Replication Competent Virus Assay

§103 §112

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 April 22, 2026. Claims 1-4, 6-10, 14-20 and 25 are pending and currently examined. Claim Objections (Previous objection- withdrawn)Claim 25 is objected to because of the following informalities: The “comprising” in claim 25 should be “comprises”. This objection is withdrawn in view of the arguments filed on July 27, 2026. 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 (Previous rejection- withdrawn) Claims 20 and 25 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out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. This rejection is withdrawn in view of the arguments filed on July 27, 2026. (Previous rejection- withdrawn) Claims 1-4, 6-10 and 14-20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. This rejection is withdrawn in view of the arguments filed on July 27, 2026. Claim Rejections - 35 USC § 112 (Scope of Enablement) The following is a quotation of the first paragraph of 35 U.S.C. 112(a): (a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention. The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112: The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention. (Previous rejection- withdrawn) Claims 1-4, 6-10 and 14-19 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling detecting HIV replication competent virus in HEK293T and C8166-45 cells using F-PERT (Fluorescence Product-Enhanced Reverse Transcriptase) assay, does not reasonably provide enablement for using a method being enabling to detect any replication competent virus in any cell culture through any testing method. This rejection is withdrawn in view of the arguments filed on July 27, 2026. 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- 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 base claim 1 is directed to a method for detecting replication competent virus in cell culture aliquots, 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, wherein each aliquot has 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 cell culture 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. 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 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). PNG media_image1.png 279 808 media_image1.png Greyscale Therefore, Sastry teaches the step a) of claim 1, where the “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 limitation of “10 ml or less”, Sastry teaches that the cell can be seeded in the 6-well plate (See page 253, 3.1.3) or 25-cm 2 flasks (See page 252; Table 2; ) that require less than 10 ml culture volume. Also, the 6-well plate is considered as “a plurality of individual cell culture aliquots” as claimed. For step b) in 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 passed 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. PNG media_image2.png 488 503 media_image2.png Greyscale As for the limitation of claim 1 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 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 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). 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). PNG media_image3.png 247 433 media_image3.png Greyscale 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 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- maintained) 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 includes 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 SEQ ID NO: 1 of claims 20 and 25 is free of art. Accordingly, claims 20 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims. Claim 25 is allowable. Responses to Applicant’s Remarks Applicant’s arguments filed on July 27, 2026, have been received and fully considered. Applicant’s argument regarding the objection is considered. The objection is withdrawn. Applicant’s arguments regarding the rejection under 35 U.S.C. § 112 (b) is considered. The rejection is withdrawn. Applicant’s argument regarding the rejection under 35 U.S.C. § 112 (a) (Scope of Enablement) is considered. The rejection is withdrawn. Applicant’s argument regarding the rejection under 35 U.S.C. § 103 is not found persuasive. 1). Applicant argued that the Response to Applicant's Remarks never explains how Sastry discloses or suggests managing the combined aqueous volume of a plurality of individual aliquots during the assay. Instead, it dismisses Applicant's explanation of the claim language because the explanatory phrases themselves do not appear in the claim. That does not address the substance of Applicant's argument (See Remarks, page 10, paragraph 2). The argument is not persuasive. Based on the description above, Sastry teaches the base claim 1-a) to c). As for the argued phrases such as "configuring the assay as a plurality of discrete culture aliquots," "controlling relationships between multiple parallel culture volumes," or "a discrete cell culture reaction chamber.", they are not presented in the instant claims and not disclosed in the instant specification. In addition, applicant does not clarify how to do the “configuring” and “controlling”. Accordingly, one of ordinary skill in the art would not know the metes and bounds of the exact means of the argued phrases. Nevertheless, Sastry teaches the 6-well plate culture that can be considered as “a plurality of individual cell culture aliquots” as claimed. As for the argument of “The Response to Applicant's Remarks never explains how Sastry discloses or suggests managing the combined aqueous volume of a plurality of individual aliquots during the assay”, which is also not persuasive. First, Sastry teaches the cell passage can be scaled down (See page 252, paragraph 2 and Table 2). Second, Sastry teaches making the cell culture size down during the cell passage (See page 252, Table 2 and paragraph 2) and teaches a plurality of individual aliquots by stating using 6-well plates (See e.g., page 253, 3.1.3.). At the same time, Sastry’s “scaling down of large flasks with later passages” for the RCL assay can be evidenced by prior art references in the fields. For example, iCorre ((Hum Gene Ther. 2016 Feb;27(2):202-10, hereinafter, “Corre”) teaches how the passage can be scaled down. Corre teaches “RCL-Pooling Assay, a Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling” (Hum Gene Ther. 2016 Feb;27(2):202-10). They teach a method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized ‘‘RCL-pooling assay’’ ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity (See Abstract; Figure 1, page 206 and below). PNG media_image4.png 517 831 media_image4.png Greyscale Third, the instant specification discloses the “combine” as “the cells from two parent aliquots may be harvested and combined ("pooled") and a proportion of the combined cells may be used to re-seed one daughter aliquot. In another example, the cells from two parent aliquots may be harvested but not combined, and a proportion of each of the parent cells may be added to one daughter aliquot to effect pooling of the cells from those parents. Pooling therefore reduces the overall number aliquots after each passage” (See [0193]). Fig. 3 of the instant specification shows a comparable method as taught by Corre for reducing the volume (See instant specification, [0081] and below). PNG media_image5.png 399 644 media_image5.png Greyscale 2). Applicant argued “the Office Action does not respond to Applicant's explanation that the volumes reported in Sastry's Table 1 are test sample volumes, not culture volumes. Sastry expressly states that "[t]he size of the amplification phase depends on the volume of the sample being tested" and that Table 1 "indicates the number of cells that will be needed based on the test volume." Sastry at 251 ( emphasis added). The cited values therefore describe the amount of test article introduced into the assay, not the total aqueous volume of an individual cell culture aliquot. Rather than addressing this distinction, the Office Action states only that "one of skilled in the art can adjust the volume to '10 ml or less' based on the needs." Office Action at 19. The ability to adjust a culture volume does not establish that Sastry teaches or suggests configuring the assay using a plurality of individual aliquots, each having an aqueous volume of 10 mL or less, as required by claim 1. The argument is not persuasive. Sastry teaches that the cell can be seeded in the 6-well plate (See page 253) or can be passed into 25-cm 2 flasks (See page 252; Table 2), where the culture volume is less than 10 ml. Sastry also teaches determining the total number of 6-well plates required for the assay. Three wells will be needed for each test article. For the control samples 30 wells (6 plates) will be needed (See page 253, 1.1.3). Here the 6-well plate can be considered as “a plurality of individual cell culture aliquots” as claimed. The teaching of the claimed “each aliquot has an aqueous volume of 10 ml or less” does not depend on the Table 1 as argued. In addition, Table 1 is used in the office action as the size of cell-seeding vs. the test articles. 3). Applicant argued that the same reasoning applies to the feature that "the combined aqueous volume of the plurality of individual cell culture aliquots is reduced by at least 50%." , and no evidence is cited that reducing the aggregate culture volume of a plurality of aliquots during a replication competent virus assay was recognized in the art as a result-effective parameter, much less one that would have been expected to preserve assay sensitivity. Nor does the Office Action identify any disclosure in Sastry suggesting that the total volume of multiple aliquots should be deliberately reduced during the assay (See Remarks, page 10, paragraph 4 and page 11, paragraph 1). The argument is not persuasive. (i). Table 2 teaches at least 50% scale down of cell culture volume as described above (from 300 cm2 to 75 cm2). At the same time, (ii). The cell culture claimed in the base claim 1 is a broad claim with generic cell culture, however, the “aggregate culture” as argued is specific for "non-adherent cells" that is also disclosed in the instant specification (See instant specification, [0151]). At the same time, Sastry uses a same permissive “non-adherent cell” of C8166-45 as Applicant used, so, a similar result-effective parameter would be recognized to the researchers in the art, such as “The cells will often have a preferred range of densities for optimal growth and subculture (referred to as "passaging" herein) will normally try to keep the cells in this range” as disclosed in the instant specification (See instant specification, [0151]). Nevertheless, Sastry teaches “In these assays, test material is placed on a permissive cell line, and the cells are passaged for a minim um of 3 weeks (amplification phase)” (See page 245, paragraph 3), where the process of passaging cells implies a cell-aggregation reduction. Furthermore, Corre also uses the C8166-45 as the permissive cell line and teaches that “This assay is based on the amplification of the putative RCL after infection of a highly permissive C8166-45 T-cell line. Then, by subsequently culturing these cells over several passages, it is possible to detect a single infection event by observing HIV-1 p24 protein levels increasing over the background level” (See Corre, page 203, left column, paragraph 3). Accordingly, culturing “non-adherent cell” by passaging and dilute cells is a routine technique in the art. Additionally, the instant specification only disclosed the “aggregate culture” once, and the disclosure is for taking a small volume of the parent culture and diluting it in fresh growth medium (See [0151]), which is not the same as the argument of “reducing the aggregate culture volume of a plurality of aliquots during a replication competent virus assay”. (iii). As for the argument of “ Nor does the Office Action identify any disclosure in Sastry suggesting that the total volume of multiple aliquots should be deliberately reduced during the assay”, Sastry teaches the volume of cell culture can be scale down (See Table 2; page 252 and paragraph 2). Corre teaches that a scale down of culture volume is achievable while the sensitivity of the assay remaining at a similar level, for example the P24 detection simulated-pooling methods (See Corre, Figures 3 and 4). 4). Applicant argued the responses on the Declaration filed on April 22, 2026, in the previous office action filed on 05/05/2026 (See Remarks, page 11). (i). The previous responses to the Declaration Under 37 CFR 1.132 are proper and maintained. (ii). Applicant argued that the Declaration itself constitutes factual evidence regarding the knowledge and expectations of persons of ordinary skill in the art. The Office Action neither rebuts that evidence nor explains why the unrebutted testimony regarding the technical expectations in the field should be disregarded (See Remarks, page 11, paragraph 2). The argument is not persuasive. The “ knowledge and expectations of persons of ordinary skill in the art” cannot be considered as the “ factual evidence” at this situation. Both Sastry and the instant application use a 6-well plate to grow the virus, and both Sastry and the instant application use PCR and ELISA to detect the RCL. It is obvious that a small volume culture can satisfy the sensitivity of detection with PCR-based or ELISA-based essays. For example, iiSchalk teaches a rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR (J Virol Methods. 2007 Nov;145(2):89-95), where the cell culture is performed using the 96-wells plate (35,000 cells/well) and volume of the cell culture is 100ul (See page 91, left column, paragraph 2). Conclusion Claims 1-4, 6-10, 14-20 are not allowed. Claim 25 is allowed. 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. 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, Thomas J. Visone can be reached on (571) 270-0684. 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. /RUIXUE WANG/Examiner, Art Unit 1672 /NICOLE KINSEY WHITE/ Primary Examiner, Art Unit 1672 i Corre is cited solely to respond to applicant’s argument and not to reject any claim. ii Schalk is cited solely to respond to applicant’s argument and not to reject any claim.