DETAILED ACTION
Disposition of Claims
Claims 1-20 were pending. Claims 1-12 and 21 have been cancelled. Amendments to claim 13 are acknowledged and entered. Claims 13-20 will be examined on their merits.
Examiner’s Note
All paragraph numbers (¶) throughout this office action, unless otherwise noted, are from the US PGPub of this application US20230038151A1, Published 02/09/2023. Amendments to the specification presented on 10/01/2025 are acknowledged and entered.
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Response to Arguments
Applicant's arguments filed 10/01/2025 regarding the previous Office action dated 07/03/2025 have been fully considered. If they have been found to be persuasive, the objection/rejection has been withdrawn below. Likewise, if a rejection/objection has not been recited, said rejection/objection has been withdrawn. If the arguments have not been found to be persuasive, or if there are arguments presented over art that has been utilized in withdrawn rejections but utilized in new rejections, the arguments will be addressed fully with the objection/rejection below.
Specification
(Objection withdrawn). The objection to the abstract of the disclosure is withdrawn in light of the amendments to the abstract.
Claim Objections
(Objection withdrawn). The objection to Claims 1 and 13 is withdrawn in light of the amendments to the claims.
Claim Interpretation
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art.
Claim 13 is drawn to a method of determining a viral titer in a biological sample, consisting essentially of:
a) obtaining the biological sample which contains a virally-transduced cell;
b) mechanically disrupting the virally-transduced cell of the biological sample with glass beads to generate a crude lysate;
c) conducting droplet digital polymerase chain reaction (ddPCR) on nucleic acid molecules in the crude lysate from the disrupted virally-transduced cell; and
d) calculating the viral titer.
Further limitations on the method of claim 13 are wherein the virally-transduced cell is a mammalian cell (claim 14), wherein the mammalian cell is a human cell (claim 15) or a Chinese hamster ovary (CHO) cell (claim 19), wherein the human cell is a human embryonic kidney (HEK) cell (claim 16), wherein the viral titer is an adeno-associated virus (AAV) viral titer (claims 17 and 20) or a lentivirus viral titer (claims 18 and 20).
Claim Rejections - 35 USC § 102
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
(Rejection withdrawn.) The rejection of Claims 1-2, and 4-8 under 35 U.S.C. 102(a)(1) as being anticipated by Lock et. al. (US20190002841A1, Pub. 01/03/2019; hereafter “Lock”) as evidenced by Lock et. al. (Lock M, et. al. Hum Gene Ther Methods. 2014 Apr;25(2):115-25. Epub 2014 Feb 14.; hereafter “Lock-2014”) is withdrawn in light of the cancellation of said claims.
(Rejection withdrawn.) The rejection of Claims 1-2, 4-8, and 10-11 under 35 U.S.C. 102(a)(2) as being anticipated by Drouin et. al. (WO2020072849A1, Pub. 04/09/2020, Priority 10/04/2018; hereafter “Drouin”) is withdrawn in light of the cancellation of said claims.
(New rejection – necessitated by amendment.) Claims 13-16 and 18 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Basha et. al. (Basha IHK, et. al. Micromachines (Basel). 2017 Aug 30;8(9):266.; hereafter “Basha”.)
The Prior Art
Basha teaches a variety of point-of-care (POC) and high throughput molecular diagnostic methods in order to determine diagnosis of viruses (entire document; see abstract.) Basha teaches techniques for lysis of cells infected by viruses (p. 3, “2. Lysis Techniques”), nucleic acid extraction from the cell lysate (p. 8, “Nucleic Acid Extraction”), and amplification of said nucleic acid (p. 11; “Amplification”). Basha teaches mechanical lysis techniques of cells (p. 4, “2.2. Mechanical Lysis”), wherein the mechanical lysis includes shearing, shocking, grinding, or beating (p. 4, ¶4). Basha teaches that bead-based cell lysis can disrupt the cellular membrane (¶3, p. 5) using zirconia or silica (glass) beads (Fig. 2B). Basha teaches that after lysis, in these POC devices, the crude lysate can be released and subjected to nucleases to inhibit nucleic acid destruction (¶5, p. 8). Basha teaches that amplification prior to detection is required because the amount of nucleic acid extracted is typically insufficient for direct detection (p. 11, ¶1), and that digital amplification of nucleic acids is a novel approach that integrates amplification and detection to generate additional information about the quantity of genomic material present in the sample (Sect. 4.3, starts at p.18). Basha teaches that digital PCR was more sensitive than other detection methods to quantify the viral titer of human cytomegalovirus (HCMV) in a sample (p. 18, ¶3), and that droplet-based digital detection (ddPCR) can be integrated into microfluidic devices (p. 18, ¶3 to p. 20), especially ones with self-priming compartmentalization (SPC), which eliminates the need for valves and pumps (p. 19, ¶3-4). While Basha notes that purification of the crude lysate improves amplification and detection, it is not required (p. 8, ¶5). Basha therefore teaches the limitations of instant claim 13, and anticipates the invention encompassed by said claim.
Basha teaches mechanical lysis of mammalian cells, such as white blood cells (WBCs) from whole blood, HeLa cells, HEK293, and HaCaT cells (pp. 4-6; instant claims 14-16). Basha teaches that viruses such as HCMV and HIV can have their copy number determined (p. 23, ¶2; instant claim 18). Basha teaches that CHO cells can be lysed with electrical means (p. 8, ¶2).
Basha teaches the limitations of instant claims 13-16 and 18, and anticipates the invention encompassed by said claims.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
(Rejection withdrawn.) The rejection of Claims 3, 9, and 12-20 under 35 U.S.C. 103 as being unpatentable over Drouin as applied to claims 1-2, 4-8, and 10-11 above and over Lock as evidenced by Lock-2014 as applied to claims 1-2, and 4-8 above and further in view of Goldberg et. al. (US20180057808A1, Pub. 03/01/2018; hereafter “Goldberg”) is withdrawn in light of the amendments to the claims.
(New rejection – necessitated by amendment.) Claims 17 and 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Basha as applied to claims 13-16 and 18 above, and further in view of Ai et. al. (Ai J, et. al. Hum Gene Ther Methods. 2017 Jun;28(3):139-147. Epub 2017 Apr 13.; hereafter “Ai”.)
The Prior Art
The teachings of Basha have been set forth supra. While Basha teaches methods of isolating viral nucleic acid from virally-transduced cells, and teaches methods of mechanical disruption of cells, such as the use of glass beads for “bead beating” techniques to disrupt the cells. While Basha teaches different types of cells that are infected/transduced by different viruses throughout the review and teaches the electrical means for disruption of CHO cells, Basha never directly teaches the mechanical disruption of CHO cells, nor does Basha teach the isolation of adeno-associated virus (AAV). However, the use of ddPCR to assess viral titer, such as AAV viral titer, from crude cell lysates was known or suggested in the art, as taught by Ai.
Ai teaches increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. However, a reproducible test for determining AAV titer is lacking (entire document; see abstract.) While Ai teaches the freeze/thaw disruption of AAV-infected cells and performing the qPCR directly on the crude cell lysate (“Materials and Methods: Small-scale vector production” to “Quantitative PCR”) is equally reproducible as isolating the viral nucleic acid from the cell lysate (Fig. 2), Ai also teaches that the use of ddPCR has emerged as a powerful technique for the absolute quantification of purified rAAV, and that this alternative method would abolish the need to compare to a standard curve for accurate quantitation and would further improve the sensitivity of in-process titration of crude lysates following their pretreatment protocol (p. 146, left col., ¶2).
Given the teachings of Basha, one of skill in the art would be apprised as to methods of disrupting virally-transduced cells using mechanical means and then testing said cell lysates using ddPCR to determine the viral titer. Give the teachings of Ai, one of skill in the art would be further apprised as to the non-necessary step of purifying the viral nucleic acid from the crude cell lysate, and a skilled artisan would have motivation to apply the viral titer techniques to ddPCR analysis. While Basha teaches CHO cells that were electrically disrupted, it would be obvious to try and disrupt said cells using mechanical means, as they were a common cell line utilized in viral in vitro studies. Given what was known in the art at the time of filing, it would be a routine optimization to use bead beating to mechanically lyse transduced cells, such as CHO cells, infected/transduced with such viruses as HIV and AAV, thus rendering the limitations of instant claims 17 and 19-20 obvious to a skilled artisan, given the teachings of Basha and Ai.
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Basha in order to utilize specific types of cells or viruses in the methods of mechanical disruption of infected cells. One would have been motivated to do so, given the suggestion by Ai that ddPCR on crude cell lysates could improve their methods for determining viral titer reproducibly and quickly. There would have been a reasonable expectation of success, given the knowledge that these methods and kits for performing such methods were known in the art, as taught by Basha, and also given the knowledge that Basha teaches that transduced cells may be lysed using any known mechanical lysis method in the art before performing ddPCR to determine viral titer in the sample. Thus the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Conclusion
No claims are 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 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 RACHEL B GILL whose telephone number is (571)272-3129. The examiner can normally be reached on M to F 8:00 AM to 5:00 PM Eastern.
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/RACHEL B GILL/
Primary Examiner, Art Unit 1671