DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 23, 2026, has been entered.
Claims 1, 2, 4, and 5 are cancelled.
Claims 3 and 6-24 are pending. Claims 18-20 are withdrawn.
Claims 3, 6-17, and 21-24 are examined on the merits.
Notice Re: Prior Art Available Under Both Pre-AIA and AIA
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.
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 3, 7, 9, 10, 12-14, 16, 21, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Beebe (US 2012/0315704. Previously cited) in view of Dzekunov (US 2004/0115784).
Beebe discloses a method of enhancing gene expression in a cell using nanosecond pulse electric fields (nsPEFs) (paragraph [0074]). Cells are placed in the presence of polynucleotides being introduced into the cells, the cells and polynucleotides are exposed to relatively long pulses in the millisecond range, and then the cells are exposed to nsPEF pulses to facilitate transfer of the polynucleotides into the nucleus (paragraph [0074]). In a preferred embodiment, the gene delivery is performed using a pulse generator that provides classical electroporation pulses (in the microsecond or the millisecond range) to open the plasma membranes and nsPEF pulses to open the nucleus (paragraph [0078]). A pause, i.e. time between the pulse types, is provided to separate the long pulses (classical electroporation pulses) and short pulses (nsPEFs) (paragraphs [0078] and [0080]). The pause can vary between 0.1 second to several minutes or hours (paragraphs [0078] and [0080]). Example 1 teaches an embodiment in which cells are treated with a combination of a long pulse (classical plasma membrane electroporation pulse) followed 30 minutes later by a short nsPEF pulse, in the presence of a plasmid (paragraphs [0099]-[0100]).
Given these teachings, Beebe teaches a method meeting limitations of the claimed invention since Beebe teaches an electroporation method comprising:
subjecting a sample comprising one or more intact cells to a first electrical pulse (the long pulse) having a first field strength (inherently possessed by the long pulse) and a first pulse duration sufficient to load the cells with a first agent (a polynucleotide or plasmid) according to a first protocol;
pausing between the first electrical pulse (the long pulse) and the second electrical pulse (the nsPEF pulse); and
subjecting the sample to a second electrical pulse (the nsPEF pulse) having a second field strength (inherently possessed by the nsPEF pulse) and a second pulse duration sufficient to load the cells with a second agent (the polynucleotide or plasmid, meeting the claimed limitation of ‘the first and second agents are the same agent’ of instant claims 7 and 16) according to a second protocol;
wherein the first pulse duration is different from the second pulse duration.
Beebe differs from the claimed invention in that Beebe does not expressly disclose:
the sample is allowed to recover for at least about 24 hours between the long pulse (directed to the claimed ‘first electrical pulse’) and the nsPEF pulse (directed to the claimed ‘second electrical pulse). As indicated in the instant specification, “Recovering the sample or allowing the sample to recover means culturing the cells of the sample in any of the cell-culture vessels and cell culture media disclosed herein under conditions such as those disclosed herein that are appropriate and sufficient to facilitate restoration or return of the cells to an improved or desired state or condition” (paragraph [144]); and
the first electrical pulses or the second electrical pulse is delivered by an electroporation system comprising: a chamber having at least two electrodes separated by a spacer, a first port for allowing the sample to enter the chamber, and a second port for allowing the sample to exit the chamber.
Regarding difference (1) (the recovery time):
As pointed out above, Beebe teaches that the pause between the two pulse types can vary between 0.1 second to several minutes or hours (paragraphs [0078] and [0080]). Beebe also teaches that one or more embodiments of their invention allow modification of at least some factors or conditions to enhance the transfection/expression efficiency of the pulse generator based on classical electroporation factors, wherein the factors/conditions include time between pulse types (paragraph [0091]). It is further stated that the conditions can be readily determined by those of skill in the field of the invention without undue experimentation (paragraph [0091]).
Before the effective filing date of the claimed invention, it would have been a matter of routine experimentation to have varied the pause between the pulse types to at least about 24 hours when practicing the invention of Beebe for the predictable result of electroporating the cells for delivering the agent (polynucleotide or plasmid) into the cells, because Beebe teaches that the pause could be in hours and the pause is one of the conditions which can be modified to enhance the transfection/expression efficiency of the pulse generator without undue experimentation. It is noted that “[W]here 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.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Since Beebe teaches an embodiment in which the cells are in a growth medium (paragraph [0082]), then it would have been obvious to provide the cells in a growth medium when performing the method rendered obvious by Beebe. Since the cells are in a growth medium during the pause of at least about 24 hours rendered obvious by Beebe, then the cells necessarily are cultured during the pause. As such, the sample is allowed to recover according to paragraph [144] of the instant specification.
Regarding difference (2) (the electroporation system):
Dzekunov discloses techniques for streaming electroporation and electroporation apparatus (abstract; paragraph [0030]). The method and apparatus are suitable for the electrical treatment of cells or particles, especially for the introduction of biologically active substances into various types of living cells by means of electrical treatment (paragraph [0003]). The electroporation apparatus includes a channel, an inlet, an outlet, and a pair of electrodes (paragraph [0030]). A suspension comprising particles can be introduced into the flow channel through the inlet flow portal, and the suspension can be withdrawn from the flow channel through the outlet flow portal (paragraph [0063]). See Figures 3, 4, and 4A. Referring to Figures 4, 4A, and 4B, the electrodes 12 and 14 are typically separated by one or more electrode gap spacers 18 and 20. The electroporation apparatus of Dzekunov is directed to the claimed electroporation system since it comprises a chamber (flow channel) having at least two electrodes separated by a spacer (the gap spacer), a first port (the inlet flow portal) for allowing the sample to enter the chamber, and a second port (the outlet flow portal) for allowing the sample to exit the chamber.
Before the effective filing date of the claimed invention, it would have been obvious to the person of ordinary skill in the art to use the electroporation apparatus of Dzekunov to apply the first electrical pulse or the second electrical pulse, when performing the method rendered obvious by Beebe. It would have been an obvious matter of simple substitution of one known means for applying electroporation pulses to cells for another for the predictable result of performing the method of introducing polynucleotides into cells. Since the electroporation apparatus of Dzekunov is suitable for electroporation of cells for the purpose of introducing biologically active substances into cells, there would have been a reasonable expectation of applying one of the pulses of Beebe according to the parameters taught by Beebe.
Thus, Beebe in view of Dzekunov renders obvious instant claims 3 and 7.
Regarding instant claims 9 and 14, Beebe discloses that the cells are loaded with a polynucleotide or plasmid which are directed to a nucleic acid. Furthermore, Beebe teaches an invention in which a desired agent is introduced into a cell, wherein the desired agent can be a nucleic acid (e.g., polynucleotide), a polypeptide, or a protein (paragraphs [0009] and [0040]-[0041]; claim 2 of Beebe). Thus Beebe in view of Dzekunov renders obvious performing the claimed invention in which a nucleic acid, a polypeptide, or a protein is loaded into one or more intact cells (first and second agents are the same agent). As such, instant claims 9 and 14 (protein embodiment) are rendered obvious.
Regarding instant claims 12, 13, and 16, Beebe discloses that the polynucleotide of their invention includes DNA, cDNA, and RNA sequences of all types (paragraph [0042]). Thus instant claims 12, 13, and 16 are rendered obvious.
Regarding instant claim 10, see the preceding paragraph. Beebe further discloses that the polynucleotides of their invention can be DNA constructs such as expression vectors (paragraph [0043]). Thus instant claim 10 (DNA that is a vector) is rendered obvious.
Regarding instant claims 21 and 23, Beebe teaches that the field amplitudes for the long pulses can range from 0.1 to 5 kV/cm, preferably 0.1 to 1 kV/cm, while the field amplitudes for the nsPEF pulses can range from 1 to 1000 kV/cm, preferably 10 to 350 kV/cm (paragraph [0074]). The preferred field amplitude ranges for the long pulses and the nsPEF pulses do not overlap, with the preferred field amplitude range for the nsPEF pulses being greater than the preferred field amplitude range for the long pulses. It would have been prima facie obvious to perform the invention rendered obvious by Beebe and Dzekunov with long pulses and nsPEF pulses having the preferred field amplitudes. In doing so, then the ‘first field strength’ is different from the ‘second field strength.’ As such, instant claims 21 and 23 are rendered obvious.
Claims 6, 8, 11, 15, 22, and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Beebe and Dzekunov as applied to claims 3, 7, 9, 10, 12-14, 16, 21, and 23 above, and further in view of DeWitt (Methods. 2017. 121-122: 9-15. Previously cited).
As discussed above, Beebe in view of Dzekunov renders obvious claims 3, 7, 9, 10, 12-14, 16, 21, and 23.
The references differ from claims 6 and 22 in that they do not expressly disclose that their method is a method of serially editing cell genes.
The references differ from claim 8 in that they do not expressly disclose that the first agent loaded into cells using the long pulse (directed to the claimed ‘first electrical pulse’) is different from the second agent loaded into cells using the nsPEF pulse (directed to the claimed ‘second electrical pulse’); that is, Beebe does not disclose that the first and second agents are different agents.
The references differ from claim 11 in that they do not expressly disclose that the protein loaded into the cells by their method is a ribonucleoprotein and comprises a Cas9 protein and a guide RNA. The references differ from claims 15 and 24 in that they do not expressly disclose that the protein loaded into the cells by their method is a ribonucleoprotein.
DeWitt discloses delivery of recombinant Cas9 protein and guide RNA (gRNA) as a preformed ribonucleoprotein (RNP) complex, stating that it is a powerful and general approach to genome editing (abstract). RNP delivery avoids many of the pitfalls associated with mRNA, DNA, or viral delivery (page 9, last paragraph). DeWitt points out that a donor DNA carrying desired sequence changes can be included with the RNP to program precise sequence introduction or replacement (abstract). The RNP, when paired with a DNA donor, comprises a “total package” that does not require the cellular environment to synthesize Cas9 and single guide RNA (sgRNA), and ensure temporal coordination of the editing reagents (page 9, last paragraph). DeWitt teaches a method for introducing a Cas9 RNP (ribonucleoprotein comprising a Cas9 protein and guide RNA) and donor DNA into cells using electroporation. In particular, Donor teaches that a mixture of Cas9 RNP and donor DNA was delivered to cells by electroporation (Figure 2 on page 11; page 12, left column, second and third paragraphs). DeWitt discloses that RNP can be co-delivered or sequentially delivered with donor DNAs (page 14, last paragraph).
Before the effective filing date of the claimed invention, it would have been obvious to the person of ordinary skill in the art to substitute the agent delivered into the cells with a ribonucleoprotein comprising a Cas9 protein and guide RNA, and a donor DNA, when performing the method rendered obvious by Beebe in view of Dzekunov for the predictable result of introducing the ribonucleoprotein and donor DNA into cells. One of ordinary skill in the art would have been motivated to do this because it would have permitted a powerful and general approach to genome editing which avoids many of the pitfalls associated with mRNA and DNA delivery. There would have been a reasonable expectation of delivering the ribonucleoprotein and donor DNA into cells with the method rendered obvious by Beebe because Beebe discloses their method as being suitable for introducing proteins and nucleic acids into cells, and because electroporation had been successfully used for delivering Cas9 RNP and donor DNA into cells, as demonstrated by DeWitt. Therefore, instant claims 11, 15, and 24 (see the reasoning applied for the rejection of instant claims 21 and 23 over Beebe and Dzekunov above) are rendered obvious.
Regarding instant claims 6, 8, and 22, before the effective filing date of the claimed invention, it would have been obvious to the person of ordinary skill in the art to sequentially deliver the ribonucleoprotein (comprising a Cas9 protein and guide RNA) and donor DNA, specifically providing these agents for loading at distinct electroporation steps (a first of the two agents during the long pulse delivery; a second of the two agents during the nsPEF pulse delivery) when performing the method rendered obvious by Beebe, Dzekunov, and DeWitt. One of ordinary skill in the art would been motivated to do this because DeWitt discloses that RNP can be sequentially delivered with donor DNAs (page 14, last paragraph). In doing so, then the ‘first agent’ and the ‘second agent’ of the method rendered obvious by Beebe, Dzekunov, and DeWitt are different agents. Thus, instant claim 8 is rendered obvious. Since the ribonucleoprotein and donor DNA are sequentially delivered, then Beebe in view of Dzekunov and further in view of DeWitt renders obvious a method of serially editing cell genes. Thus, instant claims 6 and 22 (see the reasoning applied for the rejection of instant claims 21 and 23 over Beebe above) are rendered obvious.
Claims 6, 8, 17, and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Beebe and Dzekunov as applied to claims 3, 7, 9, 10, 12-14, 16, 21, and 23 above, and further in view of Cabaniols (WO 2018/007263. Previously cited).
As discussed above, Beebe and Dzekunov renders obvious claims 3, 7, 9, 10, 12-14, 16, 21, and 23. The references differ from claims 8 and 17 in that they do not expressly disclose that the first agent loaded into cells using the long pulse (directed to the claimed ‘first electrical pulse’) is different from the second agent loaded into cells using the nsPEF pulse (directed to the claimed ‘second electrical pulse’); that is, Beebe does not disclose that the first and second agents are different agents.
The references differs from claims 6 and 22 in that they do not expressly disclose that their method is a method of serially editing cell genes.
Cabaniols discloses a method of sequential gene editing using several rounds of electroporation (page 4, lines 17-18). Their method is a method for introducing genetic modifications at different loci of a primary immune cell comprising (a) subjecting said primary immune cell to a first electroporation step to introduce at least a first sequence-specific reagent into the immune cell; (b) cultivating said primary immune cell thereby enabling said first sequence-specific reagent to modify its genome at a first locus; (c) subjecting said primary immune cell to at least a second electroporation step to introduce at least a second sequence-specific reagent into the cell (claim 1 of Cabaniols). For the electroporation steps, Cabaniols indicates that in general, the suspension of cells undergoes one or more pulsed electric fields (page 19, lines 7-8). Cabaniols also teaches that in step (b) the primary immune cell is cultivated from 12 to 72 hours, preferably 24 to 48 hours (claim 2 of Cabaniols). Example 2 of Cabaniols teaches sequential transfection of cells at a 40 hour interval with two TALEN mRNA which are mRNA encoding TRAC TALEN® and PD1 TALEN® (page 43, lines 9-21; page 44, lines 1-5). These two mRNA are directed to first and second agents that are different agents.
Also, Cabaniols teaches that the first and/or second sequence-specific reagent is a polynucleotide or polypeptide encoding a rare-cutting endonuclease (claims 6 and 7 of Cabaniols). Also, the endonuclease reagent, which is the sequence-specific reagents of Cabaniols (page 14, lines 6-9), can be RNA (more particularly mRNA), proteins, or complexes mixing proteins and nucleic acids (e.g. ribonucleoproteins) (page 14, lines 17-21). Cabaniols also teaches an embodiment in which the first and/or second sequence-specific reagent is a conjugate of RNA guide and a Cas9 polypeptide (claim 8 of Cabaniols). Thus the first and/or second sequence-specific reagent of Cabaniols can be a ribonucleoprotein comprising Cas9 polypeptide (i.e. Cas9 protein) and guide RNA.
Before the effective filing date of the claimed invention, it would have been obvious to the person of ordinary skill in the art to modify the method rendered obvious by Beebe in view of Dzekunov by introducing a first sequence-specific reagent, such as mRNA encoding TRAC TALEN®, into cells during the application of the long pulse and introducing a second sequence-specific reagent, such as mRNA encoding PD1 TALEN®, into cells during the application of the nsPEF pulse, wherein the cells are immune cells. One of ordinary skill in the art would have been motivated to do this because it would have permitted sequential gene editing which Cabaniols discloses as improving the genetic modification of primary human cells, especially immune cells originating from individual donors or patients (page 4, lines 1-3). One of ordinary skill in the art would have been motivated to introduce mRNA encoding TRAC TALEN® and mRNA encoding PD1 TALEN® into immune cells when practicing the method rendered obvious by Beebe and Dzekunov because they express TALEN® which are nucleases useful in producing allogenic therapeutic T-cells (page 1, second-to-last paragraph). There would have been a reasonable expectation of introducing these two different agents by the method rendered obvious by Beebe and Dzekunov because Beebe discloses their method as being suitable for introducing nucleic acids into cells. Therefore, instant claims 8 and 17 are rendered obvious. Since the method rendered obvious by Beebe in view of Dzekunov and further in view of Cabaniols permits sequential gene editing, then the method is directed to a ‘method of serially editing cell genes’ as recited in instant claim 6. Therefore, instant claims 6 and 22 (see the reasoning applied for the rejection of instant claims 21 and 23 over Beebe and Dzekunov above) are rendered obvious.
Response to Arguments
Applicant’s arguments, filed January 23, 2026, with respect to the objections to the claims and the rejections under 35 U.S.C. 103 have been fully considered and are persuasive. In particular, the amendments to the claims have overcome the claim objections and those rejections. Therefore, those objections and rejections have been withdrawn.
However, upon further consideration, a new ground(s) of rejection is made in view of the newly cited reference Dzekunov, as necessitated by the amendments to the claims.
Conclusion
No claims are allowed.
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/SUSAN E. FERNANDEZ/Examiner, Art Unit 1651