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 .
Application Status
This action is written in response to applicant’s correspondence received 06/23/2026. Claims 1-2, 4-5, 8, 10-14, 28, 30, 36-38, 41, 60, 64, 68, and 91-107 are currently pending. Claim 11 is withdrawn from prosecution as being drawn to non-elected subject matter. Claims 106 and 107 are newly added. Accordingly, claims 1-2, 4-5, 8, 10, 12-14, 28, 30, 36-38, 41, 60 64, 68, and 91-107 are examined herein. The restriction requirement mailed 12/26/2023 is still deemed proper.
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 factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4-5, 12-14, 28, 30, 36-38, 41, 60, 91-93, and 96-97 are rejected under 35 U.S.C. 103 over Lada (Lada et al. Measurement of Integrated HIV DNA by Pulse-Field Gel Electrophoresis and ddPCR. Poster Presentation. CROI conference, Boston, 2016.;of record, applicant’s submission) in view of Charrier (Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction. Gene Therapy (2011) 18, 4 79-487.; of record, applicant’s submission).
Regarding claim 1, Lada teaches a method for assessing genomic integration of a transgene, the method comprising separating a high molecular weight fraction of DNA of greater than 10kb (15kb+)from isolated DNA, and from the high weight fraction, determining, by PCR, the copy number of the transgene (i.e., gene which does not naturally occur in that cell) sequence, wherein the PCR is carried out using a pair of primers that are complementary to or are capable of specifically amplifying sequences of the transgene sequence (i.e., viral transgenes such as gag):
PNG
media_image1.png
172
599
media_image1.png
Greyscale
PNG
media_image2.png
283
595
media_image2.png
Greyscale
While Lada does not explicitly teach determining the copy number per diploid genome, they do teach calculating the copy number per 1E6 cells using human cells and cell lines. The ordinary artisan would have been able to calculate copies per diploid genome based on the number of cells assayed.
Lada does not teach that, prior to the separating, a polynucleotide comprising the transgene sequence encoding the recombinant protein has been introduced, via transduction with a viral vector, into at least one cell of the one or more cells to produce at least one engineered cell. The broadest reasonable interpretation of an engineered cell encompasses cells which have been genetically engineered to express transgenes, but excludes cells infected with naturally-occurring viruses without human intervention. Lada does not teach engineered cells. Instead, Lada teaches the measurement of integrated HIV DNA in infected CD4+ T cells of patients undergoing antiretroviral therapy (Background).
However, Lada does teach that the method has an advantage over ddPCR alone because ddPCR does not distinguish between integrated and unintegrated molecular forms (Background). Lana further teaches that the method provides a sensitive and precise approach to the measurement of integrated viral (HIV) DNA with sufficient throughput for translational research studies (Cocnlusions). Therefore, Lada provides a teaching, suggestion or motivation to apply the method to other cells and viruses.
Charrier teaches the quantification of lentiviral vector (HIV-1-derived, rHIV) copy numbers in genetically corrected hematopoietic stem cells (Title, p. 479). They further provide a teaching, suggestion or motivation to precisely and sensitively quantify vector copy number, noting (Id.):
The biological potency of the vector is expected to correlate positively with the frequency of transduced cells and also with the number of integration per cell unless transgene silencing is observed, as suggested in some studies. At the same time, genotoxicity related to the number of vector insertions per cell can result from inappropriate transgene expression in cells or from effects of elements contained in the integrated cassette, to thus requiring strict control of the number of gene insertion per cell. It is therefore important to determine the distribution of vector copies in the infected cell population at the single cell level to assess the efficacy and safety, that is, the therapeutic window of integrative vectors.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of measuring integrated HIV DNA using PFGE and ddPCR, as taught by Lada, to measure integrated gene therapy viral vectors in engineered cells, as taught by Charrier. The ordinary artisan would have been motivated to do so based on the combination of Lada’s disclosures that the method was sensitive and precise, and on Charrier’s disclosures that sensitive, precise methods of measuring integrated viral vectors were needed in the field of stem cell gene therapy. The ordinary artisan would further have had a reasonable expectation that Lada’s method could successfully have been applied to HIV-derived lentiviral vectors based on Lada’s disclosure that the method was effective, precise and sensitive when applied to genome-integrated exogenous genes.
Regarding claim 2, Lada teaches a step of isolating the DNA from the cells (see above in Methods).
Regarding claim 4, by teaching the method being applied to CD4+ T cells of HIV positive patients undergoing antiretroviral therapy, Lada teaches that the one or more cells comprises a population of cells in which a plurality of cells comprises the transgene (HIV) sequence.
Regarding claim 5, Lada teaches calculating the copy number per a certain number of cells, as discussed above, and it would have been well within the means of the ordinary artisan to calculate a mean copy number per genome based on that information.
Regarding claim 12, Lada teaches wherein the one or more cells is a primary cell obtained from a sample from a subject (see above).
Regarding claims 13 and 14, Lada teaches wherein the one or more cells is an immune cell (T cell).
Regarding claims 28 and 91, Lada teaches wherein the separating is carried out by pulse field gel electrophoresis (see above).
Regarding claim 30, the claim recites the limitations of claims 28 and 5 in combination, which are taught by Lada as described above.
Regarding claims 36-38, Lada teaches wherein the cutoff between the low and high molecular weight fractions is about 15 kb, and therefore teaches that the high molecular weight fraction is greater than about 15, about 17.5, or about 20 kb.
Regarding claim 41, Lada teaches wherein the PCR is ddPCR (see above).
Regarding claim 60, Charrier teaches wherein the viral vector is a lentiviral vector (see above).
Regarding claim 92, Lada teaches wherein the copy number is determined per cell expressing the viral protein.
Regarding claim 93, Lada teaches wherein the one or more cells are viable cells (long-lived CD4+ T cells isolated from living subjects, and thus presumed to be viable).
Regarding claim 96, Lada teaches wherein the primary cell is a T cell.
Regarding claim 97, Lada teaches wherein the copy number is determined per cell, as described above.
Claims 8 and 106-107 are rejected under 35 U.S.C. 103 as being unpatentable over Lada and Charrier, as applied to claims 1-2, 4-5, 12-14, 28, 30, 36-38, 41, 60, 91-93, and 96-97 above, further in view of Zhang et al. (The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events. Genetic Vaccines and Therapy 2004, 2:6.).
Lada and Charrier render obvious the invention of claim 1, from which the instantly rejected claims depend, as described above.
Lada and Charrier do not teach wherein the one or more cells has not been incubated at a temperature that is at or about 37C for more than 96 hours following transduction.
Zhang teaches a series of experiments examining various aspects of lentiviral vector transduction, including the determination of transduction efficiency (i.e., transgene integration and expression of EGFP) at 48 hours after transduction (p. 3/10). It is noted that the 48 hour post-transduction time point falls within the windows of less than 3 or 4 days recited in claims 107 and 106, respectively, and is consistent with the limitation of culturing not more than 96 hours (4 days) as recited in claim 8.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method as taught by Lada and Charrier to comprise a step of determining transduction efficiency (i.e., transgene integration) within fewer than 4 days. Based on common sense and sound scientific reasoning, the ordinary artisan would have been motivated to validate transduction efficiency as soon as possible after transduction, to save experimental time and cost. As taught by Zhang, vector integration was measurable 48 hours after transduction, which would have given the ordinary artisan a reasonable expectation that, using known methods, validation would have been effective at those time points.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Lada and Charrier, as applied to claims 1-2, 4-5, 12-14, 28, 30, 36-38, 41, 60, 91-93, and 96-97 above, further in view of Xu et al. (of record).
Lada and Charrier render obvious the invention of claim 1, from which the instantly rejected claims depend, as described above.
Lada and Charrier do not teach wherein the one or more cells has been cryopreserved prior to the separating of the high molecular weight fraction.
As discussed in the final rejection of 02/25/2025, Xu teaches that cryopreservation of CAR T cells is necessary for their clinical application, and measurement of transduction efficiency can be done after cryopreservation to assess those parameters after resuscitation (Abstract)
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method as taught by Lada and Charrier to comprise a cryopreservation step before assessing the integration of the transgene. The ordinary artisan would have predicted, based on Xu’s teachings, that this would have permitted the successful preservation of the cells without negatively impacting the cells’ functionality, and that it could be applied to CAR T cells, thus facilitating their preservation and functionality until such time as it was appropriate to begin assessment of integration and/or use the cells in a clinical setting.
Claims 64, 68, 94-95, and 98-105 are rejected under 35 U.S.C. 103 as being unpatentable over Lada and Charrier, as applied to claims 1-2, 4-5, 12-14, 28, 30, 36-38, 41, 60, 91-93, and 96-97 above, further in view of Hartmann (Clinical development of CAR T cells—challenges and opportunities in translating innovative treatment concepts. EMBO Mol Med (2017) 9: 1183–1197.).
Lada and Charrier render obvious the invention of claim 1, from which the instantly rejected claims depend, as described above.
Lada and Charrier do not teach wherein the transgene expresses a recombinant receptor (claims 64, 94, 98) which is a CAR T cell receptor (claims 68, 95, 99, 100-105).
Hartmann teaches various factors in the clinical development of CAR T cells (Title). They further teach that, in the manufacture of CAR T cells, the “need for GMP-compliant manufacturing may constitute a specific hurdle in the timely translation to the clinic” (p. 1192). They note that, “Transduction efficiency, transgene expression levels, and copy numbers per cell are additional factors, which can all directly impact on efficacy and safety.”
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for assessing transgene integration and copy number in engineered cells, as taught by Lada and Charrier, by applying it specifically to engineered CAR T cells for clinical applications. As taught by Hartmann, transduction efficiency (i.e., the number of cells which successfully integrate the transgenic CAR) and copy numbers per cell are important factors in the efficacy and safety of CAR T cells. This would have motivated the ordinary artisan to measure those parameters in CAR T cells, while Lada and Charrier provide a reasonable expectation that the method of Lada could be applied in both primary and engineered immune cells.
Response to Arguments
Applicant's arguments filed 06/23/2026 have been fully considered but they are not persuasive for the reasons that follow.
Applicant argues that, “Charrier does not teach or suggest separating a high molecular weight fraction of DNA from engineered cells and then determining, from that fraction, the copy number of the transgene sequence integrated into the genome as recited in the claims as a whole.” (p. 13). Applicant further argues that e.g., “Lada does not determine copy number…nor provide any reason for doing so”. On page 19, Applicant argues that, “Lada’s method is different from the claimed method” in that it “does not teach engineered cells, does not teach introducing a polynucleotide comprising a transgene sequence encoding a recombinant protein by transduction with a viral vector, does not teach separating high molecular weight DNA from such engineered cells, does not teach determining copy number per diploid genome or per cell or per cell expressing the recombinant protein, and relates to an entirely different technology context”. These arguments all address the references individually, but do not address the teachings provided by the combination of the references. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). As discussed in the rejection of the claims under 35 USC above, Lada teaches a method for assessing genomic integration and copy number of an HIV provirus, provides a motivation to apply that method for assessing genomic integration by teaching that it is sensitive and accurate for that purpose, and Charrier provides a motivation to apply said method to other contexts such as assessing genomic integration and determining the copy number per cell of lentiviral vectors because it is, “important to determine the distribution of vector copies in the infected cell population at the single cell level to assess the efficacy and safety, that is, the therapeutic window of integrative vector”. In combination, the references provide two analogous applications in which it is desirable to assess integration of a transgene, and Lada provides a way to carry out that assessment. Charrier is merely cited to provide a motivation for the ordinary artisan to use that method to measure a transduced viral vector in an engineered cell as opposed to the naturally occurring viral vector in an isolated cell. Absent evidence to the contrary, and given the teachings of the prior art, the ordinary artisan would have had a reasonable expectation that Lada’s method for assessing genomic integration of an HIV provirus (i.e., exogenous gene(s) or transgene(s)) by separating the high molecular weight fraction prior to ddPCR could have successfully been applied to lentiviral vectors such as Charrier’s, and would have been motivated to apply it.
Contrary to Applicant’s assertion that Lada’s poster relates to a much different technological context (p. 13), both Lada and Charrier are analogous in that they concern assessing genomic integration of exogenous genes in a cell, and both express a need in the art to perform that assessment. What differs is the source of the exogenous gene. Applicant has not provided evidence that the source of the exogenous gene results in structural differences which would have rendered a method of separating the genomic DNA from the non-genomic DNA and measuring the copy number of the transgene in one or both, as in Lada, inoperable.
Applicant further notes various specific method steps disclosed by Charrier, and argues that POSITA would have found no reason or motivation to combine Lada’s method with Charrier’s method, and that modifying critical parameters such as Charrier’s genomic DNA extraction increases the complexity and variables at play without any recognized benefit (pages 14-15). This argument is not persuasive because, as noted above, Charrier is cited merely to provide a motivation to apply Lada’s method to assessing genomic integration of a lentiviral vector, and the rejection does not rely on any of the specific method steps described by Charrier.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references (at pages 13, 14, 15, 18), the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007).
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning (at p. 18), it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Regarding claim 8, Applicant’s argument that Zhang’s teachings relate to counting EGFP positive cells or staining cells 48 hours of transduction similarly rely on the alleged deficiencies of Zhang’s methodology. As discussed above with regards to Charrier, this argument is not persuasive because Zhang, like Charrier, is not cited to provide the particular detection methodology, but is cited merely to indicate that the ordinary artisan would have been motivated to measure transduced cells within a short time frame after transduction with viral vectors, as discussed in the rejections above.
Regarding the remaining rejections of the dependent claims, Applicant’s arguments that the cited references do not address the deficiencies of Lada in view of Charrier are not persuasive for the reasons articulated above.
Conclusion
No claim is allowed at this time.
Applicant's amendment to add new claims 106 and 107 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 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 AMANDA M ZAHORIK whose telephone number is (703)756-1433. The examiner can normally be reached M-F 8:00-16:00 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, Neil Hammell can be reached at (571) 270-5919. 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.
/AMANDA M ZAHORIK/Examiner, Art Unit 1636
/BRIAN WHITEMAN/Primary Examiner, Art Unit 1636
