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 .
Claim Status
Claim 5 is amended.
Claims 1-2, 5-6 and 8-14, and 17-21 are under examination.
Edited Rejection Necessitated by Amendment
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, following the discussion of claim 1 above, Atkinson, Yakobson, Russell, and Studzinski collectively render obvious all of the claim limitations of claim 5 for the reason set forth above with respect to claim 1. The cited combination teaches culturing recombinant AAV producer cells in the presence of DNA synthesis inhibitor/cell synchronization agent to improve AAV production. Specifically, Atkinson et al teach that altering the growth conditions of producer cells by employing stress conditions, including DNA synthesis inhibitors, enhances recombinant AAV production. Yakobson et al further demonstrate that synchronization of producer cells with a DNA synthesis inhibitor promotes AAV replication in producer cells, while Russell evidences that thymidine was a well-known DNA synthesis inhibitor and synchronization agent. On the other hand, Studzinski et al teach that the concentration of thymidine necessary to achieve effect synchronization is dependent upon the cell type and that optimization of the thymidine concentration is a result-effective variable that would have been arrived at through routine optimization.
Regarding the additionally recited limitation that culturing in a medium containing thymidine in a concentration of 3mM to 10 mM produces at least 1.5 fold more AAV virions than culturing in an otherwise identical control medium lacking thymidine would have been an expected result of practicing the obvious method. This is because Atkinson et al expressly teach that subjecting producer cells to stress conditions can dramatically enhance recombinant AAV production and discloses increases in production of at least 2-fold, at least about 3-fold, at least about 5-fold, and at least 10-fold relative to production in the absence of the stress condition. ( See Atkinson et al [0239]). Thus, it would have been obvious to employ thymidine as the DNA synthesis inhibitor in view of the combined teachings of Atkinson, Yakobson, Russell, and Studzinski, the recited comparison to control culture medium lacking thymidine merely reflects the expected improvement resulting from the use of the stress condition taught by Atkinson. Accordingly, the recited production of at least 1.5 fold more AAV than control medium not containing thymidine represents the expected performance of the obvious method and does not patentably distinguish the claimed method from the combined teachings of the cited prior art.
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 20/05/2026 have been fully considered but they are not persuasive.
Applicant argues that Russell is directed to transduction efficiency rather than to AAV production, and therefore, is not relevant to the claimed method for producing recombinant AAV virions. Applicant further argues that one with ordinary skill in the art would not have been motivated to combine Atkinson, Yakobson, Russell, and Studzinski because Russell concerns a different biological process performed in different cell type.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive. This is because the rejection does not rely upon Russell for teaching that thymidine increases AAV production. Rather, Atkinson is relied upon for teaching a method for producing recombinant AAV in producer cells and for expressly suggesting enhancing viral production by altering the growth conditions of the producer cells through the use of agents that inhibit cellular growth or metabolism, including DNA synthesis inhibitors such as hydroxyurea. Yakobson et al further demonstrate that synchronization of producer cells with DNA synthesis inhibitor (hydroxyurea) promotes the production AAV by rending the producer cells permissive for AAV replication. Accordingly, the motivation to employ a cell blocking or DNA synthesis inhibiting agent in AAV producer cells is provided by the combined teachings of Atkinson and Yakobson. Russell, is relied upon to demonstrate that thymidine was a known DNA synthesis inhibitor and cell synchronization agent. Russell demonstrates that thymidine was recognized in the art as an agent capable of inhibiting DNA synthesis and synchronizing cells, thereby Russell is cited as an evidence to demonstrate that thymidine was a known alternative to other DNA synthesis inhibitors, such as hydroxyurea. The office action does not rely on Russell as evidence that thymidine increases AAV production, but rather as evidence that thymidine is a member of the class of the known DNA synthesis inhibitor/cell synchronization agents that were already suggested by Atkinson and exemplified by Yakobson. Studzinski, on the other hand, is relied upon to teach that the concentration of thymidine required to achieve cell synchronization is dependent upon the cell type and demonstrated that concentrations within or overlapping the claimed range are effective for achieving substantial cell synchronization. Thus, Studzinski establishes that the concentration of thymidine is a result effective variable that would have been optimized through routine experimentation for the particular producer cell employed. Accordingly, one with ordinary skill in the art would have been motivated to substitute the known DNA synthesis inhibitor thymidine for the DNA synthesis inhibitor hydroxyurea suggested by Atkinson and employed by Yakobson, while optimizing the thymidine concentration as taught by Studzinski, with a reasonable expectation of successfully producing recombinant AAV. Therefore, the rejection under 35 U.S.C 103 is maintained.
Applicant further argues that Atkinson et al merely provides a list of possible stress conditions and only briefly mention hydroxyurea among numerous stress-inducing agents. Applicant also argues that one with ordinary skill in the art would not have understood Atkinson to teach hydroxyurea or DNA synthesis inhibitor increase viral production because working examples do not evaluate DNA synthesis inhibitors.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive. This is because Atkinson et al expressly teach enhancing AAV production by altering the growth conditions of producer cells through the application of stress conditions that inhibit cellular growth and/or metabolism. For example Atkinson et al state “ that stressing the producer cells, either metabolically or by other means as described below, can dramatically enhance the production of AAV vector. In some embodiments, the stress condition enhances production of rAAV vector (as compared to production without imposing a stress condition(s)) at least about 2-fold, ..etc” and expressly identifies metabolic inhibitors of DNA synthesis, including hydroxyurea, as a suitable stress agent . [See Atkinson et al [0239-0240]). Therefore, Atkinson provides an express teaching that DNA synthesis inhibitors are suitable agents for modifying producer-cell growth conditions to enhance AAV production. The argument that Atkinson identifies DNA synthesis inhibitors among several alternative stress agents does not diminish the reference express disclosure. Furthermore, Applicant are reminded that the reference is not limited to its working examples, and all teachings of the reference must be considered for what they reasonably convey to the one of ordinary skill in the art. In other words, the absence of an experimental example utilizing hydroxyurea does not negate the explicit teachings of Atkinson that DNA synthesis inhibitors are suitable stress agents for enhancing AAV production.
Applicant further argues that Yakobson teaches poor AAV production because hydroxyurea treated cells exhibited lower replication than cells coinfected with helper adenovirus, thereby allegedly teaching away from the claimed invention eliminating any reasonable expectation of success.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive. This is because Yakobson demonstrates that pretreatment of producer cells with hydroxyurea synchronizes the cells and render them permissive for AAV DNA replication in the absence of helper virus. Although Yakobson reports that hydroxyurea-mediated synchronization was less efficient than adenovirus coinfection, Yakobson nevertheless establishes that cell synchronization through DNA synthesis inhibitor is capable of supporting AAV replication and virus production. The argument that one disclosed method is less efficient than another preferred method does not constitute a teaching away. A refence teaches away when only discourages a person of ordinary skill in the art from following the claimed approach or suggest that the approach is unlikely successful. Yakobson et al does neither. Rather, Yakobson demonstrates that hydroxyurea-induced synchronization is viable alternative of supporting AAV production, though at a reduced efficiency relative to helper-virus infection. Also, the claimed method does not require that prior art disclose the best or most efficient method. Rather, it is sufficient that prior provides reasonable expectation that the proposed modification would function for the intended purpose. In this rejection, Yakobson et al is cited to confirm that synchronization of producer cells with a DNA synthesis inhibitor support AAV production supporting Atkinson’s explicit teachings that manipulation of producer cells growth condition through DNA synthesis inhibitor can be employed to enhance recombinant AAV production. Accordingly, Yakobson et al do not teach away from the claimed method, nor does it undermine the rationale for combining the cited prior art.
Applicant also argues that the working examples demonstrate unexpected results because the claimed thymidine method produces substantially greater AAV production than would have been expected from Yakobson’s hydroxyurea experiments. Applicant further argue that Yakobson represent the closest prior art and therefore provides the proper comparator for evaluating unexpected results.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive. This is because evidence of unexpected results must be commensurate in scope with the pending claims and must establish that the claimed results would have unexpected over the closest prior art. The evidence relied upon by Applicant compares thymidine to hydroxyurea, which are different DNA synthesis inhibitors having different chemical structures, different mechanism of action, and different biological properties. A difference in performance between two different compounds does not, by itself, establishes that the claimed method possesses unexpected properties over the teachings of the combined prior art. Moreover, the pending claims encompass culturing eukaryotic cells with thymidine at concentrations ranging from about 3 mM to about 10 mM, irrespective of cell type, vector construct, or production conditions. Applicant has not demonstrated that the alleged unexpected improvement is reasonably attributable to the full scope of the claim.
Conclusion
No claim is allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to FATIMAH KHALAF MATALKAH whose telephone number is (703)756-5652. The examiner can normally be reached Monday-Friday,7:30 am-4:30 pm EST.
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/FATIMAH KHALAF MATALKAH/Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638
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