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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/19/2026 has been entered.
Claim Status
Claims 1-2, 5, 11,13-14, and 20 are amended.
Claims 3-4, and 15-16 are cancelled.
Claims 1-2, 5-6 and 8-14, and 17-21 are under examination.
Rejections Withdrawn
Claim Rejections - 35 USC § 103
The rejection of claims 1-6, and 8-21 under 35 U.S.C. 103 as being unpatentable over Atkinson et al (US 2010/0248355 A1) in view of Yakobson et al (Journal of Virology,1987), and Senis et al ( Biotechnology Journal, 2014) is withdrawn in view of Applicants claim amendment. Applicant amended claim 1 to incorporate features ("from about 3 mM to about 10 mM") and ("thymidine").
Response to Amendments
Applicant’s arguments have been carefully considered and found persuasive as noted above. The new ground of rejection below addresses the deficiencies raised by Applicant with respect to the amended claims.
New Ground of 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.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-2, 5-6 and 8-14, and 17-21are rejected under 35 U.S.C. 103 as being unpatentable over Atkinson et al (US 2010/0248355 A1) in view of Yakobson et al (Journal of Virology,1987), Russell et al ( PNAS, 1995), Studzinski et al ( Journal of Cell Physiology, 1969), and Senis et al ( Biotechnology Journal, 2014).
Regarding claims 1-2, and 13-14 , Atkinson et al teach a method and composition for producing high titer of substantially purified population of Adeno-associated virus (AAV) that can be used for gene delivery. The method of Atkinson et al comprises the steps of:
a) providing an AAV producer cell that comprises:
(i) one or more AAV packaging genes, wherein each said AAV packaging gene encodes an AAV replication (rep) or encapsidation (cap) protein;
(ii) a recombinant AAV (rAAV) pro-vector that comprises a heterologous non-AAV polynucleotide flanked by at least one AAV inverted terminal repeat (ITR); and
(iii) a helper virus for AAV;
b) incubating the producer cell provided in step a) under conditions that are permissive for
replication of AAV.
Atkinson et al also suggest altering the growth conditions of the AAV producer cells to enhance viral production. Atkinson et al, for example, suggest adding to the culturing medium an agent that inhibits cellular growth (i.e. a cell cycle blocker) or metabolism, such as hydroxyurea, methotrexate, or aphidicolin. (See abstract, [0088]-[0090], and [0240]).
Atkinson et al do not teach culturing producer cells in a medium containing 3-10 mM thymidine.
Yakobson et al supplement the method of Atkinson et al by teaching a method for packaging adeno-associated Virus (AAV) in mammalian cell lines without the addition of a helper virus. The method of Yakobson et al involves pretreating the producer cells (i.e. Chinese hamster cell lines (OD4)) with 1 mM hydroxyurea prior to infection to promote cell synchronization. (See abstract). Yakobson et al demonstrated that when the OD4 cells were cultured in a medium containing 1 mM hydroxyurea prior to infection, approximately 90% of the cells were found to be arrested in S phase. (See Fig.6a). Yakobson et al demonstrate that synchronizing the producer cell with the cell cycle blocking agent (hydroxyurea) at a concentration of 1 mM promotes the production of AAV virions in the absence of a helper virus. (See Fig.2).To summarize, the method of Yakobson et al provides an ordinary skill in the art with the experimental basis to envision producing AAV utilizing a synchronizing agent (i.e. cell cycle blocking agent), such as hydroxyurea, to replace the need for using helper virus in supplementing such needs.
It is submitted that the method of Yakobson et al relied on using 1 mM hydroxyurea to promote cell synchronization, but fail to teach culturing producer cells in a medium containing 3-10 mM thymidine, which is likewise a synchronizing agent.
Russell et al supplement Atkinson and Yakobson by demonstrating that synchronizing agents that function by inhibiting DNA synthesis improve the effectiveness of AAV transduction. Russell et al utilized the AAV-LAPSN vector, which contains the human placental alkaline phosphatase (AP) and neomycin phosphotransferase genes, to transduce primary human fibroblast in order to study the effects of different DNA synthesis inhibitors on the transduction efficiency of AAV vector. Russell et al demonstrate that treating cells with DNA synthesis inhibitors, such as 40 mM of hydroxyurea or 1 mM of thymidine, efficiently increases the transduction effectiveness of the AAV vector. ( See Fig. 1, and Fig 3.B). Russell et al conclude that prior exposure of stationary cultures (i.e. nondividing cells) to drugs that inhibit DNA synthesis increases transduction by AAV vectors, and suggest employing this method to gene transfer protocols to improve prospects for gene therapy using AAV vectors. ( See abstract, and right column on page 5720-1st paragraph). To summarize. Russell et al also provide an ordinary skill in the art with the experimental basis to envision culturing cells in a medium containing DNA blocking agent, such as thymidine, to increase the efficiency of cell transduction by AAV vectors, as well as provide a suggestion to utilize the aforementioned method to improve gene therapy using AAV vectors.
It is submitted that Russell et al teach utilizing 1 mM thymidine to promote cell synchronization, but fail to teach culturing cells in a medium containing 3-10 mM of thymidine, as required by the instant claims.
Studzinski et al supplement the aforementioned prior arts by demonstrating that the amount of thymidine required to inhibit cell division by 90% or more is dependent on cell type. In particular, Studzinski et al demonstrate that the concentration of thymidine required to inhibit cell division by more than 90% in randomly growing cultures of HeLa S3 cells is 2 mM, whereas the HeLa-wild cultures requires 5 mM thymidine. ( Fig.2-3). In other words, Studzinski et al demonstrate that depending on the cell type a 2-5 mM of thymidine is needed to reach 90% cell synchronization, this reads on the instant claims. The teachings of Studzinski clearly suggest that the concentration of thymidine required for synchronization is a result effective variable. Therefore, an ordinary skill in the art upon reviewing Studzinski et al would be motivated to use routine experimentation to discover the optimum concentration of thymidine needed to obtain 90% or more of cell synchronization for the specific cell type.
According to the MPEP, "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum of workable ranges by routine experimentation. The "discovery of an optimum value of a result effective variable in a known process is ordinarily within the skill of the art."
Therefore, instant claims are combining prior art elements according to known methods to yield predictable results, namely the predictable result being the use of 3-10 mM of thymidine to increase the virus titer. Because Atkinson et al teach a method for packaging AAV in mammalian cell and suggest culturing the producer cells in a medium containing a cell cycle blocking agent to boost viral production, but fail to teach utilizing 3-10 mM of thymidine to block cell cycle. Yakobson et al teach that synchronizing the producer cell with cell cycle inhibitor (i.e. hydroxyurea) supports the production of AAV virions in the absence of a helper virus. Russell et al also demonstrate that synchronizing cells with a 1 mM of thymidine, significantly increases the transduction efficiency of AAV vector, and strongly suggest the utilization of synchronizing agents to improve gene therapy by AAV vector. Studzinski et al demonstrate that depending on the cell type, 2-5 mM of thymidine is recommended to obtain 90% or more cell synchronization. Thus an ordinary skill in the art who had viewed Atkinson could have come across Yakobson, Russell, and Studzinski and immediately noticed the strong possibility that using thymidine, instead of the hydroxyurea, to synchronize producer cell would have the predictable result of generating a high AAV titer. See MPEP 2143 (I)(A).
Regarding claims 5, Atkinson et al do not explicitly state that the increase in the viral titer is 1.5-fold or higher ; however the outcome is presumed to be inherent because the combined teachings of Atkinson, Yakobson, Russell, and Studzinski render obvious culturing the producer cells in a medium containing 1-5 mM of the cell cycle blocking agent thymidine, as this advantage would flow naturally from following the combined teachings of the recited prior arts.
Regarding claims 6 and 21, Atkinson et al also utilize mammalian host cells for packaging the AAV particles. (See [0088]).
Regarding claims 8 and 17, Atkinson et al teach that the heterologous gene product can be a polynucleotide encoding a polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function. (See [0118]).
Regarding claims 9 and 18, Atkinson et al also disclose that the heterologous gene product can be a nucleic acid (i.e. polynucleotide) that is used to decrease gene expression, for example, by supplying a therapeutic polynucleotide comprising a sequence capable of forming a stable hybrid with either the target gene or RNA transcript (i.e.siRNAs). ( See [0263]).
Regarding claims 10 and 19, Atkinson et al do not teach heterologous gene products that code for both a polynucleotide and a polypeptide.
Sensi et al teach how to generate a population of AAV carrying a heterologous genes that encode for both gRNA ( a polynucleotide) and Cas9 ( a polypeptide). Sensi et al present a proof-of-concept for the use of adeno-associated virus (AAV) vectors for the robust and specific delivery of the two essential CRISPR components , i.e. Cas9 and gRNA, paving the way for the utilization of the CRISPR-mediated gene editing in a variety of cells and organisms.
Therefore, it would have been prima facie obvious for one with ordinary skill in the art at the time the invention was filed to combine the teachings of Atkinsons and Sensi to package an AVV vector carrying heterologous genes encoding for nucleic acid and a protein. Atkinson teach a method for packaging AAV vector with a heterologous gene using a mammalian cell line. Sensi et al teach a method for packaging AAV carrying heterologous genes encoding both a nucleic acid and a protein. Thus one would have been motivated to combine the teachings of Atkinson and Sensi because, together, they provide one with ordinary skill in the art with the experimental basis to envision packaging AAV vectors carrying heterologous genes encoding both polynucleotide and a polypeptide, and with a higher expectation of success. Combining prior art elements according to known methods to yield predictable results. See MPEP 2143 (I)(A).
Regarding claims 11 and 20, following the discussion above, the combined teachings of Atkinson, Yakobson, Russell, and Studzinski render obvious claim 1. Yakobson et al teach a producer cell comprising of eukaryotic cells (i.e. Chinese hamster OD4 cells) with no nucleic acid encoding adenovirus polypeptide.
Regarding claims 12, The method of Atkinson et al also involves method of purifying the population of rAAV particles from the culture medium. (See claim 8, page 38).
Response to Arguments
Applicant's arguments filed 01/23/2026 have been fully considered but they are not persuasive.
Applicants argue that even if one considered swapping in thymidine for hydroxyurea based on the combination of Atkinson and Yakobson, they would not have used a concentration of about 3 mM to about 10 mM (or 4 mM) as recited in the independent claims. This is because Applicants are relying on the teachings of prior arts, which deemed the use of 2mM of thymidine as a synchronizing agent to be a high concentration.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive. This is because Applicants amended claim 1 to include the features of using “ thymidine” at a concentration of “ 3-10 mM”, while Atkinson in view of Yakobson do not teach these limitations, however the new ground of rejection over Atkinson in view of Yakobson, Russell, and Studzinski covers all of the added claim limitations ( as discussed above).
Applicant also argue unexpected results. According to Applicant, the methods of instant claims achieved a far greater result than would have been expected. For example, Applicants cite the results shown in Fig.6, which show that AAV production using thymidine instead of helper was nearly equivalent to using helper. Applicant goes on to explain that those results are unexpected in light of Yakobson et al teachings, which state that, although hydroxyurea-pretreatment was able to substitute for helper virus, the results came at a steep price: 10-fold fewer cells supported AAV replication than when using helper virus. In other words, Yakobson et al teach that the use of a helper virus still, by far, the most efficient method for producing AAV.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive. This is because Applicants compare the results achieved by the use of thymidine to those of hydroxyurea. Yakobson et al, for example, utilized hydroxyurea as a synchronizing agent to demonstrate that AAV vector can be produced without the need for a helper virus. This differs from instant claims which are directed to the use of 3-10 mM of thymidine. In addition, Yakobson et al utilized only 1 mM of hydroxyurea, but prior art by Russell et al demonstrated that it can be utilized at a much higher concertation to synchronize cell and to subsequently enhance AAV transduction efficiency. For example, Russel et al show that utilizing 40 mM of hydroxyurea increased the transduction efficiency of AAV-LAPSN by more than 300- fold. ( See Fig. 1 supplemented by Russell). Russel et al also showed that the transduction efficiency was proportional to the concentration of hydroxyurea used. For example, the higher the concentration the higher the transduction efficiency. ( See Fig.3A supplemented by Russell et al). Therefore, Yakobson et al statement that “the use of a helper virus still, by far, the most efficient method for producing AAV” only applies to 1 mM of hydroxyurea and cannot be used as a basis to claim unexpected results when using a different synchronizing agent at a different concentration. Also, as previously noted, Studzinski demonstrated that the amount of thymidine required to inhibit cell division by 90% or more is dependent on cell type. Therefore, to establish unexpected results over the claimed range of 3-10 mM of thymidine, applicants should compare a sufficient number of tests both inside and outside the claimed range to show the criticality of the claimed range. In re Hill,284 F.2d 955, 128 USPQ 197 (CCPA 1960).
Conclusion
No claim is allowed.
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/FATIMAH KHALAF MATALKAH/Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638