Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Detailed Action
This action is in response to the papers filed March 2, 2026.
Claim Amendments
Applicant's amendment to the claims filed 03/02/2026 is acknowledged.
Claims 1-21, 30-73, 84-85, and 102 are cancelled.
Claims 22-29, 74-83, 86-101, 103-106 are pending and under examination.
Priority
The instant application 15/780,542 was filed on 05/31/2018. This application is a national stage of international application PCT/US2016/064414 filed 12/01/2016, claiming priority based on U.S. Provisional Patent Application No. 62/261,815 filed 12/01/2015.
Withdrawal of Prior Claim Rejections/Objections
Rejections and/or objections not reiterated from the previous Office action mailed 08/28/2025 are hereby withdrawn. The following rejections and/or objections are either newly applied or are reiterated and are the only rejections presently applied to the instant application.
Regarding the previous rejection under 35 U.S.C. 112(a), for failing to comply with the written description requirement, the rejection has been withdrawn in view of applicant’s amendment to the claims and arguments filed on 03/02/2026, and in accordance with guidance provided by MPEP 2163.05(III).
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 106 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.
This rejection is newly applied, necessitated by amendment.
Claim 106 recites a step (e) of producing a “plasmid/PEI HEK cell culture” mixture, and then a step (f) of “adding a Free PEI solution to said plasmid/PEI HEK cell culture produced in step (e) at the same time as ... step (e).” In remarks filed 03/02/2026, applicant argues that the claim now clarifies “the addition of a free PEI solution that is distinct from the plasmid/PEI solution previously mixed with the cells” and “the addition of a separate solution beyond that mixture.” See, pg. 15 of applicant’s reply.
Accordingly, claim 106 recites, in step (f), that a separate and distinct solution of Free PEI is added to the “plasmid/PEI HEK cell culture” mixture produced in the previous step (e) at the same time that said “plasmid/PEI HEK cell culture” mixture is produced in step (e). The recitation is found to be indefinite, for the following reasons:
In this case, a separate and distinct solution of Free PEI cannot be added to a “plasmid/PEI HEK cell culture” mixture at the same time that said “plasmid/PEI HEK cell culture” mixture is formed, as claimed. Rather, the solution of Free PEI can only be added to the “plasmid/PEI HEK cell culture” mixture after said “plasmid/PEI HEK cell culture” mixture is formed, i.e., after step (e), because, otherwise, there is no existing “plasmid/PEI HEK cell culture” mixture to which a separate and distinct solution of Free PEI may be added. Therefore, the recitation does not appear to be logically sound. How can one of ordinary skill in the art add a solution of Free PEI to a “plasmid/PEI HEK cell culture” mixture at the same time that said “plasmid/PEI HEK cell culture” mixture is formed? Logically, the “plasmid/PEI HEK cell culture” mixture must be formed first before any solution of Free PEI may be added to it.
For these reasons, one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
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.
Claims 22-29, 74-83, 86-92, 94-101, 103-106 are rejected under 35 U.S.C. 103 as being unpatentable over Park et al. (2006) “Scalable production of adeno‐associated virus type 2 vectors via suspension transfection” Biotechnology and bioengineering, 94(3), 416-430; in view of Yue et al. “Revisit complexation between DNA and polyethylenimine — Effect of uncomplexed chains free in the solution mixture on gene transfection”, Journal of Controlled Release 155 (2011) 67–76; and as evidenced by Shin et al. (2011) “Recombinant adeno-associated viral vector production and purification” In Joseph X. DiMario (Eds.), Myogenesis: Methods and Protocols, Chapter 15. pp. 267-284, Totowa, NJ: Humana Press.
This rejection is newly applied, necessitated by amendment.
With respect to claims 22-29, and 106, Park discloses a method for producing transfected cells that produce recombinant adeno-associated virus (AAV) vector comprises a nucleic acid that encodes for a protein or is transcribed into a transcript of interest. See, e.g., Abstract. Park discloses providing a transfection mixture comprising
(a) one or more plasmids comprising nucleic acids encoding AAV packaging proteins and nucleic acids encoding helper proteins (see, e.g., pg. 417, “rAAV was produced by the dual transfection method employing one of the vector plasmids (pAAV-GFP, pAAV-RFP, pTR-UF6, or pTR-RFP) and the helper plasmid pDG. To evaluate rAAV yield in a different transfection system, pAAV-hrGFP and pTR-UF6 were used as vector plasmids for the dual and triple transfection systems. The helper plasmid pDG was used for the dual transfection method and helper plasmids pAAV-RC and pHelper for the triple transfection method.”);
(b) a plasmid comprising a nucleic acid that encodes a protein of interest or is transcribed into a transcript of interest (see, e.g., pg. 417, “pAAV-hrGFP encodes the humanized recombinant green fluorescent protein (hrGFP or simply GFP) derived from Renilla reniforms, and pTR-UF6 a blue fluorescent protein (BFP) (Zolotukhin et al., 1999). The RFP gene, encoding a red fluorescent protein (RFP) isolated from a sea anemone-relative Discosoma sp., ...”); and
(c) a polyethylenimine (PEI) solution (see, e.g., pg. 418, “Linear PEI used for transfection was obtained from Polysciences (MW 25,000; Warrington, PA), and PEI-mediated transfection was carried out following a procedure ...”),
wherein said plasmids are in a molar ratio range of 1:1 (pg. 418, “Six micrograms of plasmids containing equimolar amounts of the vector and the helper plasmids were diluted in 150 mL of 150 mM sodium chloride solution and mixed with 20 μL of 10 μM PEI solution (Polysciences, ratio of PEI nitrogens to DNA phosphate [N/P]=6).”).
Park further discloses contacting cells with the transfection mixture and incubating for 6 hours to produce transfected cells. See, e.g., pg. 418, “The DNA-PEI complex was mixed with the cell suspension and this mixture was distributed into each well. ... The culture was maintained at 37°C in a humidified 5% CO2 atmosphere for 6 h unless otherwise specified. After transfection, the culture was replaced ...”
Park further discloses harvesting the transfected cells and medium, followed by isolating and purifying the AAV vector from the harvested cells and medium. See, e.g., pg. 419, “Two or 3 days post-transfection, the culture was harvested and stored at -70°C until further purification was performed” and pg. 419, “After transfection, time-course samples were taken from the culture and cells were harvested ... Physical particles of rAAV, in the crude lysate or in the purified fractions, ...” and pg. 420, “Two-step column chromatography was performed on a Biologic Duo-Flow Workstation system (Bio-Rad) in order to purify rAAV from crude cell lysates.”)
Claim 106 recites that the cells are “HEK cells.” Park discloses that the cells are HEK293 cells. See, e.g., Abstract and pg. 417, “... human embryonic kidney 293 cells (HEK 293 ...”. Accordingly, Park teaches a HEK cell line, as claimed in claim 106.
Timing of introducing Free PEI to the plasmid/PEI cell culture:
Claims 22, 25-26, 28-29 recite a step of adding Free PEI to the plasmid/PEI cell culture produced in the previous step. Therefore, claims 22, 25-26, 28-29 require that the Free PEI is added to the cell culture after contacting the cells with the transfection mixture (plasmid/PEI mixture). Claim 106 differs from claims 22, 25-26, 28-29 by reciting that the Free PEI may be added “at the same time as, or after” the step of contacting the cells with the transfection mixture (plasmid/PEI mixture).
Park discloses that the molar ratio of nitrogen (N) in Total PEI to phosphate (P) in plasmid is 6:1 (N/P=6) in the transfection mixture (see, e.g., pg. 418, “20 μL of 10 μM PEI solution (Polysciences, ratio of PEI nitrogens to DNA phosphate [N/P]=6)”). One of ordinary skill in the art would have understood that free PEI chains (i.e., “Free PEI”) exists when N/P is greater than 3, as instructed by Yue (Yue, Abstract, “confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ~3, but the PEI/DNA polyplexes have a high in-vitro gene transfection efficiency only when N:P≥10.”; pages 70-71, joining paragraph, “… further confirming that DNA is fully condensed by PEI only when N:P≥3. For N:P=10, ~70% of the PEI chains are free in the solution mixture of PEI and DNA”). Thus, since Park discloses using an N/P=6 and Yue teaches that free PEI exists when N/P≥3, the transfection mixture taught by Park necessarily comprises “Free PEI”(i.e., free PEI chains). For these reasons, Park is found to teach that the Free PEI is added to the culture at the same time as the transfection mixture (plasmid/PEI mixture) is contacted with the cells, as claimed by claim 106.
However, Park does not teach a step of adding “Free PEI” to the culture after contacting the cells with the transfection mixture (plasmid/PEI mixture), as recited in the claims 22, 25-26, 28-29. That is, the difference between the Park disclosure and the method of claims 22, 25-26, 28-29 is that Park adds Free PEI to the cell culture at the same time as the plasmid/PEI mixture is contacted with the cells; whereas, the claims recite that the Free PEI is added to the cell culture after the plasmid/PEI mixture is contacted with the cells.
MPEP 2144.04 instructs that changes in the sequence of adding ingredients or order of performing process steps are prima facie obvious in the absence of new or unexpected results. Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.).
In addition, Yue is considered relevant prior art for teaching a method of transfecting cells (see Abstract) comprising the steps of:
contacting cells with a transfection mixture comprising PEI and plasmid (plasmid/PEI mixture);
adding free PEI to the plasmid/PEI cell culture to produce a free PEI/plasmid/PEI cell culture (page 71, last paragraph, “Furthermore, we decided to add the 7 portions of free PEI chains at different times; namely, hours before or after the administration of the PEI/DNA polyplexes (N:P=3), so that the final and total N:P ratio remains 10. We define t=0 for the simultaneous addition of the PEI/ DNA polyplexes (N:P=3) and 7 portions of free PEI chains”); and
incubating the free PEI/plasmid/PEI cell culture for at least 4 hours to produce transfected cells (Figure 6 on page 72; and page 72, first full paragraph, “Fig. 6 also reveals that in the delayed addition of free PEI chains, i.e., t>0 … incubation time was fixed to be 48 h, starting from the polyplex administration.”).
In particular, Yue teaches that the free PEI can be added to the plasmid/PEI cell culture before, at the same time as, or after the plasmid/PEI mixture is contacted with said cells. Figure 6 shows the effect on transfection efficiency of adding free PEI at different times is known and predictable:
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Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the AAV production method taught by Park, which adds free PEI at the same time as the cells are contacted with the plasmid/PEI mixture, by rather adding the free PEI after the cells are contacted with the plasmid/PEI mixture, as taught by Yue, with a reasonable expectation of success because Yue teaches that the free PEI can be added before, at the same time as, or after cells are contacted with the plasmid/PEI mixture, and the effect of transfection efficiency is known and predictable (Figure 6, reproduced above). One of ordinary skill in the art would have recognized that such a modification is a simple rearrangement of Park’s order of adding ingredients or performing process steps, which achieves known and predictable results as shown by Yue (e.g., Figure 6, reproduced above). A discussed above, changes in the sequence of adding ingredients or order of performing process steps are prima facie obvious in the absence of new or unexpected results, as instructed by MPEP 2144.04.
Please further note that the prior art recognizes that the addition of free PEI chains significantly enhances transfection efficiency (see Yue, Figure 13 on page 74; see also page 74, col. 1, “it is worth noting that this reduced transfection efficiency is still ~50-fold higher than that without the addition of free PEI chains (N:P=3), clearly indicating that the free PEI chains mainly play their role inside the cells.”).
The percent amount of Free PEI in Total PEI:
Claims 22, 25-26, 28-29, and 106 recite that the amount of Free PEI is about 33% to about 50% of Total PEI by weight.
"[W]hen, as by a recitation of ranges or otherwise, a claim covers several compositions, the claim is ‘anticipated’ if one of them is in the prior art." Titanium Metals Corp. v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985) (citing In re Petering, 301 F.2d 676, 682, 133 USPQ 275, 280 (CCPA 1962)) (emphasis in original) (Claims to titanium (Ti) alloy with 0.6-0.9% nickel (Ni) and 0.2-0.4% molybdenum (Mo) were held anticipated by a graph in a Russian article on Ti-Mo-Ni alloys because the graph contained an actual data point corresponding to a Ti alloy containing 0.25% Mo and 0.75% Ni and this composition was within the claimed range of compositions.). "If the prior art discloses a point within the claimed range, the prior art anticipates the claim." UCB, Inc. v. Actavis Labs. UT, Inc., 65 F.4th 679, 687, 2023 USPQ2d 448 (Fed. Cir. 2023). See MPEP 2131.03.
In this case, for AAV production, Park discloses using an N/P=6 (pg. 418). Since free PEI exists when N/P is greater than 3, the transfection mixture of Park comprises 6 parts total PEI, wherein 3 parts are DNA/PEI polyplexes and 3 parts are free PEI. Therefore, N/P ratio of 6 is equivalent to 3 parts free PEI in 6 total PEI, which results in (3 parts)/(6 parts) = 50% free PEI to total PEI by weight. Accordingly, Park teaches 50% free PEI to Total PEI by weight, as recited by the ranges of claims 22, 25-26, 28-29, and 106, for AAV production.
Yue teaches that N/P = 10, wherein 7 parts are free PEI (e.g., Figure 6). Therefore, the amount of free PEI is 7/10 = 70% total PEI by weight. Yue further reports performing cell transfection using N/P ratios ranging from 3 to 10 (fig. 3d). N/P ratios ranging from 3 to 10 is equivalent to an amount of free PEI ranging from 0% (0/3) to 70% (7/10) total PEI, which overlaps with the range of about 33-50% free PEI to total PEI.
The courts have held that "where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). In this case, Park teaches 50% free PEI to Total PEI by weight, which lies within the ranges of claims 22, 25-26, 28-29, and 106, for AAV production, and Yue teaches 0%-70% free PEI to Total PEI by weight, which overlaps with the ranges of claims 22, 25-26, 28-29, and 106.
For these reasons, claims 22-29, and 106 would have been prima facie obvious over the prior art.
Regarding dependent claims 74 and 79, Park discloses, in one example, that six micrograms (6 μg) of plasmids containing equimolar amounts of the vector and the helper plasmids were mixed with 20 μL of 10 μM PEI, and the PEI had a molecular weight of 25,000 g/mol (MW 25,000). See page 417, left column. Accordingly, the weight of PEI was (25000g/mol)(10μmol/L)(20μL)(1mol/106μmol) (1L/106μL)( 106μg/1g)=5μg. Therefore, the weight ratio of PEI to plasmid was 5μg to 6μg, i.e., a weight ratio of 5:6 or 0.83:1.
Regarding dependent claims 75 and 78, Park further discloses that the cells are in contact with the transfection mixture for 6 hours (see, e.g., pg. 418, “The DNA-PEI complex was mixed with the cell suspension and this mixture was distributed into each well. ... The culture was maintained at 37°C in a humidified 5% CO2 atmosphere for 6 h unless otherwise specified.”).
Regarding dependent claim 76, Park discloses, in one example, that transfection is performed in a six-well culture plate. See page 418, left column.
Regarding dependent claims 77 and 90, Park discloses using the helper plasmid pDG or the helper plasmids pAAV-RC and pHelper (pg. 417, right column). These plasmids encode AAV capsid proteins (cap genes) and rep genes for packaging. For example, Park also detected AAV2 capsid protein using antibodies (pg. 420). As further evidence, see page 278 of Shin: “Besides adenoviral helper genes, pDG also carries AAV-2 rep and cap genes.” See also pg. 416 of Park, disclosing “the most widely used strategy to generate rAAV is transfecting cells with plasmids encoding a gene of interest, the rep-cap sequences, and the minimal adenoviral genes required for the helper effect.”
Regarding dependent claim 80, Park discloses that the PEI is linear PEI. See pg. 418, left column. The recitation that the linear PEI was “hydrolyzed” is directed to a product-by-process limitation. "[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985). See MPEP 2113. In this case, the recitation indicates that the linear PEI was synthesized by a hydrolysis reaction but does not positively limit the structure of the linear PEI itself. For these reasons, the hydrolyzed linear PEI of claim 80 is found to be the same or obvious from the linear PEI taught by Park.
Regarding dependent claim 81, Park discloses that the linear PEI has a molecular weight of 25000 Da (or 25000 g/mol). See pg. 418, left column.
Regarding dependent claim 82, Park discloses that the molar ratio of nitrogen (N) in Total PEI to phosphate (P) in plasmid is 6:1 (N:P) in the transfection mixture (see, e.g., pg. 418, “20 μL of 10 μM PEI solution (Polysciences, ratio of PEI nitrogens to DNA phosphate [N/P]=6)”).
Regarding dependent claim 83, the claim recites that the plasmid/PEI mixture of step (d) is incubated for a period of time in the range of about 30 seconds to about 4 hours. Park discloses the plasmid/PEI mixture was incubated for 10 minutes (min) before transfection (pg. 418, right column).
Regarding dependent claim 86, Park discloses that the cells are in suspension culture. See, e.g., Abstract; pg. 417, left column; and pg. 418.
Regarding dependent claim 87, Park teaches that the cell culture media contains 5% fetal bovine serum. See page pg. 417, left column. Park does not teach serum-free media for cell culture, as claimed in claim 87.
However, Yue teaches that the cells are grown and maintained in serum-free culture medium before and after contact with the transfection mixture. Serum-free media improved transfection efficiency relative to media containing serum (10% FBS). See Figure 14; see page 74, col. 1-2, joining paragraph.
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the AAV production method of Park by using serum-free media conditions, as taught by Yue, with a reasonable expectation of success because Yue teaches serum-free media improved transfection efficiency relative to media containing serum (fig. 14; pg. 74, col. 1-2, joining paragraph).
Regarding dependent claims 88 and 103, the claims recite that the cells are at a density in the range of about 1 x 105 to 1 x 108 cells/mL (claim 88) or about 0.1 x 106 to 5.0 x 106 cells/ml (claim 103) prior to contact with the transfection mixture. Park discloses that transfection was carried out when the cell density reached 5-6 x 105 cells/mL, which lies inside the ranges of claims 88 and 103.
MPEP 2144.05 instructs that differences in concentration will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration is critical. "[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). In this case, Park discloses that transfection was carried out when the cell density reached 5-6 x 105 cells/mL, which lies inside the ranges of claims 88 and 103. In addition, the cell density is a result-effective variable because the production of AAV particles in a cell culture is expected to change with the number of cells modified via transfection to produce said AAV particles, and therefore one of ordinary skill in the art would have been led to optimize the cell density by routine experimentation. For these reasons, as those outlined above, the limitations of claims 88 and 103 would have been prima facie obvious over the prior art.
Regarding dependent claim 89, the claim recites that the viability of the cells is about 60% or greater when contacted with the transfection mixture.
Park does not report on cell viability. Yue reports that, after transfection, cell viability was well above 70% (page 71, col. 2, “It is also worth noting that in the general N:P range for transfection, i.e., N:P≤10, both the PEI/DNA dispersion and free PEI chains exhibit relatively low cytotoxicity, with the cell viability well above 70%”). Accordingly, cell viability was at least above 70% prior to transfection in Yue, which lies within the range recited by claim 89.
"[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). In this case, cell viability was at least above 70% prior to transfection in Yue, which lies within the range recited by claim 89. In addition, the cell viability is a result-effective variable because the cell culture viability represents the number of cells permissible to transfection and production of said AAV particles, and therefore one of ordinary skill in the art would have been led to optimize the cell density by routine experimentation. For these reasons, as those outlined above, the limitations of claim 89 would have been prima facie obvious over the prior art.
Regarding dependent claim 91, Park discloses using the helper plasmid pDG or the helper plasmids pAAV-RC and pHelper (pg. 417, right column). These plasmids encode adenovirus E2 or E4 or VA RNA for helper proteins or helper function. As further evidence, see page 278 of Shin: “We have used the Stratagene pHelper plasmid, which expresses adenoviral E2a, E4, and VA RNA (33). ... Besides adenoviral helper genes, pDG also carries AAV-2 rep and cap genes (35). ... The AAV-2 helper plasmid (pAAV-RC) can be purchased from Stratagene.” See also pg. 416 of Park, disclosing “the most widely used strategy to generate rAAV is transfecting cells with plasmids encoding a gene of interest, the rep-cap sequences, and the minimal adenoviral genes required for the helper effect.”
Regarding dependent claim 92, Park discloses that the cells are HEK293 cells. See, e.g., Abstract and pg. 417, “... human embryonic kidney 293 cells (HEK 293 ...”. Accordingly, Park teaches a mammalian cells, as claimed in claim 92.
Regarding dependent claim 94, Park discloses wherein the total amount of plasmid comprising the nucleic acid that encodes a protein of interest and plasmids comprising nucleic acids encoding AAV packaging proteins and nucleic acids encoding helper proteins is 6.7 mg in 2 L of cell culture (pg. 418, right column), which is equivalent to 3.35 µg per mL of cells.
Regarding dependent claim 95, Park discloses the amounts of the plasmids were “equimolar” (pg. 418), which means a ratio of 1:1.
Regarding dependent claim 96, Park discloses plasmids (pDG, pAAV-RC, pHelper) comprising a first plasmid comprising the nucleic acids encoding AAV packaging proteins and a second plasmid comprising the nucleic acids encoding helper proteins or helper function. As further evidence, see page 278 of Shin: “We have used the Stratagene pHelper plasmid, which expresses adenoviral E2a, E4, and VA RNA (33). ... Besides adenoviral helper genes, pDG also carries AAV-2 rep and cap genes (35). ... The AAV-2 helper plasmid (pAAV-RC) can be purchased from Stratagene.” See also pg. 416 of Park, disclosing “the most widely used strategy to generate rAAV is transfecting cells with plasmids encoding a gene of interest, the rep-cap sequences, and the minimal adenoviral genes required for the helper effect.”
Regarding dependent claim 97, Park discloses that the amounts of the plasmid comprising the nucleic acid that encodes a protein of interest to the first plasmid comprising the nucleic acids encoding AAV packaging proteins to the second plasmid comprising the nucleic acids encoding helper proteins were “equimolar” (pg. 418), which means a ratio of 1:1:1.
Regarding dependent claims 98-99, Park discloses an AAV vector, which comprises at least an AAV2 serotype and an expression control element, e.g., CMV promoter. See, e.g., Abstract; pg. 417, right column. Moreover, one of ordinary skill in the art would have understood that AAV vectors contain ITR sequences flanking the genome. As further evidence, see pg. 267 of Shin: “At the ends of the AAV genome are the inverted terminal repeats (ITRs).”
Regarding dependent claims 100-101, Park teaches wherein the AAV vector comprises a CMV promoter (pg. 417, right column), which is a constitutive promoter as claimed in claim 100. Since constitutive promoters are unregulated and not tissue-specific, the CMV promoter would be expressed in the liver, as claimed in claim 101.
Regarding dependent claim 104-105, the claims recite subculturing the cells for 2-5 days (claim 104) or 3-4 days (claim 105) prior to transfection.
Park discloses performing transfection 1 or 2 days following inoculation in 2 L culture (pg. 418, right column), and 2 days fall inside the range 2-5 days as claimed in claim 104.
1 or 2 days, as taught by Park, is outside the claimed range of 3-4 days recited by claim 105. A prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985). "[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). In this case, two days of subculturing, as taught by Park, is close to 3-4 days of subculturing, as claimed in claim 105. Moreover, Park discloses that the inoculum was prepared via cell culture of undisclosed duration (pg. 418, “the inoculum was prepared in 3–7 L spinner-flask cultures”), which suggests a longer subculturing time to scale up to 2 L culture. The subculturing time is a result-effective variable because the cells require time to multiply and reach a desired cell density for transfection, and therefore one of ordinary skill in the art would have been led to optimize the cell density by routine experimentation. For these reasons, absent a secondary consideration, 3-4 days of subculturing prior to transfection, as claimed in claim 105, would have been prima facie obvious over the prior art.
Claim 93 is rejected under 35 U.S.C. 103 as being unpatentable over Park et al. (2006) “Scalable production of adeno‐associated virus type 2 vectors via suspension transfection” Biotechnology and bioengineering, 94(3), 416-430; Yue et al. “Revisit complexation between DNA and polyethylenimine — Effect of uncomplexed chains free in the solution mixture on gene transfection”, Journal of Controlled Release 155 (2011) 67–76; and Shin et al. (2011) “Recombinant adeno-associated viral vector production and purification” In Joseph X. DiMario (Eds.), Myogenesis: Methods and Protocols, Chapter 15. pp. 267-284, Totowa, NJ: Humana Press. [evidentiary reference], as applied to claims 22-29, 74-83, 86-92, 94-101, 103-106 above; in further view of Durocher et al. “Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells”, Journal of Virological Methods 144 (2007) 32–40.
This rejection is newly applied, necessitated by amendment.
The Durocher disclosure:
Durocher is relevant prior art for teaching a method for producing transfected cells that produce recombinant AAV vector comprising a nucleic acid that encodes a protein, comprising:
providing plasmids comprising nucleic acids encoding AAV packaging proteins (section 2.2, “The pAAV-ACG2 vector contains AAV2 rep and cap genes”) and nucleic acids encoding helper proteins (section 2.2, “The pHelper vector (Stratagene, Cedar Creek, TX) contains the helper functions of adenovirus (E2A, E4, VA RNA)”);
providing a plasmid comprising a nucleic acid that encodes a protein of interest (section 2.2, “The pAAV-GFP vector was obtained following the insertion of the cDNA encoding the Green Fluorescent Protein (GFP) into the pAAV-MCS vector”);
providing a solution comprising polyethylenimine (PEI) solution (section 2.3, “The transfection mix was done in 0.1 mL of LC-SFM/10mM Hepes using 1 µg of DNA and 2 µg of PEI (25 kDa, linear) per 0.5×106 cells”);
mixing the plasmids with the PEI solution to produce a plasmid/PEI mixture (section 2.3, “The transfection mix was done in 0.1 mL of LC-SFM/10mM Hepes using 1 µg of DNA and 2 µg of PEI (25 kDa, linear) per 0.5×106 cells”),
wherein said plasmids are in an equal molar ratio (section 3.1, “A molar ratio of 1:1:1 of three plasmids [pAAV-GFP, pAAV-ACG2, pHelper] represents the optimal proportion and was used in the following studies”; see also Figure 4 on page 38, “A transfection mix composed of three plasmids (pAAV-GFP, pAAVACG2, pHelper) at a 1:1:1 weight ratio and PEI (DNA:PEI = 1:2)was transferred to the bioreactor”);
contacting cells with the plasmid/PEI mixture to produce a plasmid/PEI cell culture to produce transfected cells (section 2.3, “DNA-PEI complexes were added dropwise to the cells”);
harvesting the transfected cells to produce a cell harvest (section 2.6, “For 12 well plate experiments, transfected cells were harvested and centrifuged to obtain the cell pellet. The cell pellet was then resuspended …”); and
isolating the recombinant AAV vector form the cell harvest thereby producing recombinant AAV vector comprising a nucleic acid that encodes a protein of interest (section 2.6, “Resuspended cell pellets were subjected to three freeze–thaw cycles (freezing on dry ice followed by thawing in a 37◦C water bath) to release the virus. Cellular debris were removed by centrifugation. The supernatants were stored at −80◦C and used for viral titer determination.”; see also Figure 1 on page 36).
Durocher further teaches wherein said cells are at a density of 0.5×106 cells/mL when contacted with the plasmid/PEI mixture (section 2.4, “Bioreactors were seeded at 0.25×106 cells/mL and transfected with the DNA/PEI complexes 24 h post-inoculation, when a density of 0.5×106 cells/mL was typically reached as described in small scale production of rAAV2 vectors.”).
Durocher further teaches wherein viability of the cells when contacted with the plasmid/PEI mixture was greater than 60% (section 3.3, “It is worth mentioning that for the cells transfected at a density of 2×106 mL−1, viability at 96 hpt was lower (69%) compared to transfections performed at 0.5×106 (76%) and 1.0×106 (83%) cells/mL”).
Durocher further teaches an equal molar ratio of the plasmids (section 3.1, “A molar ratio of 1:1:1 of three plasmids [pAAV-GFP, pAAV-ACG2, pHelper] represents the optimal proportion and was used in the following studies”; see also Figure 4 on page 38, “A transfection mix composed of three plasmids (pAAV-GFP, pAAVACG2, pHelper) at a 1:1:1 weight ratio and PEI (DNA:PEI = 1:2)was transferred to the bioreactor”).
Durocher further teaches wherein the cells are subcultured to a cell density of 0.5×106 cells/mL prior to contact with the transfection mix (section 2.3, “293F cells were seeded at 0.5×106, 1.0×106 and 2.0×106 cells/mL in 19 mL of FreeStyle medium and transfected with 1 mL of transfection mix”; see also section 2.4, “Bioreactors were seeded at 0.25×106 cells/mL and transfected with the DNA/PEI complexes 24 h post-inoculation, when a density of 0.5×106 cells/mL was typically reached as described in small scale production of rAAV2 vectors”).
Durocher further teaches wherein said cells are grown and maintained in a serum-free culture medium before and after contact with plasmid/PEI mixture (page 34, first paragraph, “The objective of this study was to develop a scalable method for production of rAAV in suspension, under serum-free conditions. Here, successful productions of rAAV-2 in bioreactors using triple transfection with serum-free media using cell lines, HEK293EBNA-1 (293E) and HEK293F (293F), respectively, are demonstrated. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production”; section 2.4, “Bioreactors were seeded at 0.25×106 cells/mL and transfected with the DNA/PEI complexes 24 h post-inoculation”).
Regarding dependent claim 93, Park discloses that the cells are HEK293 cells. See, e.g., Abstract and pg. 417. Yue teaches that the cells are 293 T cells (section 2.8 on page 69). Accordingly, Park and Yue teach HEK cell lines, but Park and Yue do not teach HEK293E cells and HEK293F cells, as claimed in claim 93.
However, Durocher teaches that the cells are HEK293E cells and HEK293F cells (ABSTRACT, “This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium”; page 34, first paragraph, “Here, successful productions of rAAV-2 in bioreactors using triple transfection with serum-free media using cell lines, HEK293EBNA-1 (293E) and HEK293F (293F), respectively, are demonstrated”).
Therefore, prior to the effective filing date of the instantly claimed invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the AAV production method of Park by using HEK293E cells or HEK293F cells instead, as taught by Durocher, with a reasonable expectation of success because “choosing cells that are able to grow in suspension is an important prerequisite for scaling up” (Durocher, pg. 33), and using an established suspension-adapted cell line, HEK293E or HEK293F, the need to “wean” HEK293 to a ‘‘suspension-friendly’’ culture mode could be avoided (Park, pg. 420, right column), thereby saving time and resources.
Response to arguments:
Applicant’s remarks filed 03/02/2026 have been carefully considered, but are not found persuasive for the following reasons:
Applicant argues that one of ordinary skill in the art would not be motivated to arrive at the claimed methods requiring free PEI in the range of about 33-50% because Yue teaches that high in vitro gene transfection efficiency only occurs when free PEI is at least 70% of total PEI. Therefore, applicant believes that the technical effect when free PEI is 50% and 33% of total PEI is completely unexpected in view of the cited art. See pg. 16 of applicant’s reply.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In this case, the primary reference, Park, is directed to rAAV production, as claimed, and also discloses free PEI is 50% of total PEI, as claimed. Accordingly, the technical effect when free PEI is 50% and 33% of total PEI is expected at least in view of Park.
Applicant further argues that the instant claims provide a solution for significantly enhancing rAAV production, which would have been superior and unexpected in view of the prior art. In particular, applicant argues that the specification shows cell transfection efficiency and rAAV production was significantly improved when free PEI was added to the cell culture relative to a cell culture which did not include free PEI. See page 16 of applicant's reply.
The examiner does not find this argument persuasive because (1) the primary reference Park already teaches the addition of free PEI to the transfection mixture, as claimed, and (2) the enhanced transfection efficiency and rAAV production by the addition of free PEI molecules would have been an expected result in view of the prior art. For example, Yue recognized that the addition of free PEI chains significantly enhances transfection efficiency. See figure 13 on page 74; see also page 74, col. 1, "it is worth noting that this reduced transfection efficiency is still ~50-fold higher than that without the addition of free PEI chains (N:P=3), clearly indicating that the free PEI chains mainly play their role inside the cells." Consequently, by improving the transfection efficiency with the addition of free PEI, as suggested by Yue, the number of cells transfected with the plasmids encoding the rAAV elements is increased, thereby enhancing rAAV production from said increased number of transfected cells.
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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/JAMES JOSEPH GRABER/Examiner, Art Unit 1631