pulse) on the stable transfection frequency for HeLa or HUT-78 cells was investigated. Cells were transfected with plasmids containing the promoter and drug resistant genes pRSVgpt or pRSVneo by electroporation. The stable transfection frequency was assayed using the marker rescue technique. The transfection frequency increases with increasing values of E tau 1/2. For a given pulse length, the transfection frequency is proportional to the power of the pulse (E2 tau 1/2). Pulses with half decay times of 2.2 to 4.6 ms appear to be more efficient than 0.275 to 0.31 ms for stable transfection of HeLa cells.
Thus, in view of Heiser et al and Kubiniec et al, and Takahashi et al, one of ordinary skill in the art would have been motivated to use a time constant from about 0.1 msec to about 10 msec and including about 3 msec for the rationale of optimizing transfection efficiency and cell viability (e.g., “stable transfection”). Generally, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
Further, regarding claims 17-18, and 25-26, Dzekunov et al discloses methods of optimizing capacitance (see Table 3) but does not explicitly disclose being delivered from a capacitor having a capacitance of between 5-50 µF regarding claims 17, and 25-26, and of between 10-25 µF regarding claim 18.
Heiser disclose that the pulses are delivered from a capacitor having a capacitance of 25 µF which meets the limitation of between 5-50 µF regarding claim 17 and 25-26 and of between 10-25 µF regarding claim 18. For example, in Table 1 Heiser shows that “capacitance” in µF can be 25 µF. Heiser specifically teaches protocols to optimize electroporation results in section “3.2.1 Protocol 1” teaching (per instant claims 7-8) the transfection medium can be isotonic and a growth medium or a high conductance medium, specifically suggesting embodiments of 25 µF capacitance and 150/200/250 Volts in HEPES or phosphate-buffered sucrose (e.g., Protocol 1, #3, lines 6-7 and #6, lines 1-3).
Further, regarding claims 15 and 25, Heiser discloses that the field strength is applied at a voltage of between 150-250 V. In page 5, Heiser disclose a specific embodiment for HuT 78 (T- cell), having a time constant of 4 ms and a Field strength of 750 V/cm and a voltage of 150 V (See page 5, second row).
One of ordinary skill in the art would have been motivated to use pulses delivered from a capacitor having a capacitance of between 5-50 µF regarding claim 17 and of between 10-25 µF regarding claim 18 in the electroporation methods of Dzekunov et al for the rationale of testing for optimization of transfection efficiency and mammalian cell-type viability. It would have been obvious to one of ordinary skill in the art because Heiser et al discloses that it is standard lab procedure to optimize parameters for methods of electroporating cargo, including DNA, into mammalian cells. (See entire document). Further, Heiser disclose it is standard lab practice to optimize particular combinations of parameters including the electroporation buffer, capacitance, temperature, addition of DEAE-dextran, growth state of cells, and time constant and field strength. (See entire document). Further, Dzekunov et al discloses methods of optimizing capacitance (see Table 3). Generally, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
Regarding claim 23, Dzekunov et al does not explicitly disclose that an injected charge is between 1 mC and 30 mC.
However, Heiser et al discloses embodiments of 25 µF capacitance and 150/200/250 Volts which meets the limitation that an injected charge is between 1 mC and 30 mC. Note that 1mC - 30 mC may be stated as 1 millicoulomb to 30 millicoulomb. The combination of Heiser et al of 25 µF capacitance and 150/200/250 Volts renders obvious the limitation of an injected charge is between 1 mC and 30 mC.
The level of skill in the art of electroporation of cargo into mammalian cells was very high before the effective filing date of the presently claimed invention.
One of ordinary skill in the art having the cited references before the effective filing date of the presently claimed invention would have been motivated to optimize the combination of parameters of electroporation media, temperature, capacitor and capacitance, field strength, transfection efficiency and cell viability, time constant, voltage, and number of pulses for the rationale provided in the cited references, particularly Heiser, to optimize transfection efficiency and cell viability and Dzekunov et al in para 0096, 0145, 0162, and Figure 35 & 52 showing cell viability versus field strength & CLL-B cells). In view of the preferred embodiment of Heiser et al of 25 µF capacitance and 150/200/250 Volts one of skill in the art would have been motivated to an injected charge is between 1 mC and 30 mC to optimize conditions for transfection efficiency and viability as suggested by Heiser and Dzekunov et al. Generally, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
In view of the high skill level in the art, it is considered that one of ordinary skill in the art would have had a reasonable expectation of success to combine the elements of the cited references, and using ordinary optimization of such parameters, to arrive at the presently claimed invention.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-3, 6, 8-11, 13-20, 22-23, and 25-26 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-16 of U.S. Patent No. 11,939,585. Although the claims at issue are not identical, they are not patentably distinct from each other because the patented claims anticipate the instant claims.
Regarding instant claims 1-2, 8, 13, 16, and 20, patented claim 1 recites a method of transfecting immune competent mammalian cells with an RNA comprising:
disposing the cells with the RNA in a transfection medium,
applying 2, 3 or 4 pulses to the cells at a field strength of 1,200 V/cm and a time constant from about 0.5 msec to about 5 msec to produce transfected cells, where the transfected cells have a viability of at least 70%.
Thus patented claim 1 anticipates instant claims 1-2, 8, 13, 16, and 20.
Regarding instant claim 3, patented claim 3 recites NK cells, T-cells, B-cells, and macrophages.
Regarding instant claim 6, patented claim 2 recites an isotonic medium and a growth medium or a high conductance medium.
Regarding instant claim 9, patented claim 4 recites a time between each pulse is between 1-15 seconds.
Regarding instant claim 10, patented claim 5 recites a time between each pulse is between 2-10 seconds.
Regarding instant claim 11, patented claim 6 recites a gap width of about 0.1 cm to about 0.4 cm.
Regarding instant claims 14-15, instant claim 8 recites the time constant is about 1 msec to about 3 msec, and instant claim 9 recites the field strength is applied at a voltage of between 150-250 V.
Regarding instant claims 17-18, instant claims 10-11 recites the pulsed are delivered from a capacitor having a capacitance of between 5-50 and 10-25 µF, respectively.
Regarding instant claim 19, patented claim 12 recites culturing the transfected cells after pulsing.
Regarding instant claim 22-23, patented claims 13-14 recites transfection efficiency of at least 80% and that an injected charge is between 1mC and 30 mC, respectively.
Regarding instant claim 25, patented claim 15 recites a method of transfecting immune competent cells (comprising NK cells, T-cells, B-cells, and macrophages) with an RNA, comprising: disposing the cells with the RNA in a transfection medium;
applying at least two pulses to the cells at a voltage of 150-250 V, a gap width of about 0.1 cm to about 0.4 cm, and a time constant from about 0.5 msec to about 10 msec to produce transfected mammalian cells, where pulses are delivered from a capacitor having a capacitance of between 5-50 µF.
Thus patented claim 15 anticipated instant claim 25.
Regarding instant claim 26, patented claim 16 recites a method of transfecting immune competent cells with an RNA, comprising: disposing the cells with the RNA in a transfection medium; applying two, three, or four pulses to the cells at a field strength of 600-1,400 V/cm and , a time constant from about 0.5 msec to about 10 msec to produce transfected mammalian cells, where pulses are delivered from a capacitor having a capacitance of between 5-50 µF.
Thus patented claim 16 anticipated instant claim 26.
Claims 1-3, 6, 8-11, 13-20, 22-23, and 25-26 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-17 of U.S. Patent No. 10,724,043. Although the claims at issue are not identical, they are not patentably distinct from each other because the patented claims anticipate the instant claims.
Regarding instant claims 1-2, 8, 13, 16, and 20, patented claim 1 recites a method of transfecting immune competent mammalian cells with a cargo RNA comprising:
disposing the cells with the RNA in a transfection medium, applying 2, 3 or 4 pulses to the cells at a field strength of 800-1,200 V/cm and a time constant from about 1 msec to about 5 msec, where the transfected cells have a viability of at least 70%.
Thus patented claim 1 anticipates instant claims 1-2, 8, 13, 16, and 20.
Regarding instant claim 3, patented claim 2 recites NK cells.
Regarding instant claim 6, patented claim 3 recites an isotonic medium and a growth medium.
Regarding instant claim 9, patented claim 5 recites a time between each pulse is between 1-15 seconds.
Regarding instant claim 10, patented claim 6 recites a time between each pulse is between 2-10 seconds.
Regarding instant claim 11, patented claim 7 recites a gap width of about 0.2 cm.
Regarding instant claims 14-15, patented claim 8 recites the time constant is between 1.5 msec to 2.5 msec, and instant claim 9 recites the field strength is applied at a voltage of between 150-250 V.
Regarding instant claims 17-18, patented claims 11-12 recites the pulsed are delivered from a capacitor having a capacitance of between 5-50 and 10-25 µF, respectively.
Regarding instant claim 19, patented claim 13 recites culturing the transfected cells after pulsing.
Regarding instant claim 22-23, patented claims 14-15 recites transfection efficiency of at least 80% and that an injected charge is between 1mC and 30 mC, respectively.
Regarding instant claim 25, patented claim 1 recites a method of transfecting immune competent mammalian cells with a cargo RNA comprising: disposing the cells with the RNA in a transfection medium, applying 2, 3 or 4 pulses to the cells at a field strength of 800-1,200 V/cm and a time constant from about 1 msec to about 5 msec, where the transfected cells have a viability of at least 70%. Further, patented claim 9 depends from patented claim 1 and recites a voltage of 150-250 V. Patented claim 7 recites a gap width of about 0.2 cm. Further, patented claims 11-12 recite the pulsed are delivered from a capacitor having a capacitance of between 5-50 and 10-25 µF, respectively. Thus the combination of patented claims 1, 7, 9, and 11-12 render obvious instant claim 25 because each of claims 7, 9, and 11-12 depend from claim 1 and it would have been obvious to combine the elements of claims 1, 7, 9, and 11-12 because these are the preferred embodiments for a successful transfection/electroporation of immune competent cells.
Regarding instant claim 26, patented claim 17 recites a method of transfecting immune competent cells with an RNA, comprising: disposing the cells with the RNA in a transfection medium; applying two, three, or four pulses to the cells at a field strength of 600-1,400 V/cm and, a time constant from about 1 msec to about 5 msec, where pulses are delivered from a capacitor having a capacitance of between 5-50 µF.
Thus patented claim 17 anticipated instant claim 26.
Conclusion
No claim is allowed.
Related prior art which may be applied in a future office action if appropriate:
AU 2011281121 A1 to Elroy-Stein et al (published 02/21/2013).
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE S HIBBERT whose telephone number is (571)270-3053. The examiner can normally be reached M-F 8:00-5:00.
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CATHERINE S. HIBBERT
Primary Examiner
Art Unit 1658
/CATHERINE S HIBBERT/Primary Examiner, Art Unit 1658