DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
1. The Amendment filed January 26, 2026 in response to the Office Action of October 28, 2025, is acknowledged and has been entered. Claims 1-4, 7, 12-16, 18-22, 28-32, 34, 57, 61-63, 71, 100, and 101 are pending. Claims 1, 13, 28, 32 are amended. Claims 34, 57, 61-63, 71, 100, and 101 remain withdrawn. Claims 1-4, 7, 12-16, 18-22, and 28-32 are currently being examined.
Maintained Rejections
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.
2. Claim(s) 1-4, 7, 12-16, 18-20, and 28-30 remain rejected under 35 U.S.C. 103 as being unpatentable over Grieger et al (Molecular Therapy, 2016, vol. 24, no. 2; 287-297); in view of Karbowniczek et al (Cell & Gene Therapy Insights, 2017, p. 1-8), as evidenced by Adgene TRE:GFP plasmid, printed October 2025.
Grieger teaches a method of producing a recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences comprising:
culturing human embryonic HEK293 cells in suspension (p. 292, col. 2 to p. 293, col. 1; Figure 5; Materials And Methods);
transfecting the HEK293 cells with triple plasmids:
a first nucleic acid sequence encoding AAV replication helper proteins (named XX680 containing E4, E2A, and VA);
a second nucleic acid sequence encoding AAV replication (rep) and AAV capsid (cap) genes (named pXR2 containing AAV2 rep/cap genes); and
a third nucleic acid sequence comprising at least one inverted terminal repeat (ITR) sequence and a heterologous transgene operably linked to CMV or a TRe or Tet regulatory element (i.e., TReGFP, which contains AAV2 ITR’s flanking GFP transgene, as evidenced by Adgene TRE:GFP plasmid) (Materials And Methods);
incubating the transfected cell line for about 48 to 144 hours (Materials And Methods; p. 292, col. 2 to p. 293, col. 1; Table 3);
lysing the cells and purifying the nucleic acid sequences (p. 289, col. 2 to p. 290, col. 1; Figure 3; Materials And Methods); and
producing the rAAV (Figures 3-6; Tables 1-3; Materials And Methods);
wherein the total amount of nucleic acid transfected from (i), (ii), and (iii) per 1x106 cells is 1 or 1.5 µg, which is less than 2µg (p. 289, col. 1; Figure 2a);
wherein the ratio of (i):(ii):(iii) is any of 2:1.5:1 or 1:1:1 or 1:1:2 which fall into the scope of the claimed ratios recited in instant claim 1 of “about 0.5-1.75”: “about 0.75-2.25”: “about 0.5-1.75”, and instant claim 13 of “about 1”: “about 1-1.6”: “about 1” (p. 288, col. 2; Figure 1a; Materials And Methods);
wherein the HEK293 cells are transfected with a fully hydrolyzed linear polyethylenimine (PEI Max) cationic polymer at a ratio of PEI Max to DNA at 2:1 (p. 288, col. 2; Materials And Methods);
wherein about a liter of medium was added to the transfected cells in order to bring the volume up to 4, 10 or 20 liters for the WAVE bioreactor bags (p. 295, Materials and Methods),
wherein the PEI was added to DNA, and incubated at room temperature for 10-15 minutes (which is about 10 minutes as recited in instant claim 28 and 29), then the transfection cocktail was added to the cells for transfection (p. 295, col. 2 at top);
wherein the rAAV yield produced was at greater than 9.3x1013 vector genomes (vg) / 3.0x109 viable cells transfected (Tables 2 and 3; p. 293, col. 1; Figure 5);
wherein prior to transfection, the suspension of HEK293 cells was cultured to increase volumes in order to create a scalable manufacturing process, and cell lines comprised a density of 3.5x106 viable cells/ml (p. 295, col. 1).
Grieger teaches there is a need to improve on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors (abstract). Grieger teaches (abstract):
Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1×105 vector genome containing particles (vg)/cell or greater than 1×1014 vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1–6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1×1013 vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.
Grieger does not teach:
that the third nucleic acid sequence (iii) is configured as a closed ended linear duplexed vector (claim 1);
that each of the nucleic acid sequences (i)-(iii) are configured as a closed ended linear duplexed vector (claim 15);
the suspension of HEK293 cells is progressively cultured to an increased volume between about 50ml – 2000 liters volume prior to transfection (claim 20); and
the cationic polymer is added to DNA at a ratio of 2.2:1 polymer:DNA (claim 29).
Karbowniczek teaches AAV represents one of the most promising delivery vehicles for genetic medicines. However, the manufacture of plasmid DNA for the production of AAV presents a number of significant challenges, including scalability fidelity, high costs, and long lead times for GMP (Good Manufacturing Practices) production (abstract). Karbowniczek teaches a rapid, in vitro enzymatic technology for multi-gram scale GMP manufacture of DNA that addresses all of the issues of DNA manufacture for AAV production (abstract). Karbowniczek teaches their method generates covalently closed, linear DNA constructs known as doggybone™ DNA or dbDNA™. The process is rapid, cost effective, of high fidelity and eliminates antibiotic resistance genes, which eliminates packaging of these sequences. Karbowniczek teaches dbDNA™ can resolve a number of significant challenges in the production of AAV vectors at both clinical and commercial scale (abstract; p. 2-3).
Karbowniczek teaches their method of producing a recombinant adeno-associated virus (rAAV) lacking prokaryotic sequences comprises:
culturing human embryonic HEK293 cells;
transfecting the HEK293 cells with triple plasmids:
a first nucleic acid sequence encoding AAV replication helper proteins;
a second nucleic acid sequence encoding AAV replication (rep) and AAV capsid (cap) genes (p. 2, col. 1); and
a third nucleic acid sequence comprising at least one inverted terminal repeat (ITR) sequence and a heterologous transgene (payload) operably linked to a regulatory element, wherein the third nucleic acid sequence is configured as a closed-ended linear duplexed vector, “doggybone™” or “dbDNA™” (Figure 2; p. 3);
wherein the nucleic acids sequences are devoid of eukaryotic and prokaryotic cellular modification of DNA because the production is abiological; and
wherein the DNA is transfected into cells utilizing fully hydrolyzed linear PEI at as 1:1 PEI:DNA ratio (p. 6)
Karbowniczek teaches how to make dbDNA™ (Figures 1, 2, and 4). Karbowniczek demonstrates fully processed dbDNA™ manufacturing run up to 500 ml scale using fully scalable upstream system and downstream purification, providing evidence of linear scalability of the dbDNA™ (Figure 3; p. 5). The closed linear hairpin ends of the dbDNA™ provide the advantages of making the DNA resistant to exonuclease activity, allowing for simple purification, and improving stability and duration of expression (p. 4). Karbowniczek teaches upstream process improvements have demonstrated increases in final process yields in excess of 1g/L (p. 5, col. 2). Their process has been successfully prosecuted with hundreds of AAV constructs containing ITRs (p. 6, col. 1). The cost of composition of this completely abiological process contrasts significantly with that for the manufacture of plasmid DNA. Because the process is abiological, the requirement for large, dedicated facilities with high capital requirements is removed. As a result, in excess of 90% of the cost of a batch of dbDNA™ comprises the cost of reagents and variable material costs (p. 6, col. 1). Karbowniczek successfully exemplifies methods for generating AAV2 particles comprising dbDNA™, using Helper, RepCap and Payload, by transfection of HEK293 cells using PEI (p. 6, col. 2 to p. 7, col. 1; Figure 6). Karbowniczek teaches: “Comparable total and genomic titers were achieved for pDNA and dbDNA™- produced AAV confirming the substitutability of using dbDNA™ in AAV manufacture” (p. 7, col. 1). Karbowniczek teaches (p. 7 Discussion):
With the anticipated success of AAV gene therapy, the ability to manufacture large quantities of DNA at GMP quality is critical to scaling these products to meet demand. Traditional methods of pDNA production are time consuming and costly due to the need for large-scale fermentation and downstream processing to make gram quantities of GMP DNA. AAV production delays due to a bottleneck in pDNA supply could be highly deleterious to discovery and clinical development efforts.
The technology to produce dbDNA™ is able to meet these needs. The enzymatic dbDNA™ technology has been shown to be scalable, rapid, amenable to GMP production and is low cost. The case study shown here demonstrates that the technology can amplify and maintain ITR-containing AAV payload sequences, and that when combined and optimized within existing platforms, triple-dbDNA™ transfection produces functional AAV at titers at least comparable to those produced with an equivalent plasmid DNA system. Additional studies have been performed at larger scale and with further purification that further confirm the utility and benefits of dbDNA™ in the AAV system [Manuscript in Preparation].
In conclusion, the novel technology developed for dbDNA™ production represents an important advance that can enable the success of AAV gene therapy.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to utilize the dbDNA™ format (close-ended linear duplexed rAAV vector) technology of Karbowniczek for the rAAV vectors in method of Grieger. One would have been motivated to because: (1) both Grieger and Karbowniczek recognize the need to produce a scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors; (2) Karbowniczek additionally recognizes the need to improve cost, time, and fidelity of production to provide GMP; and (3) Karbowniczek teaches the solution to these needs and problems by utilizing dbDNA™ format for rAAV vectors. One of ordinary skill in the art would have a reasonable expectation of success to utilize the dbDNA™ format taught by Karbowniczek for the rAAV vectors in method of Grieger, because: (1) Karbowniczek demonstrates comparable total and genomic titers were achieved for pDNA (plasmid DNA) and dbDNA™- produced AAV confirming the substitutability of using dbDNA™ in AAV manufacture; and (2) Karbowniczek demonstrates the advantages and success utilizing dbDNA™ technology to increase linear scalability of the dbDNA™, reduce time to production, and reduce costs for production.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to progressively culture the HEK293 cells to an increased volume between about 50ml – 2000 liters volume prior to transfection in the method of Grieger. One would have been motivated to, and have a reasonable expectation of success to, because both Grieger and Karbowniczek recognize the need to increase scalability of production, and Karbowniczek demonstrates successful production HEK293 cells for transfection increased to a volume of 500 ml.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to combine the PEI with DNA at a ratio of 2.2:1. One would have been motivated to, and have a reasonable expectation of success to, because both Grieger and Karbownicz teach the known, established function of combining PEI with DNA is for transfection of HEK293 cells, and both Grieger and Karbownicz demonstrate successfully transfecting cells at various PEI:DNA ratios of 1:1 and 2:1, providing a reasonable expectation to success to slightly vary the amount of PEI with the DNA and predictably transfect the HEK293 cells.
3. Claim(s) 21, 22, and 31 remain rejected under 35 U.S.C. 103 as being unpatentable over Grieger et al (Molecular Therapy, 2016, vol. 24, no. 2; 287-297); and Karbowniczek et al (Cell & Gene Therapy Insights, 2017, p. 1-8) as applied to claims 1-4, 7, 12-16, 18-20, and 28-30 above, and further in view of Chahal et al (Journal of Virological Methods, 2014, 196:163-173); Blessing et al (Molecular Therapy: Methods & Clinical Development, 2019, 13:14-26); and Strobel et al (Human Gene Therapy Methods, 2019, 30:23-33).
Grieger and Karbowniczek (the combined references) teach a scalable method for producing rAVV particles in HEK293 suspension for gene therapy, including large volumes of 4, 10 or 20 liters, as set forth above.
Grieger and Karbowniczek (the combined references) do not teach:
the cell culturing volume comprises a concentration of amino acid from about 1mM to about 20mM (claim 21).
The cell culture volume is 5 liters and comprises about 10mM amino acid (claim 22);
the temperature of the HEK293 culture suspension is increased to 37ºC at about 12-36 hours prior to transfection (claim 31).
Chahal teaches a protocol for successfully transfecting HEK293 cell suspension with AAV vectors, wherein the protocol is intended for use in gene therapy. Chahal teaches their HEK293 cells were propagated at 37ºC prior to and after transfection (sections 2.2 – 2.4). Chahal teaches supplementing HEK293 culture medium with 4mM L-glutamine (amino acid) (section 2.2), wherein cultures were produced at a volume of 2 liters (p. 169, col. 2).
Blessing teaches a method for scalable production of AAV vectors in HEK293 culture. HEK293 cells were cultured at 37ºC prior to and after transfection (Table 1; p. 20, col. 1-2). HEK293 culture medium was supplemented with 4mM of commercial amino acid GlutaMAX (p. 20, col. 1).
Strobel teaches scalable, flexible AAV vector production using frozen stocks of HEK293 cells. HEK293 cell stocks were thawed and cultivated with 2mM L-Glutamine at 37ºC prior to transfection with the transgene plasmid + helper plasmid + Rep/Cap plasmid, and subsequently incubated at 37ºC (p. 24-26; Figure 1). HEK293 cells were seeded in 1,050 mL (~1 liter) of DMEM (comprising amino acids) + GlutaMAX-I (amino acid) culture medium at 37ºC with prior to transfection (p. 26, col. 1).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to culture the HEK293 cells at 37ºC and add about 4-10mM of amino acid to about 2 to 5 liters of volume prior to transfection in the method of the combined references. One would have been motivated to, and have a reasonable expectation of success to, because Chahal, Blessing, and Strobel teach and demonstrate routine protocol to culture HEK293 cells for transfection at 37ºC prior to and after transfection, and to supplement media with additional amino acid, particularly commercially available L-glutamine or GlutaMAX. Given the established function of adding amino acid to HEK293 cell culture to support the process of transfection, and given its commercial availability, it is well within the level of the skilled artisan to increase the amount of amino acid to about 10 mM in a 5-liter volume of cell culture and for the amino acids to predictably serve the same function.
Response to Arguments
4. Applicants argue that claim 1 is amended to recite the ratio of a):b):c) is about 0.5-1.75: about 0.75-2.25: about 0.5-1.75 (weight:weight:weight). Applicants argue that Figure 1 and paragraph [0358] demonstrate the specific ratio of transfection composition instantly claimed results in increased specific (vg/cell) productivity of the clDNA to 2-2.5 times as compared to the pDNA as starting material. Applicants argue that Tables 6 and 7 disclose the resulting clDNA derived vectors show increased infectivity compared to pDNA controls, as indicated by a lower vg/TCID50 ratio +. Applicants argue that Figures 2 and 3 demonstrate superior vector yield of titer when using a ratio of stable cationic polymer to total amount of nucleic acid of 2:1 to 3:1.
Applicants argue that Grieger merely mentions transfection with plasmid DNA at a ratio of 2:1.5:1 AA680:AAV rep/cap helper:TR plasmid. Applicants argue that the instant specification discloses at [0357] and [0362] that clDNA transfection optimization shows quadratic effect. Applicants argue that Greiger does not recognize this and there is no expectation of success for the specifically claimed vector ratio to achieve high titer rAAV without undue experimentation. Applicants argue that none of the secondary references remedy this deficiency.
5. The arguments have been carefully considered but are not persuasive. Applicants have not persuasively argued the specific ratio of the a):b):c) vectors claimed is the novel, sole, or critical attributing factor for the argued superior result of increased viral genome yield. Applicants are arguing unexpected or superior results for assays conducted in the instant application that are affected by numerous variables and conditions outside of vector ratio and vector type. For example, Applicants argue that Figure 1 of the instant application demonstrates increased specific (vg/cell) productivity of the clDNA to 2-2.5 times as compared to the pDNA as starting material, and the specification discloses very specific assay conditions to achieve these results (Example 1):
[0347] The PRO10™ cell line (AskBio, NC, USA) used to manufacture recombinant adeno-associated viral vectors (rAAV) is a suspension-adapted, serum-free cell line derived from the human embryonic kidney cell line 293 (HEK293). The PRO10™ Viral vector manufacture is a batch process carried out at mid- to high-range cell densities and employs a triple transfection method via condensation of the requisite plasmid (pDNA) or closed linear (cl) DNA substrate with linear Polyethylenimine MAX in a cocktail of production media. Both cell growth and production medias are chemically defined with no animal derived components. Each DNA molecule provides a key element for the recombinant AAV production. The first provides Adenovirus helper (Ad helper) proteins for efficient replication and packaging of the vector but lacks essential Adenoviral structural and replication genes to generate an Adenovirus. The second is an AAV8 or, AAVrh10 Trans construct (packaging construct) containing the AAV2 rep gene and AAV8 or, AAVrh10 capsid (cap) protein gene. The third construct is the therapeutic transgene encoding, AAV vector construct and contains the adeno-associated virus 2 inverted terminal repeat (ITR) sequences flanking (5' to 3') the gene of interest. The construct used for all experiments was the dual GFP and Luciferase reporter. Additionally, subsequent studies utilized two therapeutic transgene cassettes comprising CYP and GAA transgenes.
[0348] Initial experiments were conducted applying Design of Experiments (DoE) methodology in a traditional, non-block approach at bench scale (31.25 mL - 2 L) to identify and optimize critical parameters relating to production by simultaneously examining the factors clDNA concentration, ratio of clDNA to transfection reagent. All small-scale experiments were controlled by side-by-side vector production using an optimized triple-plasmid transfection system. Additional factors that will be evaluated include, but are not limited to, media, cell density, time of transfection, transfection volume, temperature, and other cell-dependent or cell-independent factors.
[0349] Small-scale transfected cultures were incubated for approximately 72 hrs post-transfection (hpt) and then harvested by mechanical cell lysis. Total vector production was assessed via vector genome (vg) quantification using the in-house qPCR-based DNase Resistant Particle (DRP) method specific to the viral ITRs. Yields typically range from 4-6 × 1011 vg/mL, as indicated by qPCR Yields were further assessed by observing transgene-targeted qPCR as well as total viral particle (capsids) per mL (vp/mL) via ELISA. Relative packaging efficiency is also modeled by observing the A260/280 ratio at harvest of affinity-purified lysates via SEC-HPLC.
[0350] The primary aim of the small-scale screening experiments was to identify near-optimal transfection conditions for the 50 L scaled portion of the experimental plan. For both the pDNA and clDNA runs, cells were thawed, cultured and progressively expanded until inoculation into the 50 L production bioreactor. The cell culture expansion process continued in the production bioreactor prior to transient transfection being performed. The transfected cell culture was incubated in the production bioreactor for approximately 72-hpt. At harvest, the transfected cell culture was lysed and clarified via depth and membrane filtration followed by purification. Purification consists of capture chromatography, gradient ultracentrifugation, ion exchange chromatography, ultrafiltration/diafiltration (UF/DF), and a 0.2 µm filtration step. Table 3 provides characterization testing for rAAV vector produced by pDNA and clDNA, respectively.
Detailed Process Description for 50L SUB Upstream Operations
[0351] To generate a 50 L batch, cells were thawed, cultured and progressively expanded until inoculation into the 50 L production bioreactor. The cell culture expansion process continued in the production bioreactor prior to transient transfection being performed. Currently, the seed train growth media is supplemented with L-Glutamine to a final concentration of 10 mM, which is used for recovery of frozen cell stocks as well as inoculum expansion up to 5 L suspensions using a 10 L WAVE bag bioreactor. The media used in the WAVE suspension was supplemented with 0.2% PLURONIC™ acid. The growth media used following seed of the ThermoFisher 50 L single-use, stirred-tank bioreactor (SUB, STR) is composed of the see train growth media supplemented with about 1 to 100 mM GLUTAMAX™, about 0.01% to 10% PLURONIC™ acid (ThermoFisher, Waltham, MA), and about 0.001% to 1% FOAMAWAY™ (Gibco, Waltham, MA). GLUTAMAX™ is a stabilized dipeptide source of L-glutamine designed to prevent degradation and reduce toxic buildup of excess ammonia.
[0352] Transient transfection to produce AAV was carried out at cell densities between 3.25 - 4.25 × 106 viable cells/mL3 via condensation of three clDNA and linear Polyethylenimine MAX (Polysciences Inc., Warrington, PA) (PEI Max). The transfection cocktail constitutes 10% (v/v) of the culture volume (5 L). Condensation was carried out in a custom 10 L WAVE Rocker bag equipped with tubing mated for the 50 L SUB. The transfection cocktail was prepared by first adding 4 L of media to the rocker bag at 25° C. with gentle rocking (8° angle, 25 RPM). To prevent the bag from deflating, an air overlay is applied at 0.2 LPM. The plasmids (Table 2) were then added, followed by a 1 L chase with media.
PNG
media_image1.png
194
470
media_image1.png
Greyscale
[0353] Following the media chase, PEI was added over the course of 1 minute and chased with 1L of media. The cocktail was incubated for 7 minutes, and then transferred to the SUB. The transfection-cell suspension is incubated for three hours and quenched by a 10% (v/v) volume of chemically defined, serum-free HEK293 media supplemented with 10 mM L-Glutamine.
SUB Control Parameters
[0354] The current large-scale manufacturing platform utilized a Finesse G3Pro Universal Controller outfitted with a ThermoFisher jacketed 50 L SUB. The single-use vessels were equipped with a 3-blade, 45° pitch, axial impellor, dual-sparger (Frit-Drilled-Hole) design, along with primary Finesse TruFluor pH/DO single-use probe sheaths as well as secondary Pall Kleenpak connections for reusable pH/DO probe inserts. The day before media charge, the bag was installed and inflated with an air overlay at 10 LPM. The optical/reusable DO probe was connected to the transmitter. On the day of charge, the DO probe was calibrated using a 2-pt slope calibration. Following media addition, both single-use and reusable pH probes were standardized using an offline sample on a calibrated blood-gas analyzer.
[0355] The SUB temperature was ramped to 37° C. the day before inoculation. The media was then conditioned by saturating with a continuous drilled-hole air sparge at a flow rate of 0.5 LPM (0.025 VVM). Prior to inoculation, both single-use and reusable DO probes were standardized to 100% air saturation using a 1 pt calibration.
[0356] Following inoculation, the controller was set to administer a continuous drilled-hole air sparge at a rate of 0.5 LPM, and the headspace was swept with an air overlay of 1 LPM. DO was controlled via O2 gas cascade and designed to maintain the set point by increasing O2 flow rate to the frit sparger from 0.00 to 5.00LPM (0-100% DO output / 0-100% MFC-3 output). pH was controlled on the high end (7.0 -14) by increasing CO2 gas flow to the frit sparger from 0.00 to 2.00 LPM (0-(-100)%) output / 0-100% MFC-4 output); however, a base supply was not used to control pH on the low end, but rather, it was allowed to drift naturally.
ResultsDoE Evaluation of Total clDNA (µg)Per 1 × 106 Viable Cells and PEI:DNA Ratio
[0357] In a DoE setting, µg DNA per 1 × 106 viable cells and PEI:DNA ratio were studied in a range between 0.5 - 2 µg and 1 - 3, respectively. The design was a custom response surface model (RSM) with three levels for each factor allowing for the interpretation of both linear and quadratic effects. Duplicate center points in the design space were used to estimate the significance of each effect.
[0358] Total vector production at harvest was evaluated via ITR-qPCR (FIG. 1). The data indicates 2-2.5 times increase in specific (vg/cell) productivity using the clDNA as starting material compared to the pDNA as starting material.
PNG
media_image2.png
752
756
media_image2.png
Greyscale
The instant specification discloses there are several factors that affect viral genome (Vg) yield, including media, cell density, time of transfection, transfection volume, temperature, other cell-dependent or cell-independent factors ([348]), PEI:DNA ratio ([360]; Figure 2), starting DNA amounts : cell density for transfection, serotype of AAV vector, and scale of production (Figure 2 and 3; [360- 363]).
MPEP 716.02 indicates that to be persuasive, unexpected results argued should be commensurate in scope with the claimed invention. MPEP 716.02 states: Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980) (Claims were directed to a process for removing corrosion at "elevated temperatures" using a certain ion exchange resin (with the exception of claim 8 which recited a temperature in excess of 100C). Appellant demonstrated unexpected results via comparative tests with the prior art ion exchange resin at 110C and 130C. The court affirmed the rejection of claims 1-7 and 9-10 because the term "elevated temperatures" encompassed temperatures as low as 60C where the prior art ion exchange resin was known to perform well. The rejection of claim 8, directed to a temperature in excess of 100C, was reversed.). See also In re Peterson, 315 F.3d 1325, 1329-31, 65 USPQ2d 1379, 1382-85 (Fed. Cir. 2003) (data showing improved alloy strength with the addition of 2% rhenium did not evidence unexpected results for the entire claimed range of about 1-3% rhenium); In re Grasselli, 713 F.2d 731, 741, 218 USPQ 769, 777 (Fed. Cir. 1983) (Claims were directed to certain catalysts containing an alkali metal. Evidence presented to rebut an obviousness rejection compared catalysts containing sodium with the prior art. The court held this evidence insufficient to rebut the prima facie case because experiments limited to sodium were not commensurate in scope with the claims.).
PNG
media_image3.png
18
19
media_image3.png
Greyscale
In the instant case, Applicants are arguing superior results that only occur for a specific rAAV production method that requires specific conditions that are not currently claimed. Claim 1 recites a method of producing any serotype of rAAV lacking prokaryotic sequences comprising:
culturing in any human embryonic cell line in suspension;
transfecting the cell line with a): b); and c) at the claimed ratios and under any culture conditions, into any cell density, at any volume, with any starting quantity of DNA, and at any DEI:DNA ratios unspecified;
incubating the transfected cell line for between 40-400 hours;
thereby producing the rAAV.
Thus, the claims encompass a broad range of variables and conditions that will affect Vg yield, and the argued superior results of the examples and figures in the specification are not expected to occur across the broad range of conditions encompassed by the claims.
In addition to the specification identifying variables that affect Vg yield (summarized above), Grieger demonstrates variables affecting Vg yield outside of vector ratios. Grieger demonstrates that total yield is affected by PEI:DNA ratio (Figure 1a); transfection cocktail volume (Figure 1b), the density of cells transfected and starting DNA concentration (Figure 2a), the passage number of cells (Figure 2b), rAAV serotype (Table 1 and Figure 5), culture volume (Table 2), and time between 48 hours and 144 hours of culture (Table 3). Greiger optimized Vg yield by experimenting and identifying vector ratios (2:1.5:1), PEI:DNA ratio (2:1); cell density (1e6 cells/ml), and starting DNA concentration (1.5 ug/ml) as providing the highest viral genome yield, therefore, achieving the highest or optimal Vg yields using these parameters is expected. Karbowniczek provides motivation and a reasonable expectation of success to swap out the pDNA with clDNA and achieve superior yield because Karbowniczek demonstrates that clDNA is superior to plasmid DNA because it is resistant to exonuclease activity, and Karbowniczek demonstrates the advantages and success utilizing dbDNA™ technology to increase linear scalability of the dbDNA™, reduce time to production, and reduce costs for production. Thus, the cited combination of references in the rejections of record provide an expected result of superior Vg yield using the claimed ratios and using a clDNA.
New Rejection
(necessitated by amendments)
Claim Rejections - 35 USC § 103
6. Claim 32 is now amended to depend from claim 22 and is rejected as set forth below.
7. Claim(s) 32 is rejected under 35 U.S.C. 103 as being unpatentable over Grieger et al (Molecular Therapy, 2016, vol. 24, no. 2; 287-297); Karbowniczek et al (Cell & Gene Therapy Insights, 2017, p. 1-8), Chahal et al (Journal of Virological Methods, 2014, 196:163-173); Blessing et al (Molecular Therapy: Methods & Clinical Development, 2019, 13:14-26); and Strobel et al (Human Gene Therapy Methods, 2019, 30:23-33), as applied to claims 1-4, 7, 12-16, 18-22, and 28-31 above, and further in view of Yang et al (AMB Expr. 2019, 9:70).
Grieger; Karbowniczek; Chahal; Blessing; and Strobel (the combined references) teach a scalable method for producing rAVV particles in HEK293 suspension for gene therapy, including large volumes of 4, 10 or 20 liters, and wherein the cell culture volume is 5 liters and comprises about 10mM amino acid, as set forth above.
The combined references do not teach the culture medium is subject to an air sparge at a flow rate between about 0.1 liters per minute (LPM or l/m) to about 1.0 LPM.
Yang demonstrates large-scale culturing of HEK293 cells in bioreactors (Figure 6) and teaches such culturing is intended for the production of viral vectors (abstract; Discussion). Yang determined aeration rates for the cell culture using an air sparger, and capped maximum air sparger rates at 1 l/m (p. 4, col. 2). Yang teaches an “important parameter for cell growth and scale up in stirred tank bioreactors is the aeration rate, which includes the flow rate or sparger rate for compressed air, oxygen, and carbon dioxide. Compressed air and oxygen provide dissolved oxygen for cell growth, and compressed air can also function to regulate culture pCO2 below inhibitory or suboptimal levels. In micro carrier culture processes, high aeration rates can result in significant stress or damage to cells, thereby impacting attachment and growth. This is especially true for the cell types that are not firmly attached to microcarriers, such as HEK293T cells” (p. 4, col. 1).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to subject to the HEK293 culture of the combined references to an air sparge at a flow rate of about 1.0 LPM or less. One would have been motivated to, and have a reasonable expectation of success to because: (1) the combined references teach scalable production to achieve large volume cultures, (2) Yang teaches an important parameter for cell growth and scale up production is the aeration rate that provides dissolved oxygen for cell growth, and regulates CO2 levels; and (3) Yang teaches limiting the aeration flow rates so as not to damage HEK293 cell culture, and demonstrates successfully culturing cells with an air sparger rate capped at 1 l/m.
8. All other rejections recited in the Office Action mailed October 28, 2025 are hereby withdrawn in view of amendments.
9. Conclusion: No claim is allowed.
Conclusion
10. THIS ACTION IS MADE FINAL. 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.
11. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Samira Jean-Louis can be reached at 571-270-3503. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Laura B Goddard/Primary Examiner, Art Unit 1642