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 .
The amendment filed September 30, 2025, has been received and entered.
Claim 20 is new.
Claims 1-20 are pending. Claims 13-19 are withdrawn.
Claims 1-12 and 20 are examined on the merits.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 8 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 8 is indefinite because it is unclear how the terms in parentheses modify the preceding symbols. For instance, it is unclear how “(m2)” modifies the symbol “S.” For the purpose of applying prior art, the symbols S, C, u, and D are being interpreted as having units of m2, m, m/s, and s-1, respectively.
Claim 8 is rendered indefinite by the recitation “a shear rate D (s-1) defined by Expression (1) is 1 s-1 or more and 5,000 s-1 or less, D = 2u × C/S.” It is unclear whether Expression (1) is D = 2u × C/S. This ground of rejection can be overcome by inserting “wherein Expression (1) is” before the recitation “D = 2u × C/S” (i.e., ‘wherein Expression (1) is D = 2u × C/S’).
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 20 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.
The limitation in claim 20 that the length of the second pair of electrodes in the flow direction is shorter than a distance between the first pair of electrodes and the second pair of electrodes is not supported by the specification as filed. In the Remarks filed September 30, 2025, Applicant cites paragraph [0022] of the specification for providing support for claim 20. It appears Applicant is citing paragraph [0022] of the published patent application, which corresponds to paragraph [0020] of the specification as filed. Paragraph [0020] of the specification states that the ratio of L1/L0 of the length L1 of the first pair of electrodes in the flow direction to the length L0 between the first pair of electrodes and the second pair of electrodes in the flow direction is preferably 1/30,000 or more and 1/10 or less. This teaching provides support for the limitation in claim 20 that the length (i.e., L1 in paragraph [0020]) of the first pair of electrodes in the flow direction being shorter than a distance (i.e., L0 in paragraph [0020]) between the first pair of electrodes and the second pair of electrodes. However, paragraph [0020] and the rest of the specification do not compare the length of the second pair of electrodes in the flow direction with the distance between the first pair of electrodes and the second pair of electrodes. Claim 1 recites that the length of the first pair of electrodes in a flow direction is shorter than the length of the second pair of electrodes in a flow direction. That limitation is supported by the specification. However, that limitation in combination with paragraph [0020] of the specification does not signify that the length of the second pair of electrodes in the flow direction is shorter than the distance between the two pairs of electrodes.
While Applicant was in possession of a portion of the claimed invention, the full scope of the claimed invention, specifically the length of the second pair of electrodes in the flow direction being shorter than a distance between the first pair of electrodes and the second pair of electrodes, is not fully described in the specification. As such, Applicant was not in possession of the full scope of the claimed invention at the time of filing. Because the specification as filed fails to provide clear support for the new claim language, a new matter rejection is clearly proper.
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 1-7, 9, 10, and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Cooper (US 2011/0104128. Previously cited) in view of Dzekunov (US 2004/0115784) and Sukharev (Biophys. Journal. 1992. 63(5): 1320-1327).
Cooper discloses devices and methods for transfection of living cells using electroporation, in particular high throughput microfluidic electroporation (abstract). The multi-stream channel of the device of Cooper can include a plurality of electrodes in a pattern that generates multiple electroporation zones in the channel (paragraph [0121]). The electroporation zones can include mechanisms to control the duration and electric voltage of electroporation so as to control the number and size of pores on a cell flowing through the channel (paragraph [0121]). For instance, Figure 6 shows a side view of a cell traveling through multiple electric fields to improve transfection efficiency (paragraph [0090]). It is evident from Figure 6 that cells flow through a flow channel comprising multiple electric field regions, wherein each electric field region is created by a pair of electrodes arranged opposite each other on opposing walls of the flow channel, meeting limitations of the first and second pairs of electrodes of instant claim 1.
In one embodiment, the cells can be mixed with the DNA and/or RNA prior to subjecting the cells to electroporation (paragraphs [0050]-[0051] and [0128]). Also, an example of the invention of Cooper is shown in Figure 16 in which cells from a patient are delivered into inlet 110, and a stream of an RNA species is delivered into inlet 120 (paragraph [0107]). Thus the cells are mixed with the RNA prior to their entry into the microfluidic electroporation units (MEU) (paragraph [0107]). DNA and RNA are directed to a ‘bioactive substance,’ with DNA directed to the ‘bioactive substance’ of instant claim 10.
Additionally, Cooper teaches that the living cells are obtained from a bodily fluid from a patient, and the patient can be a human being (paragraphs [0064] and [0080]-[0081]). Living cells from a human being are directed to an ‘organism-derived material’ which is a ‘human-derived cell,’ thereby meeting limitations of instant claim 1. In transfecting living cells obtained from a human being, then the method of Cooper is directed to a producing method for an organism-derived material into which a bioactive substance has been introduced.
In sum, Cooper meets limitations of the claimed invention in that Cooper discloses a producing method for an organism-derived material (living cells obtained from a human being) into which a bioactive substance (DNA or RNA) has been introduced, the producing method comprising:
a step of causing a suspension containing the organism-derived material (living cells obtained from a human being) before the introduction of the bioactive substance (DNA or RNA) and containing the bioactive substance to flow through a flow channel, thereby passing through a first electric field region having a first electric field intensity generated by a first pair of electrodes arranged opposite each other on opposing walls of the flow channel; and
a step of causing the suspension to pass through a second electric field region after the suspension has passed through the first electric field region, the second electric field region being generated by a second pair of electrodes arranged opposite each other on the opposing walls of the flow channel,
wherein the organism-derived material is a human-derived cell.
Cooper differs from the claimed invention in that Cooper does not expressly disclose that the second electric field region has a second electric field intensity lower than the first electric field intensity, and a length of the first pair of electrodes in a flow direction is shorter than a length of the second pair of electrodes in the flow direction.
Dzekunov discloses techniques for streaming electroporation, wherein “streaming electroporation” is a term used to refer to a sample streaming relative to an electric field that primarily determines the expose of the sample to the electric field that effects electroporation (abstract, paragraph [0052]). In streaming electroporation, biological cells are effectively “pulsed” by their defined movement across electrical field lines (paragraph [0053]). For instance, the cells pass through a pair of electrodes, and each cell is exposed to an electric field for the period of time it spends between the electrodes, which is analogous to a pulse width in a typical application (paragraph [0053]). The half-width of the approximately bell-shaped curve of the electric field intensity will depend on the rate of the passage of the cells between the electrodes and the electrode spacing and dimensions (paragraph [0069]). Furthermore, in an apparatus in which cells flow between the electrodes through a channel, positioning multiple electrode pairs in sequence in the flow can result in multiple pulses being applied to each cell (paragraph [0069]).
Sukharev speaks of electrotransfection of cells, which is transfection induced by electric field pulses (page 1320, first paragraph). In the electrotransfection experiments of Sukharev, simian Cos-1 cells were transfected with the plasmid pCH110 containing a bacterial β-galactosidase gene (lacZ) (paragraph bridging pages 1320 and 1321). In particular, aliquots of cell suspension were mixed with aliquots of plasmid, incubated, and then subjected to electric pulses (page 1321, left column, third paragraph). Some experiments involved applying a sequence of two unequal pulses, wherein there is a delay of 100 µs between pulses except for two experiments (page 1321, left column, second and third paragraphs). For preliminary electrotransfection experiments in the two-pulse mode, the first pulse was short but of high intensity, whereas the second pulses was of a lower amplitude but much longer (page 1322, right column, second-to-last paragraph; Figure 5 on page 1323).
Sukharev found that there was an increase in transfection efficiency (TE) by the second pulse when it follows the first one which is much higher than the level of TE evoked by the second pulse alone (page 1322, right column, second-to-last paragraph). Sukharev asserts that this suggests that the effects of these two pulses on cell membrane and the DNA situated nearby are different and create a sequence of events that favors DNA penetration into the cell (page 1322, right column, second-to-last paragraph). In additional experiments, Sukharev found that the first pulse of 6 kV/cm and 10 µs provides efficient and reversible poration, but is weak in electrotransfection as it induces <5% of the transfection efficiency (TE) obtained at 3.5 kV/cm and 100 µs (page 1322, right column, last paragraph). In those additional experiments, Sukharev found that applying the second pulse of 0.2 kV/cm and 10 ms immediately after the first pulse enhances transfection efficiency by one order of magnitude (paragraph bridging pages 1322 and 1323).
Before the effective filing date of the claimed invention, it would have been obvious to the person of ordinary skill in the art to include only two electroporation zones in the flow channel of Cooper by providing only two electrode pairs in the channel, modify the sizes of the electrodes of the first and second electrode pairs so that the length of the first pair of electrodes in the flow direction is shorter than the length of the second pair of electrodes in the flow direction, and modify the electric field intensities so that the second electric field intensity generated by the second pair of electrodes is low (e.g. 0.2 kV/cm) and the first electric field intensity generated by the first pair of electrodes is high (e.g. 6 kV/cm), which meets the claimed limitation of the second electric intensity being lower than the first electric field intensity. One of ordinary skill in the art would have been motivated to do this in order to improve transfection efficiency of the method of Cooper by implementing the findings in Sukharev for improving transfection efficiency by applying two electric pulses to cells to create a sequence of events that favors DNA penetration into the cell. Based on Dzekunov, flowing a cell through a channel between opposing electrodes is effectively applying an electric pulse on the cell. Therefore, there would have been a reasonable expectation of practicing the two-pulse technique of Sukharev with the method of Copper by modifying the number of electrode pairs in the method of Cooper to two and varying the lengths and electric field intensities of the electrodes in each electrode pair of Cooper to obtain the desired electric field intensities and pulse durations as taught in Sukharev for improved transfection efficiency, with the length of the electrodes correlating with the pulse duration (i.e., the longer the electrodes, the longer the pulse duration).
Therefore, Cooper in view of Dzekunov and Sukharev renders obvious instant claims 1 and 10.
Regarding instant claims 2 and 3, since the length of the first pair of electrodes in a flow direction is shorter than the length of the second pair of electrodes, then a first period during which the cells pass through the first electric field region (in this case, the entire duration of exposure to the first electric field region) is shorter than a second period during which the cells pass through the second electric field region (in this case, the entire duration of exposure to the second electric field region). Therefore, instant claim 2 is rendered obvious. Moreover, the recitations of a ‘first period’ and a ‘second period’ in instant claims 2 and 3 are broad. First, the claimed recitation ‘a first period during which the suspension passes through the first electric field region’ is any period during which the suspension passes through the first electric field region. That is, it is a period of time less than or equal to the duration of the suspension passing through the first electric field region. Likewise, the claimed recitation ‘a second period during which the suspension passes through the second electric field region’ is any period during which the suspension passes through the second electric field region. That is, the ‘second period’ is any period of time less than or equal to the duration of the suspension passing through the second electric field region. Given the breadth of the claimed ‘first period’ and ‘second period,’ then it is obvious that there are periods within the duration of the suspension passing through the ‘first electric field region’ of the invention rendered obvious by Cooper, Dzekunov, and Sukharev that are equal to or shorter than periods within the duration of the suspension passing through the ‘second electric field region’ of the invention rendered obvious by Cooper, Dzekunov, and Sukharev. Thus, instant claim 2 is rendered obvious. Likewise, given the breadth of the claimed ‘first period’ and “second period,’ then it is obvious that there are periods (each a ‘first period T1’) within the duration of the suspension passing through the ‘first electric field’ of the invention rendered obvious by Cooper, Dzekunov, and Sukharev and periods (each a ‘second period T2’) within the duration of the suspension passing through the ‘second electric field’ of the invention rendered obvious by Cooper, Dzekunov, and Sukharev that result in a ratio T1/T2 of 1/1,000 or more and 1 or less. Thus, instant claim 3 is rendered obvious.
Further regarding instant claim 3, it is noted that Sukharev discloses a first pulse of 6 kV/cm and 10 µs, and a second pulse of 0.2 kV/cm and 10 ms (abstract). In performing the method rendered obvious by Cooper in view of Dzekunov and Sukharev, it would have been obvious to obtain the electric pulses of Sukharev by adjusting the lengths of the electrodes of the first and second electrode pairs such that the cells pass through the first electrode field region for 10 µs and pass through the second electrode field region for 10 ms, in addition to modifying the electric field intensities to those disclosed by Sukharev, because it would have improved transfection efficiency. In doing so, then the ratio T1/T2 of the first period T1 (in this case, the entire duration of exposure to the first electric field region, which is 10 µs) to the second period T2 (in this case, the entire duration of exposure to the second electric field, which is 10 ms) is 1/1,000 which falls within the claimed range of ‘1/1,000 or more and 1 or less than 1,’ rendering obvious instant claim 3.
Regarding instant claim 4, Sukharev discloses a first pulse of 6 kV/cm and 10 µs, and a second pulse of 0.2 kV/cm and 10 ms (abstract). In performing the method rendered obvious by Cooper in view of Dzekunov and Sukharev, it would have been obvious to obtain the electric pulses of Sukharev by modifying the first electric field intensity generated by the first pair of electrodes to 6 kV/cm and the second electric field intensity generated by the second pair of electrodes to 0.2 kV/cm, in addition to adjusting the lengths of the electrodes of the first and second electrode pairs so that the cells pass through them at the pulse durations disclosed by Sukharev, because it would have improved transfection efficiency. In doing so, then the ratio E2/E1 of the second electric field intensity E2 to the first electric field intensity E1 is 1/30 which falls within the claimed range of ‘1/1,000 or more and less than 1.’ Thus, instant claim 4 is rendered obvious.
Regarding instant claims 5 and 6, Figure 6 of Cooper shows that the multiple electroporation zones are separated by electric field-free regions. As pointed out above, Sukharev teaches that between the two pulses, there is a delay (page 1321, left column, second and third paragraphs). When performing the method rendered obvious by Cooper in view of Dzekunov and Sukharev, it would have been obvious that there is an electric field-free region between the first pair of electrodes and the second pair of electrodes so that a delay is obtained between the two electric pulses which are effectively applied by flowing the cells between the two electrode pairs, because Sukharev teaches a delay in their two-pulse technique that improves transfection efficiency. Therefore, instant claim 5 is rendered obvious. Further regarding instant claim 6, the recitations of a ‘first period T1’ and a ‘third period T0’ are broad. First, the claimed recitation ‘a first period T1 during which the suspension passes through the first electric field region’ is any period during which the suspension passes through the first electric field region. That is, it is a period of time less than or equal to the duration of the suspension passing through the first electric field region. Likewise, the claimed recitation ‘a third period T0 during which the suspension passes through the electric field-free region’ is any period during which the suspension passes through the electric field-free region. That is, the ‘third period’ is any period of time less than or equal to the duration of the suspension passing through the electric field-free region. Given the breadth of the claimed ‘first period’ and ‘third period,’ then it is obvious that there are periods within the duration of the suspension passing through the ‘first electric field region’ of the invention rendered obvious by Cooper, Dzekunov, and Sukharev, T1, that are shorter than periods within the duration of the suspension passing through the ‘electric field-free region’ of the invention rendered obvious by Cooper, Dzekunov, and Sukharev, T0, including resulting in a ratio T1/T0 of 1/25,000 or more and less than 1. Thus, instant claim 6 is rendered obvious.
Further regarding instant claim 6, Sukharev discloses a first pulse of 6 kV/cm and 10 µs (abstract) and a delay between the two pulses of 100 µs (page 1321, left column, second paragraph). In performing the method rendered obvious by Cooper in view of Dzekunov and Sukharev, it would have been obvious to obtain the electric pulse sequence of Sukharev by adjusting the lengths of the electrodes of the first and second electrode pairs and the length between the two electrode pairs such that the cells pass through the first electrode field region for 10 µs, pass through the electric field-free region for 100 µs, and then pass through the second electrode field region for 10 ms, in addition to modifying the electric field intensities to those disclosed by Sukharev, because it would have improved transfection efficiency. In doing so, then the ratio T1/T0 of a first period T1 (in this case, the entire duration of exposure to the first electric field region) to a third period T0 during which the suspension passes through electric field-free region (in this case, the entire duration of passing through the electric field-free region) is 1/10 [Calculation: (10 µs)/(100 µs) = 1/10] which falls within the claimed range of ‘1/25,000 or more and 1 or less than 1,’ rendering obvious instant claim 6.
Regarding instant claim 7, an example of the invention of Cooper is shown in Figure 16 in which cells from a patient are delivered into inlet 110, and a stream of an RNA species is delivered into inlet 120 (paragraph [0107]). Thus the cells are mixed with the RNA prior to their entry into the microfluidic electroporation units (MEU) (paragraph [0107]). Therefore, in practicing the method rendered obvious by Cooper in view of Dzekunov and Sukharev, it would have been obvious to incorporate the teaching of Cooper of providing the cells in an inlet 110 and delivering a stream of DNA or RNA into inlet 120 for mixing prior to entry into the flow channel. As such, instant claim 7 is rendered obvious.
Regarding instant claim 9, in performing the method rendered obvious by Cooper in view of Dzekunov and Sukharev, it would have been obvious to include at least one additional electroporation zone because Cooper teaches four electroporation zones in their invention (Figure 6). Therefore, the cell suspension passes through at least one electric field region different from the first electric field region and the second electric field region, rendering obvious instant claim 9.
Regarding instant claim 20, Sukharev discloses a first pulse of 6 kV/cm and 10 µs, and a second pulse of 0.2 kV/cm and 10 ms (abstract). Using those two pulses, Sukharev also discloses testing the dependence of transfection efficiency on the time delay between two pulses (page 1323, left column, third paragraph; Figure 6). Even at 100-s interval between pulses, the transfection efficiency (TE) level is twice as high as that obtained by application of the first pulse alone. Figure 6 shows that for the delay that is between 0.01 s (i.e. 10 ms) and 1 s, which appears to be approximately 0.1 s (i.e. 100 ms), the error bar for transfection efficiency overlaps with the error bar for the optimum transfection efficiency obtained for 0.0001 s (i.e. 100 µs, 0.1 ms). In performing the method rendered obvious by Cooper in view of Dzekunov and Sukharev, it would have been obvious to obtain the electric pulse sequence of Sukharev by adjusting the lengths of the electrodes and the electric field-free region between the first pair of electrodes and the second pair of electrodes such that the cells pass through the first electrode field region for 10 µs, pass through the electric field-free region for 100 ms, and then pass through the second electrode field region for 10 ms, in addition to modifying the electric field intensities to those disclosed by Sukharev, because it would have improved transfection efficiency. Based on Dzekunov, the skilled artisan would have expected that the lengths of the electrodes of each of the electroporation zones and the length of the electric field-free region correlate with the exposure times to electric pulses and the delay time between the two pulses, respectively. Given that the duration through the electrode field-free region (100 ms) is greater than the duration through the first electrode field region (10 µs) and the duration through the second electrode field region (10 ms), then the length of the first pair of electrodes in the flow direction and the length of the second pair of electrodes in the flow direction are shorter than the distance between the first pair of electrodes and the second pair of electrodes. Therefore, instant claim 20 is rendered obvious.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper, Dzekunov, and Sukharev as applied to claims 1-7, 9, 10, and 20 above, and further in view of Mastwijk (US 6,178,880. Previously cited) and Palsson (US 5,534,423. Previously cited), and in light of Pearson (US 4,821,564. Previously cited).
As discussed above, Cooper in view of Dzekunov and Sukharev renders obvious claims 1-7, 9, 10, and 20. The references meet limitations of claim 8 in that they render obvious the suspension (cell suspension) passing through the first electric field region and the second electric field region by flowing inside a flow channel. The references differ from claim 8 in that they do not expressly disclose that the area of a cross section of the flow channel orthogonal to a flow direction of the suspension is denoted by S in unit of m2, a circumference length of the cross section of the flow channel is denoted by C in unit of m, and an average speed at which the suspension passes through the first electric field region and the second electric field region is denoted by u in unit of m/s, a shear rate D in unit of s-1 is 1 s-1 or more and 5,000 s-1 or less, wherein D is defined by the following equation: D = 2u × C/2.
Mastwijk discloses a system for treating pumpable products by electrical pulses comprising a flow channel through which product can be pumped (abstract). The invention is suitable for invoking pores in membranes of cellular structures to promote the transport of macromolecular components across the membrane (column 1, lines 12-15). As shown in Figure 1, the channel has a cylindrical cross section (column 4, lines 12-14). However, different cross-sectional shapes give similar results (column 4, lines 14-15). An increasing or decreasing electric field strength across the treatment chamber can be obtained (column 4, lines 55-57). An example of the invention of Mastwijk is shown in Figures 3-4 which provides a treatment chamber having a cylindrical cross-section and comprises 3 annular electrodes spaced apart along the treatment chamber through which the product flows (column 5, lines 8-19).
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 flow channel with a cylindrical channel when performing the method rendered obvious by Cooper in view of Dzekunov and Sukharev for the predictable result of electroporating the cells for their transfection. It would have been a matter of simple substitution of one shape of flow channel for another. There would have been a reasonable expectation of performing the method rendered obvious by Cooper in view of Dzekunov and Sukharev with an electroporation channel that is cylindrical because Mastwijk teaches that a cylindrical channel is suitable as a flow channel through which a pumpable product is treated with electrical pulses along the channel that may be used for invoking pores in membranes of cellular structures to transport macromolecular components across the membrane, and because Mastwijk teaches that different cross-sectional shapes give similar results. Since the cross section of a cylindrical channel is a circle, then the cross section of a cylindrical electroporation channel has a circumference length, i.e., C which inherently can be in any unit of length, including meters. Thus Cooper, Dzekunov, Sukharev, and Mastwijk render obvious limitations of instant claim 8.
Additionally, as evidenced by Pearson, shear rate for laminar flow in a pipe is determined by the following equation, wherein D = pipe diameter and V = velocity (column 4, lines 54-64):
γ
=
8
V
/
D
For the cylindrical electroporation channel of the method rendered obvious by Cooper, Dzekunov, Sukharev, and Mastwijk, then the shear rate is calculated below in terms of r = radius of the cylindrical electroporation channel:
γ
=
8
V
/
(
2
r) = 4V/r
For a cylinder, it is well known in the art that the area of a circular cross section is πr2, and the circumference length of said circular cross section is 2πr. Therefore, the shear rate equation of instant claim 8 for a cylindrical flow channel converts to the following:
S
h
e
a
r
r
a
t
e
=
2
u
×
C
S
=
2
u
×
2
π
r
π
r
2
=
4
u
/
r
Since the equation for shear rate for the cylindrical electroporation channel of the method rendered obvious by Cooper, Dzekunov, Sukharev, and Mastwijk, as evidenced by Pearson, is 4V/r (i.e. 4u/r) which is the same as the shear rate equation of instant claim 8 for a cylindrical channel, then the shear rate of the flow in the cylindrical electroporation channel of the method rendered obvious by Cooper, Dzekunov, Sukharev, and Mastwijk (in light of Pearson) meets the equation for shear rate D in instant claim 8. It is also well known in the art that the SI units for area, length, and velocity (i.e. speed) are m2, m, and m/s, respectively.
Further regarding the claimed shear rate, Palsson discusses genetic engineering and discloses a method of transfecting target cells in which vectors such as nucleic acids are introduced into the target cells (column 1, lines 7-9; claims 1 and 13 of Palsson). The target cells include human cells (in particular, hematopoietic stem cells of human origin) (column 4, line 64 through column 5, line 4). Palsson points out that shear sensitivity of different types of cell varies greatly (column 6, lines 59-60). Thus, the flow velocity of a fluid approaching cells has to be such that the target cells are not damaged (column 6, lines 60-62). In Palsson’s invention, the shear rate experienced by the target cells is expected to be below about 200 per second (column 6, lines 62-64).
Before the effective filing date of the claimed invention, it would have been an obvious matter of routine optimization to adjust the flow velocity of the suspension through the cylindrical electroporation channel such that the shear rate is low such as 200 s-1 (falling in the claimed range) or in the range of 1 s-1 or more and 5,000 s-1 or less, when performing the method rendered obvious by Cooper, Dzekunov, Sukharev, and Mastwijk (in light of Pearson). One of ordinary skill in the art would have been motivated to do this because flow velocity affects shear rate, which in turn affects different types of cells differently since shear sensitivity varies for different types of cells. Moreover, a shear rate of 200 s-1 was found suitable for target cells that include human cells in Palsson, which would have motivated the skilled artisan to adjust the flow velocity to obtain that shear rate.
As such, Cooper, Dzekunov, Sukharev, Mastwijk, and Palsson (in light of Pearson, cited as evidence) render obvious instant claim 8.
Claims 11 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Cooper, Dzekunov, and Sukharev as applied to claims 1-7, 9, 10, and 20 above, and further in view of Witting (Human Gene Therapy. 2012. 23: 243-249. Previously cited).
As discussed above, Cooper in view of Dzekunov and Sukharev renders obvious claims 1-7, 9, 10, and 20. The references differ from claim 11 in that they do not expressly disclose a step of culturing the transfected cells (directed to the claimed ‘organism-derived material produced by the producing method according to claim 1’), and a step of extracting a product that is produced by the transfected cells (directed to the claimed ‘the organism-derived material’). The references further differ from claim 12 in that they do not expressly disclose that the product (that is produced and extracted) is a virus.
Witting found that flow electroporation (EP) was highly suited for large-scale production of lentivirus, specifically lentiviral productions for early phase clinical gene therapy trials with potential for further scale-up (page 244, left column, second paragraph). In particular, Witting discloses suspending HEK293 FT cells in EP buffer, and adding the suspension of HEK293 FT cells and DNA (four plasmids) to a device to perform flow electroporation (page 244, left column, last full paragraph through right column, first paragraph for disclosing the cells and the plasmids; page 244, right column, third and last paragraphs for disclosing lentivirus production using EP, in particular flow EP). HEK293 FT cells are directed to human-derived cells. After the electroporation, the cells were incubated with DNase and then cultured in a bioreactor (page 244, right column, last paragraph). Afterwards, aliquots of virus-containing media were harvested, filtered, and stored (page 245, first 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 have applied the invention of Cooper, Dzekunov, and Sukharev to lentiviral production as disclosed by Witting by substituting the cells derived from a human being with HEK293 FT cells and substituting the DNA with the four plasmids taught by Witting in order to transfect HEK293 FT cells with said plasmids by the method rendered obvious by Cooper, Dzekunov, and Sukharev, culturing the transfected cells according to Witting, and obtaining the lentivirus by harvesting and filtering the virus-containing media resulting from the culture. One of ordinary skill in the art would have been motivated to do this in order to produce lentivirus, a desirable product, which is suitable for early phase clinical gene therapy trials. There would have been a reasonable expectation of producing lentivirus by this modification of the invention rendered obvious by Cooper, Dzekunov, and Sukharev because Witting demonstrated through their study that flow electroporation (which the invention of Cooper in view of Dzekunov and Sukharev is directed to) is suitable for lentivirus production. As such, the references render obvious a producing method for a product (lentivirus, directed to ‘virus’ of instant claim 12) comprising a step of culturing the transfected HEK293 FT cells (directed to ‘organism-derived material’ which is a human-derived cell) produced by the method rendered obvious by Cooper in view of Dzekunov and Sukharev (rendering obvious ‘the producing method according to claim 1’); and a step of extracting the product (by harvesting and filtering the virus-containing media) that is produced by the transfected HEK293 FT cells (claimed ‘organism-derived material’). Thus, Cooper, Dzekunov, Sukharev, and Witting render obvious instant claims 11 and 12.
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 1-6, 9, 10, and 20 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 3 and 6-13 of copending Application No. 18/489,205 in view of Dzekunov (US 2004/0115784) and Sukharev (Biophys. Journal. 1992. 63(5): 1320-1327).
Claim 6 of `205 meets limitations of the claimed invention, with one difference being that claim 6 of `205 does not recite that the biologically derived material is an organism-derived material which is a human-derived cell. However, claim 12 of `205 recites the limitation of a human derived cell as the biologically derived material. It would have been prima facie obvious to apply that limitation of claim 12 of `205 to claim 6 of `205 as well as claims 7-11 and 13 of `205. In doing so, then claim 6 of `205 in view of claim 12 of `205 renders obvious a method meeting limitations of instant claim 1 in that they render obvious a method of introducing a bioactive substance into an organism-derived material (which is a human-derived cell) (directed to a producing method for an organism-derived material into which a bioactive substance has been introduced) comprising:
a step of causing a suspension containing the organism-derived material (inherently before the introduction of the bioactive substance) and the bioactive substance to flow through a flow passage, thereby passing through a first electric field region; and
a step of causing the suspension to pass through a second electric field region (after the suspension has passed through the first electric field region since the last step of claim 6 of `205).
Though claim 6 of `205 includes an additional step of causing the suspension to pass through a cooling region, it is directed to a narrower embodiment that is wholly encompassed by the method of the claimed invention.
Claim 6 of `205 in view of claim 12 of `205 (as well as claim 12 of `205 itself) differs from the instant claims in that claim 6 of `205 does not recite that:
the first electric field region is generated by a first pair of electrodes arranged opposite each other on opposing walls of the flow passage (directed to a ‘flow channel’), and the second electric field region is generated by a second pair of electrodes arranged opposite each other on the opposing walls of the flow channel, wherein the length of the first pair of electrodes in a flow direction is shorter than the length of the second pair of electrodes in the flow direction; and
the second electric field intensity of the second electric field region is lower than the first electric field intensity of the first electric field region.
It is noted that claim 12 of `205 includes the further limitation that the bioactive substance is DNA. This meets the limitation of instant claim 10.
However, claim 3 of `205 recites an electric perforating device comprising a plurality of pairs of conductive members provided on wall surfaces of the flow passage facing each other along the flowing direction of the liquid, wherein, by supplying a pulse voltage with the voltage output portion to each of two pairs of conductive members, one of the two pairs of conductive members functions as the first electrode, and the other of the two pairs of conductive members functions as the second electrode. In performing the method of claim 6 of `205 in view of claim 12 of `205, or the method of claim 12 of `205 itself, it would have been prima facie obvious to incorporate the limitations regarding the electric perforating device of claim 3 of `205. In doing so, then the first electric field region is generated by a first pair of conductive members, directed to the claimed ‘first pair of electrodes’ arranged opposite each other on opposing walls of the flow passage, and the second electric field region is generated by the second pair of conductive members, directed to the claimed ‘second pair of electrodes’ arranged opposite each other on opposing walls of the flow passage.
Dzekunov discloses techniques for streaming electroporation, wherein “streaming electroporation” is a term used to refer to a sample streaming relative to an electric field that primarily determines the expose of the sample to the electric field that effects electroporation (abstract, paragraph [0052]). In streaming electroporation, biological cells are effectively “pulsed” by their defined movement across electrical field lines (paragraph [0053]). For instance, the cells pass through a pair of electrodes, and each cell is exposed to an electric field for the period of time it spends between the electrodes, which is analogous to a pulse width in a typical application (paragraph [0053]). The half-width of the approximately bell-shaped curve of the electric field intensity will depend on the rate of the passage of the cells between the electrodes and the electrode spacing and dimensions (paragraph [0069]). Furthermore, in an apparatus in which cells flow between the electrodes through a channel, positioning multiple electrode pairs in sequence in the flow can result in multiple pulses being applied to each cell (paragraph [0069]).
Sukharev speaks of electrotransfection of cells, which is transfection induced by electric field pulses (page 1320, first paragraph). In the electrotransfection experiments of Sukharev, simian Cos-1 cells were transfected with the plasmid pCH110 containing a bacterial β-galactosidase gene (lacZ) (paragraph bridging pages 1320 and 1321). In particular, aliquots of cell suspension were mixed with aliquots of plasmid, incubated, and then subjected to electric pulses (page 1321, left column, third paragraph). Some experiments involved applying a sequence of two unequal pulses, wherein there is a delay of 100 µs between pulses except for two experiments (page 1321, left column, second and third paragraphs). For preliminary electrotransfection experiments in the two-pulse mode, the first pulse was short but of high intensity, whereas the second pulses was of a lower amplitude but much longer (page 1322, right column, second-to-last paragraph; Figure 5 on page 1323).
Sukharev found that there was an increase in transfection efficiency (TE) by the second pulse when it follows the first one which is much higher than the level of TE evoked by the second pulse alone (page 1322, right column, second-to-last paragraph). Sukharev asserts that this suggests that the effects of these two pulses on cell membrane and the DNA situated nearby are different and create a sequence of events that favors DNA penetration into the cell (page 1322, right column, second-to-last paragraph). In additional experiments, Sukharev found that the first pulse of 6 kV/cm and 10 µs provides efficient and reversible poration, but is weak in electrotransfection as it induces <5% of the transfection efficiency (TE) obtained at 3.5 kV/cm and 100 µs (page 1322, right column, last paragraph). In those additional experiments, Sukharev found that applying the second pulse of 0.2 kV/cm and 10 ms immediately after the first pulse enhances transfection efficiency by one order of magnitude (paragraph bridging pages 1322 and 1323).
When practicing the invention rendered obvious by claim 6 of `205 in view of claims 3 and 12 of `205, or the invention of claim 12 of `205 in view of claim 3 of `205, it would have been obvious to the person of ordinary skill in the art to modify the sizes of the conductive members (directed to electrodes) of the first and second pairs of conductive members (directed to the first and second pairs of electrodes) so that the length of the first pair of conductive members in the flow direction is shorter than the length of the second pair of conductive members in the flow direction, and modify the electric field intensities so that the second electric field intensity generated by the second pair of conductive members is low (e.g. 0.2 kV/cm) and the first electric field intensity generated by the first pair of conductive members is high (e.g. 6 kV/cm), which signifies that the second electric intensity is lower than the first electric field intensity. One of ordinary skill in the art would have been motivated to do this in order to improve transfection efficiency of the method of the claims of `205 by implementing the findings in Sukharev for improving transfection efficiency by applying two electric pulses to cells to create a sequence of events that favors DNA penetration into the cells. Based on Dzekunov, flowing cells between electrodes through a channel is effectively applying electric pulses to the cells. Therefore, there would have been a reasonable expectation of practicing the two-pulse technique of Sukharev with the method of the claims of `205 by varying the lengths and electric field intensities of the conductive members (i.e. electrodes) in each pair of conductive members to obtain the desired electric field intensities and pulse durations taught in Sukharev for improved transfection efficiency, with the length of the conductive members correlating with the pulse duration (i.e., the longer the conductive member, the longer the pulse duration).
Thus claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10. Also, claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10. Likewise, claim 13 of `205 (its manufacturing method is directed to a ‘producing method’) in view of claims 3 and 12 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10.
Regarding instant claims 2 and 3, since the length of the first pair of electrodes (the first pair of conductive members) in a flow direction is shorter than the length of the second pair of electrodes (the second pair of conductive members), then a first period during which the cells pass through the first electric field region (in this case, the entire duration of exposure to the first electric field region) is shorter than a second period during which the cells pass through the second electric field region (in this case, the entire duration of exposure to the second electric field region). Therefore, instant claim 2 is rendered obvious by claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev, as well as being rendered obvious by claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev. Moreover, the recitations of a ‘first period’ and a ‘second period’ in instant claims 2 and 3 are broad. First, the claimed recitation ‘a first period during which the suspension passes through the first electric field region’ is any period during which the suspension passes through the first electric field region. That is, it is a period of time less than or equal to the duration of the suspension passing through the first electric field region. Likewise, the claimed recitation ‘a second period during which the suspension passes through the second electric field region’ is any period during which the suspension passes through the second electric field region. That is, the ‘second period’ is any period of time less than or equal to the duration of the suspension passing through the second electric field region. Given the breadth of the claimed ‘first period’ and ‘second period,’ then it is obvious that there are periods within the duration of the suspension passing through the first electric field region of the invention rendered obvious by claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev, as well as being rendered obvious by claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev that are equal to or shorter than periods within the duration of the suspension passing through the second electric field region of the invention rendered obvious by claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev, as well as being rendered obvious by claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev. Thus, instant claim 2 is rendered obvious. Likewise, given the breadth of the claimed ‘first period’ and “second period,’ then it is obvious that there are periods (each a ‘first period T1’) within the duration of the suspension passing through the first electric field of the invention rendered obvious by claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev, as well as being rendered obvious by claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev, and periods (each a ‘second period T2’) within the duration of the suspension passing through the second electric field of the invention rendered obvious by claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev, as well as being rendered obvious by claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev, that result in a ratio T1/T2 of 1/1,000 or more and 1 or less. Thus, instant claim 3 is rendered obvious.
Further regarding instant claim 3, it is noted that Sukharev discloses a first pulse of 6 kV/cm and 10 µs, and a second pulse of 0.2 kV/cm and 10 ms (abstract). In performing claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev, it would have been obvious to obtain the electric pulses of Sukharev by adjusting the lengths of the conductive members (directed to electrodes) of the first and second pairs of conductive members such that the cells pass through the first electrode field region for 10 µs and pass through the second electrode field region for 10 ms, in addition to modifying the electric field intensities to those disclosed by Sukharev, because it would have improved transfection efficiency. In doing so, then the ratio T1/T2 of the first period T1 (in this case, the entire duration of exposure to the first electric field region) to the second period T2 (in this case, the entire duration of exposure to the second electric field) is 1/1,000 which falls within the claimed range of ‘1/1,000 or more and 1 or less than 1,’ rendering obvious instant claim 3.
Regarding instant claim 4, Sukharev discloses a first pulse of 6 kV/cm and 10 µs, and a second pulse of 0.2 kV/cm and 10 ms (abstract). In performing claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev, it would have been obvious to obtain the electric pulses of Sukharev by modifying the first electric field intensity generated by the first pair of conductive members to 6 kV/cm and the second electric field intensity generated by the second pair of conductive members to 0.2 kV/cm, in addition to adjusting the lengths of the conductive members (directed to electrodes) of the first and second pairs of conductive members so that the cells pass through them at the pulse durations disclosed by Sukharev, because it would have improved transfection efficiency. In doing so, then the ratio E2/E1 of the second electric field intensity E2 to the first electric field intensity E1 is 1/30 which falls within the claimed range of ‘1/1,000 or more and less than 1.’ Thus, instant claim 4 is rendered obvious.
Regarding instant claims 5 and 6, the cooling region of the claims of `205 meets the claimed limitation of an ‘electric field-free region.’ Since the suspension passes through the cooling region after the suspension has passed through the first electric field region and before passing through the second field region in the claims of `205 (see claim 6 of `205), then the limitation of instant claim 5 is met. As such, claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev renders obvious instant claim 5; and claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev renders obvious instant claim 5. Further regarding instant claim 6, the recitations of a ‘first period T1’ and a ‘third period T0’ are broad. First, the claimed recitation ‘a first period T1 during which the suspension passes through the first electric field region’ is any period during which the suspension passes through the first electric field region. That is, it is a period of time less than or equal to the duration of the suspension passing through the first electric field region. Likewise, the claimed recitation ‘a third period T0 during which the suspension passes through the electric field-free region’ is any period during which the suspension passes through the electric field-free region. That is, the ‘third period’ is any period of time less than or equal to the duration of the suspension passing through the electric field-free region. Given the breadth of the claimed ‘first period’ and ‘third period,’ then it is obvious that there are periods within the duration of the suspension passing through the first electric field region of the claims of `205, T1, that are shorter than periods within the duration of the suspension passing through the cooling region (directed to the claimed ‘electric field-free region’), T0, including resulting in a ratio T1/T0 of 1/25,000 or more and less than 1. Thus, claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev renders obvious instant claim 6; and claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev renders obvious instant claim 6.
Further regarding instant claim 6, Sukharev discloses a first pulse of 6 kV/cm and 10 µs (abstract) and a delay between the two pulses of 100 µs (page 1321, left column, second paragraph). In performing claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev, it would have been obvious to obtain the electric pulse sequence of Sukharev by adjusting the lengths of the conductive members (directed to electrodes) of the first and second pairs of conductive members such that the cells pass through the first electrode field region for 10 µs, pass through the cooling region (directed to an electric field-free region) for 100 µs, and then pass through the second electrode field region for 10 ms, in addition to modifying the electric field intensities to those disclosed by Sukharev, because it would have improved transfection efficiency. In doing so, then the ratio T1/T0 of a first period T1 (in this case, the entire duration of exposure to the first electric field region) to a third period T0 during which the suspension passes through the electric field-free region (in this case, the entire duration of passing through the cooling region) is 1/10 which falls within the claimed range of ‘1/25,000 or more and 1 or less than 1,’ rendering obvious instant claim 6.
Regarding instant claim 9, claims 10 and 11 of `205 each recite a plurality of electric field regions including the first electric field region and the second electric field region. It would have been obvious to the skilled artisan that the plurality of electric field regions encompasses electric field regions other the first electric field region and the second electric field region. Thus each of claims 10 and 11 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev renders obvious instant claim 9.
Regarding instant claim 20, Sukharev discloses a first pulse of 6 kV/cm and 10 µs, and a second pulse of 0.2 kV/cm and 10 ms (abstract). Using those two pulses, Sukharev also discloses testing the dependence of transfection efficiency on the time delay between two pulses (page 1323, left column, third paragraph; Figure 6). Even at 100-s interval between pulses, the transfection efficiency (TE) level is twice as high as that obtained by application of the first pulse alone. Figure 6 shows that for the delay that is between 0.01 s (i.e. 10 ms) and 1 s, which appears to be approximately 0.1 s (i.e. 100 ms), the error bar for transfection efficiency overlaps with the error bar for the optimum transfection efficiency obtained for 0.0001 s (i.e. 100 µs, 0.1 ms). In performing claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev, it would have been obvious to obtain the electric pulse sequence of Sukharev by adjusting the lengths of the conductive members (directed to electrodes) and the cooling region between the first pair of conductive members and the second pair of conductive members such that the cells pass through the first electrode field region for 10 µs, pass through the cooling region for 100 ms, and then pass through the second electrode field region for 10 ms, in addition to modifying the electric field intensities to those disclosed by Sukharev, because it would have improved transfection efficiency. Based on Dzekunov, the skilled artisan would have expected that the lengths of the conductive members of each of the electric field regions and the length of the cooling region correlate with the exposure times to electric pulses and the delay time between the two pulses, respectively. Given that the duration through the cooling region (100 ms) is greater than the duration through the first electrode field region (10 µs) and the duration through the second electrode field region (10 ms), then the length of the first pair of conductive members (electrodes) in the flow direction and the length of the second pair of conductive members (electrodes) in the flow direction are shorter than the distance between the first pair of conductive members and the second pair of conductive members. Therefore, instant claim 20 is rendered obvious.
This is a provisional nonstatutory double patenting rejection.
Claim 7 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 3 and 6-13 of copending Application No. 18/489,205 in view of Dzekunov, Sukharev, and Cooper (US 2011/0104128. Previously cited).
As discussed above, claims 6-13 of `205 in view of Dzekunov and Sukharev render obvious claims 1-6, 9, 10, and 20. In particular, claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10. Also, claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10.
The claims of `205 in view of Dzekunov and Sukharev differ from instant claim 7 in that the claims of `205 do not recite that a suspension containing the bioactive substance (DNA) and the human derived cell (directed to the claimed ‘organism-derived material’) before the introduction of the bioactive substance flow through flow channels different from each other, are mixed at a combining point of the respective flow channels, and then pass through the first electric field region and the second electric field region.
Cooper discloses devices and methods for transfection of living cells using electroporation, in particular high throughput microfluidic electroporation (abstract). For instance, Figure 6 shows a side view of a cell traveling through multiple electric fields to improve transfection efficiency (paragraph [0090]). In one embodiment, the cells can be mixed with the DNA and/or RNA prior to subjecting the cells to electroporation (paragraph [0128]). Also, an example of the invention of Cooper is shown in Figure 16 in which cells from a patient are delivered into inlet 110, and a stream of an RNA species is delivered into inlet 120 (paragraph [0107]). Thus the cells are mixed with the RNA prior to their entry into the microfluidic electroporation units (MEU) (paragraph [0107]).
For the invention of claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev and the invention of claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev, it would have been obvious to the person of ordinary skill in the art to flow a suspension comprising the bioactive substance (DNA) through a flow channel and also to flow a suspension comprising the human derived cell through another flow channel such that these two flow channels connect with each other prior to delivering them into the flow passage of the claimed invention of `205. One of ordinary skill in the art would have been motivated to do this in order to mix the human derived cell with the DNA to thereby obtain a suspension comprising the human derived cell and DNA that flows through the flow passage of the method of the claims of `205 in view of Dzekunov and Sukharev. There would have been a reasonable expectation of introducing the bioactive substance (the DNA) into the human derived cell by the modification of the method rendered obvious by the claims of `205 in view of Dzekunov and Sukharev because Cooper indicates that transfection of cells can be performed by mixing the cells with DNA prior to subjecting the cells to electroporation, and because Cooper points to converging separate streams of cells and nucleic acid to mix them together prior to their entry into regions of electroporation (which the electric field regions of the claims of `205 are directed to). Thus, claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, Sukharev and Cooper renders obvious instant claim 7. Also, claim 12 of `205 in view of claim 3 of `205, Dzekunov, Sukharev, and Cooper renders obvious instant claim 7.
This is a provisional nonstatutory double patenting rejection.
Claim 8 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 3 and 6-13 of copending Application No. 18/489,205 in view of Dzekunov, Sukharev, Mastwijk (US 6,178,880. Previously cited), and Palsson (US 5,534,423. Previously cited), and in light of Pearson (US 4,821,564. Previously cited).
As discussed above, claims 3 6-13 of `205 in view of Dzekunov and Sukharev render obvious claims 1-6, 9, 10, and 20. In particular, claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10. Also, claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10.
The claims of `205 in view of Dzekunov and Sukharev differ from instant claim 8 in that they do not recite that the area of a cross section of the flow passage (directed to the claimed ‘flow channel’) orthogonal to a flow direction of the suspension is denoted by S in unit of m2, a circumference length of the cross section of the flow channel is denoted by C in unit of m, and an average speed at which the suspension passes through the first electric field region and the second electric field region is denoted by u in unit of m/s, a shear rate D in unit of s-1 is 1 s-1 or more and 5,000 s-1 or less and is defined by the following equation: D = 2u × C/2.
Mastwijk discloses a system for treating pumpable products by electrical pulses comprising a flow channel through which product can be pumped (abstract). The invention is suitable for invoking pores in membranes of cellular structures to promote the transport of macromolecular components across the membrane (column 1, lines 12-15). As shown in Figure 1, the channel has a cylindrical cross section (column 4, lines 12-14). However, different cross-sectional shapes give similar results (column 4, lines 14-15). An increasing or decreasing electric field strength across the treatment chamber can be obtained (column 4, lines 55-57). An example of the invention of Mastwijk is shown in Figures 3-4 which provides a treatment chamber having a cylindrical cross-section and comprises three annular electrodes spaced apart along the treatment chamber through which the product flows (column 5, lines 8-19).
It would have been obvious to the person of ordinary skill in the art to substitute the flow passage with a cylindrical channel when performing the method rendered obvious by the claims of `205 in view of Dzekunov and Sukharev for the predictable result of introducing the DNA (the bioactive substance) into the human derived cell (the biologically derived material, directed to the claimed ‘organism-derived material’). It would have been a matter of simple substitution of one shape of flow passage for another. There would have been a reasonable expectation of performing the method rendered obvious by the claims of `205 in view of Dzekunov and Sukharev with a flow passage that is cylindrical because Mastwijk teaches that a cylindrical channel is suitable as a flow channel through which a pumpable product is treated with electrical pulses along the channel that may be used for invoking pores in membranes of cellular structures to transport macromolecular components across the membrane, and because Mastwijk teaches that different cross-sectional shapes give similar results. Since the cross section of a cylindrical channel is a circle, then the cross section of a cylindrical flow passage has a circumference length, i.e., C which inherently can be in any unit of length, including meters. Thus the claims of `205 in view of Dzekunov, Sukharev, and Mastwijk render obvious limitations of instant claim 8.
Additionally, as evidenced by Pearson, shear rate for laminar flow in a pipe is determined by the following equation, wherein D = pipe diameter and V = velocity (column 4, lines 54-64):
γ
=
8
V
/
D
For the cylindrical flow passage of the claims of `205 in view of Dzekunov, Sukharev, and Mastwijk, then the shear rate is calculated below in terms of r = radius of the cylindrical electroporation channel:
γ
=
8
V
/
(
2
r) = 4V/r
For a cylinder, it is well known in the art that the area of a circular cross section is πr2, and the circumference length of said circular cross section is 2πr. Therefore, the shear rate equation of instant claim 8 for a cylindrical flow channel converts to the following:
S
h
e
a
r
r
a
t
e
=
2
u
×
C
S
=
2
u
×
2
π
r
π
r
2
=
4
u
/
r
Since the equation for shear rate for the cylindrical flow passage of the method rendered obvious by the claims of `205 in view of Dzekunov, Sukharev, and Mastwijk, as evidenced by Pearson, is 4V/r (i.e. 4u/r) which is the same as the shear rate equation of instant claim 8 for a cylindrical channel, then the shear rate of the flow in the cylindrical electroporation channel of the method rendered obvious by the claims of `205 in view of Dzekunov, Sukharev, and Mastwijk (in light of Pearson) meets the equation for shear rate D in instant claim 8. It is also well known in the art that the SI units for area, length, and velocity (i.e. speed) are m2, m, and m/s, respectively.
Further regarding the claimed shear rate, Palsson discusses genetic engineering and discloses a method of transfecting target cells in which vectors such as nucleic acids are introduced into the target cells (column 1, lines 7-9; claims 1 and 13 of Palsson). The target cells include human cells (in particular, hematopoietic stem cells of human origin) (column 4, line 64 through column 5, line 4). Palsson points out that shear sensitivity of different types of cell varies greatly (column 6, lines 59-60). Thus, the flow velocity of a fluid approaching cells has to be such that the target cells are not damaged (column 6, lines 60-62). In Palsson’s invention, the shear rate experienced by the target cells is expected to be below about 200 per second (column 6, lines 62-64).
It would have been an obvious matter of routine optimization to adjust the flow velocity of the suspension through the cylindrical flow passage such that the shear rate is low such as 200 s-1 (falling in the claimed range) or in the range of 1 s-1 or more and 5,000 s-1 or less, when performing the method rendered obvious by the claims of `205 in view of Dzekunov, Sukharev, and Mastwijk (in light of Pearson). One of ordinary skill in the art would have been motivated to do this because flow velocity affects shear rate, which in turn affects different types of cells differently since shear sensitivity varies for different types of cells. Moreover, a shear rate of 200 s-1 was found suitable for target cells that include human cells in Palsson, which would have motivated the skilled artisan to adjust the flow velocity to obtain that shear rate.
As such, claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, Sukharev, Mastwijk, and Palsson (in light of Pearson) renders obvious instant claim 8. Also, claim 12 of `205 in view of claim 3 of `205, Dzekunov, Sukharev, Mastwijk, and Palsson (in light of Pearson) renders obvious instant claim 8.
This is a provisional nonstatutory double patenting rejection.
Claims 11 and 12 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 3 and 6-13 of copending Application No. 18/489,205 in view of Dzekunov, Sukharev, and Witting (Human Gene Therapy. 2012. 23: 243-249. Previously cited).
As discussed above, claims 3 and 6-13 of `205 in view of Dzekunov and Sukharev render obvious claims 1-6, 9, 10, and 20. In particular, claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10. Also, claim 12 of `205 in view of claim 3 of `205, Dzekunov, and Sukharev renders obvious instant claims 1 and 10.
The claims of `205 in view of Dzekunov and Sukharev differ from instant claim 11 in that they do not recite a step of culturing the biologically derived material produced by their method (directed to the claimed ‘organism-derived material produced by the producing method according to claim 1’), and a step of extracting a product that is produced by the cells (directed to the claimed ‘the organism-derived material’). The claims of `205 in view of Dzekunov and Sukharev further differ from instant claim 12 in that they do not recite that the product (that is produced and extracted) is a virus.
Witting found that flow electroporation (EP) was highly suited for large-scale production of lentivirus, specifically lentiviral productions for early phase clinical gene therapy trials with potential for further scale-up (page 244, left column, second paragraph). In particular, Witting discloses suspending HEK293 FT cells in EP buffer, and adding the suspension of HEK293 FT cells and DNA (four plasmids) to a device to perform flow electroporation (page 244, left column, last full paragraph through right column, first paragraph for disclosing the cells and the plasmids; page 244, right column, third and last paragraphs for disclosing lentivirus production using EP, in particular flow EP). HEK293 FT cells are directed to human-derived cells. After the electroporation, the cells were incubated with DNase and then cultured in a bioreactor (page 244, right column, last paragraph). Afterwards, aliquots of virus-containing media were harvested, filtered, and stored (page 245, first paragraph).
It would have been obvious to the person of ordinary skill in the art to have applied the invention of the claims of `205 in view of Dzekunov and Sukharev to lentiviral production as disclosed by Witting by substituting the human derived cells with HEK293 FT cells and substituting the DNA with the four plasmids taught by Witting in order to transfect HEK293 FT cells with said plasmids by the method rendered obvious by the claims of `205 in view of Dzekunov and Sukharev, culture the transfected cells according to Witting, and obtain the lentivirus by harvesting and filtering the virus-containing media resulting from the culture. One of ordinary skill in the art would have been motivated to do this in order to produce lentivirus, a desirable product, which is suitable for early phase clinical gene therapy trials. There would have been a reasonable expectation of producing lentivirus by this modification of the invention of the claims of `205 in view of Dzekunov and Sukharev because Witting demonstrated through their study that flow electroporation (which the invention of the claims of `205 is directed to) is suitable for lentivirus production. As such, the claims of `205 in view of Dzekunov, Sukharev, and Witting render obvious a producing method for a product (lentivirus, directed to ‘virus’ of instant claim 12) comprising a step of culturing the HEK293 FT cells (directed to ‘organism-derived material’ which is a human-derived cell) produced by the claims of `205 in view of Dzekunov and Sukharev (rendering obvious ‘the producing method according to claim 1’); and a step of extracting the product (by harvesting and filtering the virus-containing media) that is produced by the resulting HEK293 FT cells (claimed ‘organism-derived material’). Thus, claim 6 of `205 in view of claims 3 and 12 of `205, Dzekunov, Sukharev, and Witting renders obvious instant claims 11 and 12. Also, claim 12 of `205 in view of claim 3 of `205, Dzekunov, Sukharev, and Witting renders obvious instant claims 11 and 12.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments
Applicant’s arguments, filed September 30, 2025, with respect to the rejection under 35 U.S.C. 112(b) of claim 8, the rejection under 35 U.S.C. 103 of claims 1-6, 9, and 10 as being unpatentable over Zhu in view of Muller-Hartmann, the rejection under 35 U.S.C. 103 of claim 7 as being unpatentable over Zhu and Muller-Hartmann in further view of Cooper, the rejection under 35 U.S.C. 103 of claim 8 as being unpatentable over Zhu and Muller-Hartmann in further view of Mastwijk and Palsson and in light of Pearson, the rejection under 35 U.S.C. 103 of claims 11 and 12 as being unpatentable over Zhu and Muller-Hartmann in further view of Witting, the provisional nonstatutory double patenting rejection of claims 1-6, 9, and 10 as being unpatentable over claims 6-13 of copending Application No. 18/489,205 in view of Hayakawa, the provisional nonstatutory double patenting rejection of claim 7 as being unpatentable over claims 6-13 of Application No. 18/489,205 in view of Hayakawa and Cooper, the provisional nonstatutory double patenting rejection of claim 8 as being unpatentable over claims 6-13 of Application No. 18/489,205 in view of Hayakawa, Mastwijk, and Palsson, and in light of Pearson, and the provisional nonstatutory double patenting rejection of claims 11 and 12 as being unpatentable over claims 6-13 of Application No. 18/489,205 in view of Hayakawa and Witting, have been fully considered and are persuasive.
In particular, the rejection under 35 U.S.C. 112(b) has been overcome by the amendment to claim 8. The rejections under 35 U.S.C. 103 have been overcome by the amendment to claim 1 since Zhu does not disclose that their first electric field region is generated by a first pair of electrodes arranged opposite each other on opposing walls of the flow channel, and their second electric field region is generated by a second pair of electrodes arranges opposite each other on opposing walls of the flow channel. The provisional nonstatutory double patenting rejections have been overcome by the amendment to claim 1 since the grounds of rejection do not address the new limitations added to claim 1.
Therefore, these rejections have been withdrawn. However, upon further consideration, new grounds of rejection are made in view of the amendments to the claims which necessitated new rejections under 35 U.S.C. 103 over the previously cited secondary reference Cooper in view of the newly cited references Dzekunov and Sukharev, modification of the rejection under 35 U.S.C. 112(b), and modification of the provisional nonstatutory double patenting rejections over the claims of Application No. 18/489,205 in view of the newly cited references Dzekunov and Sukharev.
To the extent Applicant’s arguments are directed to the new ground of nonstatutory double patenting rejection over the claims of Application No. 18/489,205, they are unpersuasive. In particular, Applicant asserts that 18/489,205 does not provide any teaching or suggestion of the claimed limitation of the first and second electric field regions being generated by pairs of electrodes arranged opposite each other on opposing walls of the flow channel. Applicant asserts that instead, 18/489,205 discloses electrodes disposed on a single wall surface of the channel, such as printed electrodes on a substrate. However, claim 3 of 18/489,205 recites pairs of conductive members that are directed to the first and second pairs of electrodes of the instant claims. See the new double patenting rejections over the claims of 18/489,205. As set forth in the new ground of rejection, it would have been prima facie obvious to apply that limitation to the method of claims 6-13 of 18/489,205.
Conclusion
No claims are 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.
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Sef
/SUSAN E. FERNANDEZ/ Examiner, Art Unit 1651
/DAVID W BERKE-SCHLESSEL/ Primary Examiner, Art Unit 1651