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
Acknowledgement is hereby made of receipt and entry of the communication filed on Oct. 15, 2025. Claims 1-4, 6-10, 14-20 and 25 are pending and currently examined.
Claim Objections
(New) Claim 1 is objected to because of the following informalities:
The “each aliquot with an aqueous volume of 10 ml or less” should be “wherein each aliquot with an aqueous volume of 10 ml or less”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112 (b)
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.
(Previous rejection- withdrawn) Claims 1-4, 6-10 and 14-23 were rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly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This rejection is withdrawn in view of the amendment filed on Oct. 15, 2025.
(New) Claims 1-4, 6-10 and 14-20 are 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.
The base claim 1 recites “a test sample” in two different phrases at “a method for detecting replication competent virus in a test sample …” and “c) testing a test sample from the aliquots…”, which render the claims indefinite. It is not clear if the two cited “a test sample” in different phrases are the same or not.
Accordingly, one of ordinary skill in the art will not know the metes and bounds of the claim.
For purposes of compact prosecution and applying prior art, claim 1 was interpreted herein to encompass the two cited “a test sample” are the same samples.
The claim 9 recites the phrase at “…have a combined aqueous volume of at least 115ml” that render the claim indefinite. Based on the instant specification [0192]- [0193]), the “combined aqueous volume” is a “pooled” aqueous volume. Because the base claim 1 claims “a plurality of individual cell culture aliquots”, it is not clear how many “individual cell culture aliquots” need to be combined in order to acquire at least 115 ml. One of ordinary skill in the art will not know the metes and bounds of the claim.
For purposes of compact prosecution and applying prior art, the 115 ml of combined aqueous volume was interpreted herein to any cell culture that used for detecting the replication competent virus.
It is noted any interpretation of the claims set forth above does not relieve Applicant of the responsibility of responding to this rejection. If the actual interpretation of the claims is different than that posited by the Examiner, additional rejections and art may be readily applied in a subsequent final Office action.
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.
(Previous rejection- withdrawn) Claims 20-22 were rejected under 35 U.S.C. 103 as being unpatentable over Sastry et al. (Methods Mol Biol. 2009;506:243-63) as applied to claims 1-4, 6-10 and 14-16 and 18-19 above and in view of Cornetta et al. (Mol Ther. 2011 Mar;19(3):557-66).
This rejection is withdrawn in view of the amendment filed on Oct. 15, 2025.
(Previous rejection- maintained) Claims 1-4, 6-10 and 14-16 and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Sastry et al. (Methods Mol Biol. 2009;506:243-63, hereinafter “Sastry”).
The amended base claim 1 is directed to a method for detecting replication competent virus in a test sample, the method comprising:
a) culturing for at least nine days a plurality of individual cell culture aliquots comprising virus-permissive cells and (i) a gene therapy product or (ii) end of production cells that were used to manufacture the gene therapy product, each aliquot with an aqueous volume of 10 ml or less,
b) culturing the aliquots for at least a further six days, wherein the aliquots are passaged using a dilution factor of at least 2 at each passage,
wherein the combined aqueous volume of the plurality of individual cell culture aliquots is reduced by at least 50% between the start and end of step b); and
c) testing a test sample from the aliquots at the end of step b) for the presence of replication competent virus,
wherein the method is performed before clinical release of the gene therapy product.
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Sastry teaches a method for detecting replication competent retrovirus (RCR) and Lentivirus (RCL) and discloses a molecular and biological assay for detection of RCL associated with lentiviral vectors which is outlined in the Fig. 2 (See below). In the method chapter, Sastry further discloses that in an assay for detecting RCL associated with HIV-1 based vectors, the test article is used to inoculate a cell line (C8166-45 derived from human umbilical cord blood lymphocytes) that is permissive for infection and growth of HIV-1, and the transduced cell line is cultured for 21 days to amplify any RCL in the test article. Sastry also teaches a detailed method using the C8166 cell lines to detect RCL in HIV-1 vectors, a maxim of 12 ml medium is needed in per 75 CM2 flask or 50 ml culture tube (See page 255, 3.2.2).
Therefore, Sastry teaches the step a) of claim 1, where the amended “at least nine days” is taught by “the transduced cell line is cultured for 21 days…”, the “virus-permissive cells” is C8166 cell line, the “(i) a gene therapy product” is indicated by “we describe methods capable of detecting low levels of virus contamination and discuss the current regulatory guidelines for screening gene therapy products intended for human use” (Abstract). As for the newly added limitation of “10 ml or less”, Sastry teaches that the cell can be seeded in the 6-well plate (See page 253) or 25-cm 2 flasks (See page 252; Table 1), where the culture volume is less than 10 ml.
For the claimed step b) in the base claim 1, Sastry teaches that after three weeks, cell free medium from amplified cells is used to infect naïve C8166 (Indicator) cells, which are cultured for an additional seven days and analyzed for the presence of viral markers (See page 247, paragraph 1), where the “seven days” here can be considered to teach the “a further six days” as claimed in step b). As for the dilution factor claimed, Sastry teaches a serial dilution at “… Flasks #2–5: 10−2, 10−4, and 10−6…” (See page 253, paragraph 3.1.3), which can be considered a dilution factor at 2. Sastry also teaches the cells should be passaged a minimum of five times during the three-week period and at least two times per week. The amplification kinetics do allow scaling down of large flasks with later passages, as demonstrated in Table 2 (See page 252, paragraph 2 and Table 2 below), which indicates the passage of the cell aliquots and the total volume can be reduced. Therefore, the “the total volume across the plurality of aliquots is reduced by at least 50%” as claimed can be achieved through experimental optimization based on the cell density and culture volume unless there is evidence showing that they produce unexpected results.
As for the amended limitation of c), Sastry teaches that Sensitive assays have been developed for detection of RCL indicative markers including the p24 gag antigen (by ELISA), viral reverse transcriptase (by product enhanced reverse transcriptase or PERT), psi-gag sequences that result from a recombination between vector and packaging constructs (by PCR) and the psuedotyping envelop VSV-G (by PCR) (See page 247, paragraph 1). Here the new added limitation of “testing a test sample from the aliquots at the end of step b)” is taught by Sastry’s teaching at culturing a further seven days and then the sample will be tested.
As for the newly added limitation on “wherein the method is performed before clinical release of the gene therapy product”, Sastry teaches that screening of vector prior to clinical use has generally relied on biologic assays aimed at documenting a replication competent virus, rather than surrogate markers of virus (See page 245, paragraph 2).
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Thus, the invention as a whole is clearly prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Regarding claims 2-3, Sastry teaches their detention method is for replication competent retrovirus and Lentivirus (See Abstract).
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Regarding claims 4 and 6-10, Sastry teaches the virus-permissive cells such as Mus dunni cells can be seeded at different volume based on the Flask volume and cell density (See Table 1 and below), which includes the started volume of 11ml, 10ml, 5ml or 3ml and 115ml and the cell density between 1 x 10^5 total cells/ml to about 1 x 10^7 total cells/ml (Claim 4 & 9-10). Sastry also teaches that the virus-permissive cells can be C8166 cell that is permissive for infection and growth of HIV-1 and derived from human umbilical cord blood lymphocytes (See page 247, paragraph 1), which is a non-adherent cell line (claims 7-8). Sastry teaches a method for detecting replication competent retrovirus (RCR) using biologic assays (See Table 1, page 251 and below). It is a common knowledge in the art that the RCR is the virus particles capable of infecting cells and replicating to produce additional infectious particles (Claim 6).
Regarding claims 14-16, Sastry teaches that the plate for PCR test is 6-well plates (See e.g. page 249, paragraph 2), which teaches claims 14-15. As for the limited plates with 12-well plate or a 24-well in claim 16, one of ordinary skills would be able to use any cell culture plates based on the needs.
Regarding claims 18-19, Sastry teaches that the sensitive assays have been developed for detection of RCL indicative markers including the p24 gag antigen (by ELISA), viral reverse transcriptase (by product enhanced reverse transcriptase or PERT), psi-gag sequences that result from a recombination between vector and packaging constructs (by PCR) and the psuedotyping envelop VSV-G (by PCR) (See page 247, paragraph 247).
(Previous rejection- withdrawn) Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Sastry as applied to claims 1-4, 6-10 and 14-16 and 18-19 above and in view of Swiech et al. (Methods in Molecular Biology Book 2086, published on Nov. 09, 2019, hereinafter “Swiech”).
Claim 17 is directed to a method of claim 1, wherein steps a) and b) are automated.
Sastry teaches a method for detecting replication competent virus in a test sample by culturing the virus-permissive cells such as C8166 cells and a portion of the test samples, and then testing for the presence of replication competent virus. However, it is silent on an automated method.
Swiech teaches the Chimeric Antigen Receptor T Cells: Development and Production in their book Chapter, which include the production of Lentivirus for the establishment of CART-T Cells and the replication-competent retrovirus (RCR) detection (See page Vii and Viii). Swiech also teaches a new processing option for seeking automation and controlled expansion process to ensure that critical quality attributes of the cell production (See e.g. page 141, paragraph 2). Swiech teaches that automation can improve reproducibility and reduce human errors, while functionally closing this operation helps to manage contamination risks (See page 151, Introduction). Also, Swiech teaches Determine the cell density of the culture by performing a cell count using an automated cell counter (See page 79, Method 3.1), and discloses that new GMP-compliant technology for cell expansion is the CliniMACS® Prodigy (Miltenyi Biotec), which is a one-solution, automated platform, that includes all the CAR-T cell manufacturing steps: cell selection, activation, transduction, expansion, cell washing, and final formulation in a fully closed system that can reducing operator time and the cost (See e.g page 145, paragraph 1).
It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to introduce the automated culturing system into Sastry’s research and arrive at an invention as claimed. Since the Swiech’s automated system for cell culturing is focused on the T cell culturing system, one of skill in the art would have been motivated to introduce the automated system into the C8166 cell-based RCL detection system based on the advantage described above. There would be a reasonable expectation of success to adopt the automated T cell to culturing C8166 because C8166 is derived from human umbilical cord blood lymphocytes, which is also a T cell line.
Allowable Subject Matter
The SEQ ID NO: 1 of claims 20 and 25 is free of art.
Regarding claim 25, no prior art reference has been identified teaching or suggesting a replication competent virus comprising the nucleic acid sequence according to SEQ ID NO: 1. Accordingly, the base claim 25 is allowable.
Responses to Applicant’s Remarks
Applicant’s arguments filed on Oct. 15, 2025 has been received and fully considered.
Applicant’s argument on rejections under 35 U.S.C. § 112 (b) is considered and the rejection is withdrawn.
Applicant’s argument on claims 20-22 under 35 U.S.C. § 103 is considered and the rejection is withdrawn.
Applicant’s arguments on rejection under 35 U.S.C. § 103 except claims 20-22 is not found persuasive as the follows:
1. Applicant argued a surprising/unexpected finding that individual aliquot volumes that are compatible with automation can be used to successfully amplify any replication competent virus present within the test article, while also successfully diluting out any activity from the test article that would adversely affect detection of actual replication competent virus that may be present by the end-point assay. The inventors have shown that replication competent virus can still be accurately detected, even when low individual aliquot volumes are used. (See the instant Remarks, pages 5-6). Also, applicant argued it is surprising that using a plurality of aliquots with small culture volumes (low individual aliquot volumes) retains sensitivity over the assay, as demonstrated in the present application. Indeed, there is no indication in Sastry that the required sensitivity of the disclosed assay would be maintained if smaller volumes were used (See Remarks, pages 6 and 8, paragraph 2).
Applicant’s argument is not persuasive.
1). The volume of the cell culture varies depending on the experimental goal and design. One of skilled in the art can modified an optimal culture volume based on the needs through routine experimental optimization. In addition, the base claim 1 does not limit if the method of detecting replication competent virus needs to be a regular method or automation. Therefore, an automation-based culture volume can teach the alleged individual aliquot volumes because they are compatible as allegedly stated in the instant remarks.
2). Sastry actually teaches the alleged low individual aliquot volumes (See table 1) including the dilution factors of 2 (See the rejection on the claim 1-b)) based on the different container such as using different size of culture flasks. Also, the cell of C8166 of Sastry is also used by the instant application (See instant specification [0271]), which indicates that it is obvious for one of skilled in art can reach a “alleged low individual aliquot volumes” if need with the same cell line.
3). As for the unexpected sensitivity of the small culture volume, Sastry not only teaches a 25-cm 2 flasks with less than 10 ml culture volume, but also teaches that the level of sensitivity will be based on the dilution of the virus. For example, use of virus at the TCID 50 would suggest maximal sensitivity of the assay (See page 255, paragraph 4), and “Sensitive assays have been developed for detection of RCL indicative markers …”. In addition, there is no limitation for the sensitivity in the instant claims.
4). As an initial matter, “the burden of showing unexpected results rests on one who asserts them. Thus it is not enough to show that results are obtained which differ from those obtained in the prior art: that difference must be shown to be an unexpected difference.” In re Klosak, 455 F.2d 1077, 1080 (CCPA 1972) (citation omitted). Moreover, “[i]t is well settled that unexpected results must be established by factual evidence. Mere argument or conclusory statements in the specification does not suffice.” In re De Blauwe, 736 F.2d 699, 705 (Fed. Cir. 1984) (citation omitted).
2. Applicant argued that traditional methods for testing gene therapy products before clinical release typically require fifteen or more flasks with at least 40 ml culture to initiate each assay and more than 100 flasks need to be processed over the 3–4-week time course of the assay, which is time consuming and labor intensive (See Remarks, page 6).
The argument is not persuasive because the instant claims does not limit the number of flasks and the process time for detecting the gene therapy products before clinical release. In the instant specification, it discloses that “Step c) of the methods described herein comprises culturing the aliquots for at least a further six days. For the avoidance of doubt, the aliquots may be cultured for a longer duration, for example for at least a further seven, eight, nine, ten, eleven or more days before testing for replication competent virus. The methods described herein may therefore take at least 3 weeks, e.g. 3 to 4 weeks or 4 to 5 weeks to complete (See [0211). Therefore, the weeks for cell culture process can be optimized through routine experimental expectation, especially the applicant and Sastry use the same cell line of C8166.
3. Applicant argued that Sastry describes assays that lack the advantageous features of the presently claimed method. It illustrates a typical replication competent lentivirus (RCL) detection assay in Figure 2 on page 247, and provides methods for replication competent retrovirus (RCR) testing, starting on page 251 and Sastry does not use individual cell culture aliquots each with an aqueous volume of 10 ml or less. Although Tables 1 and 3 in Sastry disclose small volumes (of 1 to 5 ml), these volumes relate to the amount of test sample ("test article") (e.g., final vector product/EOPCs) to be tested not the total volume of the cell culture aliquot (i.e., the discrete volume that is going to be cultured in the assay). (See Remarks, page 7, paragraphs 1-4).
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The argument is not persuasive.
The instant claims do not limit the methods for RCL or RCR detection. Sastry teaches the limitations for the detection as claimed in the instant application. As for the Table 1 of Sastry (See page 251 and below), it is a common knowledge in the art that the aqueous volume/cell culture of a 25-cm2 flasks with 2 × 10^5 cells per flask is less than 10 ml. This can be evidenced by Conning (https://www.corning.com/catalog/cls/documents/application-notes/CLS-AN-209.pdf), which teaches that a recommended medium volumes of 25-cm2 flasks with a similar density as claimed is 5-7.5 ml. Based on the description above, Sastry teaches the elements argued as “(1) uses small, individual cell culture aliquot volumes of 10 ml or less, (2) which small individual cell culture aliquot volumes are used in: culture step a), and in culture step b ), in which the aliquots are passaged using a dilution factor of at least 2 at each passage, and the combined aqueous volume of all aliquots being cultured at the end of step b) is 50% or less compared to the combined aqueous volume of all aliquots being cultured at the start of step b )” (See Remarks, page 7).
4. For claim 17, applicant argued that the system of Sastry is not amenable to automation because the volumes required are too large (See page 8, paragraphs 3-4).
The argument is not persuasive because Sastry teaches the required volume (10 ml less) (See Sastry’s Table 1).
Conclusion
Claims 1-4, 6-10 and 14-16 and 18-20 are not allowed. Claim 25 is 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 extension fee 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 date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to RUIXUE WANG whose telephone number is (571)272-7960. The examiner can normally be reached Monday-Friday 8:00 am-5:00 pm, EST.
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/RUIXUE WANG/Examiner, Art Unit 1672
/THOMAS J. VISONE/ Supervisory Patent Examiner, Art Unit 1672