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 .
1. Claims 1-39 and 53-56 have been cancelled. Claims 50 and 51 have been withdrawn.
Claims 40-49, 52, and 57-63 are under examination.
Claim Rejections - 35 USC § 103
2. In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
3. Claims 40-43, 46, 48, 49, 57, 59, 62, and 63 are rejected under 35 U.S.C. 103 as being unpatentable over Tremblay et al. (JBC, 2015, 290: 16142-16156), in view of all Brown et al. (Diabetes, 2000. 49: 383-391), Ohara-Imaizumi et al. (Biochem. J., 2002, 363: 73-80), Ward et al. (British J. Pharmacol., 2011 162: 1439-1252), and Regazzi et al. (EMBO J., 1996, 15: 6951-6959).
Tremblay et al. teach a non-naturally occurring nucleic acid encoding synaptotagmin I (Syt1) fused to an HA tag; the non-naturally occurring nucleic acid is used to express the fusion protein in HEK293 cells for monitoring secretory vesicle trafficking to the plasma membrane; the fusion protein localizes into the membrane of the secretory vesicles and, upon secretion, the HA tag is detectable on the cell surface in the absence of cell permeabilization (claims 40 and 62) (see p. 16144, Fig. 1; p. 16145, column 1). Since it is expressed in HEK293 cells, the non-naturally occurring nucleic acid comprises an operably-linked regulatory sequence (claim 46).
Tremblay et al. do not teach that the Syt1 fusion protein comprises a SNAP tag (claims 40 and 62). However, Tremblay et al. teach that SNAP tags could be used instead of the HA tag to follow trafficking to the plasma membrane by labeling the SNAP tags with cell-impermeable fluorescent markers (claims 40, 41, 59, 62, and 63) (see p. 16143, column 2, third paragraph; paragraph bridging p. 16143 and 16144; p. 16144). One of skill in the art would have found obvious to replace the HA tag with a SNAP tag to achieve the predictable result of obtaining a non-naturally occurring nucleic acid for monitoring trafficking to the plasma membrane.
Tremblay et al. do not teach monitoring insulin secretion in single [Symbol font/0x62]-cells by measuring endocytic uptake and accumulation of the cell impermeable marker within a single cell (claims 40, 42, 43, 48, 49, and 62). However, doing so is suggested by the prior art. For example, Tremblay et al. teach that synaptotagmins are involved in calcium-mediated secretion and endocytosis (see p. 16142, paragraph bridging columns 1 and 2). Brown et al. teach that synaptotagmin III (Syt3) is involved in insulin exocytosis from beta-cells (see Abstract; p. 383; p. 390, column 1, last paragraph). Ohara-Imaizumi et al. teach monitoring exocytosis/endocytosis of insulin secretory granule as an effective method for determining the molecular mechanism in insulin secretion; monitoring could be achieved by using labeled secretory granule membrane proteins (see Abstract; p. 73). Based on these teachings, one of skill in the art would have found obvious to modify Tremblay et al. by replacing Syt1 with Syt3 to achieve the predictable result of obtaining a nucleic acid encoding a SNAP tag-Syt3 fusion protein suitable for monitoring insulin secretion. One of skill in the art would have found obvious to further introduce the nucleic acid in β-cells with the reasonable expectation that doing so would allow for monitoring and measuring secretory vesicle trafficking to the plasma membrane of these cells (i.e., measuring insulin secretion). Furthermore, Ward et al. teach that it was common knowledge in the prior art that SNAP tagging results in the irreversibly labeling of the SNAP tags with the impermeable marker. Ward et al. teach that SNAP tagging could be used to monitor and quantify the internalization of the labeled SNAP-tagged proteins in single cells with high sensitivity due to markedly higher signal to background ratios (see Abstract; p. 1440, paragraph bridging columns 1 and 2; p. 1446, paragraph bridging columns 1 and 2; p. 1448, Fig. 6; paragraph bridging p. 1449 and 1451). Based on these teachings, one of skill in the art one of skill in the art would have known that the fluorescent impermeable marker would be irreversible bound to the SNAP-tagged Syt3 and would have reasonably concluded that the internalization of the impermeable marker could also be used to measure insulin secretion. Thus, one of skill would have found measuring secretion via quantifying the endocytic uptake of the impermeable marker as an obvious variant (claim 40).
Tremblay et al., Brown et al., Ohara-Imaizumi et al., and Ward et al. do not teach the elected species VAMP2 (claim 40). Regazzi et al. teach that VAMP2, a protein associated with the insulin secretory granules, is involved in calcium-dependent insulin exocytosis from beta-cells (see Abstract; paragraph bridging p. 6951 and 6952). One of skill in the art would have found obvious to replace Syt3 with VAMP2 to achieve the predictable result of obtaining a nucleic acid encoding a fusion protein comprising VAMP2 fused to a SNAP tag, suitable for monitoring insulin secretion. One of skill in the art would have also found obvious to introduce the nucleic acid in [Symbol font/0x62]-cells and contact the [Symbol font/0x62]-cells with a cell-impermeable fluorescent marker to achieve the predictable result of monitoring and quantifying stimulus-induced insulin secretion by measuring the fluorescence accumulated in single [Symbol font/0x62]-cells.
The limitation of a kit (claim 57) is not innovative over the prior art; kits containing instructions for transfection of cells with non-viral vectors containing nucleic acids have been used before the invention was made. One would have been motivated to assemble a kit, i.e., put the reagents in a box containing instructions how to use the reagents, because they are convenient to use and save time.
Thus, the claimed invention was prima facie obvious at the time of its effective filing date.
4. Claims 40-43, 46-49, 57, 59, and 61-63 are rejected under 35 U.S.C. 103 as being unpatentable over Tremblay et al. taken with all Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al., in further view of Lewis et al. (Human Gene Therapy, 2003, 14: 1009-1016).
The teachings of Tremblay et al., Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al. are applied as above for claims 40-43, 46, 48, 49, 57, 59, 62, and 63. Tremblay et al., Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al. do not specifically teach a selection marker, where the selection marker is an antibiotic resistance gene (claims 47 and 61). Lewis et al. teach that vectors comprising a gene of interest and the selectable marker neo each expressed from a different promoter could be used for the selection of the cells expressing the gene (see p. 1011). One of skill in the art would have found obvious to further express neo from a different promoter to achieve the predictable result of selecting the cells expressing the SNAP tag-VAMP2.
Thus, the claimed invention was prima facie obvious at the time of its effective filing date.
5. Claims 40-43, 46, 48, 49, 52, 57-59, 62, and 63 are rejected under 35 U.S.C. 103 as being unpatentable over Tremblay et al. taken with all Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al., in further view of both Simpson et al. (J. Biotechnol., 2007, 129: 352-365) and Scrace et al. (Cancer Res., 2015, 75, 15_Supplement, Abstract).
The teachings of Tremblay et al., Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al. are applied as above for claims 40-43, 46, 48, 49, 57, 59, 62, and 63. Tremblay et al., Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al. do not teach a CRISPR system (claims 52 and 58). Simpson et al. teach using siRNAs to probe for proteins involved in protein secretion, where probing takes place via identifying the correct delivery of a secretion marker to the cell surface (see Abstract; p. 353, column 2, last paragraph). Scrace et al. teach that the CRIPR/Cas9-sgRNA system is superior to siRNA with respect to gene silencing (see Abstract). Since the cells of Tremblay et al., Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al. could be used to monitor calcium-mediated insulin exocytosis by using SNAP tag-VMP2 as a secretion marker, one of skill in the art would have reasonably concluded that these cells could be also used to screen for proteins involved in this process. One of skill in the art found obvious to further include a CRIPR/Cas9-sgRNA system in the [Symbol font/0x62]-cells with the reasonable expectation that doing so would identify other proteins involved in calcium-mediated insulin exocytosis.
Thus, the claimed invention was prima facie obvious at the time of its effective filing date.
6. Claims 40-46, 48, 49, 57, 59, 60, 62, and 63 are rejected under 35 U.S.C. 103 as being unpatentable over Tremblay et al. taken with all Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al., in further view of Lu et al. (Journal of Innovative Optical Health Sciences, 2009, 2: 397-405).
The teachings of Tremblay et al., Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al. are applied as above for claims 40-43, 46, 48, 49, 57, 59, 62, and 63. Tremblay et al., Brown et al., Ohara-Imaizumi et al., Ward et al., and Regazzi et al. do not teach monitoring in vivo in an animal model (claims 44, 45, and 60). Lu et al. teach transgenic mice expressing a secretory granule resident protein linked to fluorescent proteins in the pancreatic β-cells, where the mice could be used to monitor in vivo insulin secretion (see Abstract; p. 399, column 1, first paragraph). One of skill in the art would have found obvious to express the SNAP tag-VAMP2 in the pancreatic β-cells a mouse with the reasonable expectation that doing so would result in a mouse model suitable to study insulin secretion in vivo; one of skill in the art would have found obvious to further inject the cell-impermeable marker into the mouse model with the reasonable expectation that doing so would allow monitoring insulin secretion in vivo.
Thus, the claimed invention was prima facie obvious at the time of its effective filing date.
Response to Arguments
7. The applicant argues that the examiner did not identify any reference teaching or even mentioning the endocytic uptake of a cell-impermeable marker. The applicant argues that the examiner did not identify any teaching or suggestion that the endocytosis of a SNAP tag could be used to measure secretion from a single cell.
This is not found persuasive. The examiner cited Ward, who teaches that the irreversible labeling of SNAP tags by the impermeable marker could be used to monitor and quantify the internalization (i.e., endocytosis) of the labeled SNAP-tagged proteins in single cells with high sensitivity.
The test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). The combined teachings in the prior art suggest that that secretion could be measured via quantifying the endocytic uptake of the impermeable marker.
The argument that the examiner did not consider the word “proxy” because this word is not mention in the rejection is not found persuasive. The examiner is not required to specifically mention “proxy”. This word is addressed by the statement that secretion could be measured by quantifying the endocytic uptake of the impermeable marker.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, 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).
The argument that the applicant discovered that SNAP tags are particularly amenable for quantifying secretion in a single cell via vesicle-mediated uptake is not found persuasive. This was already taught by Ward.
The applicant argues that it would not have been obvious to replace Syt1 with Syt3 because Tremblay suggests that Syt1 and Syt3 exhibit different behavior.
This is not found persuasive because. The teachings indicated by the applicant only pertain to ESyts which insert into the ER membrane, but lack the capability of inserting into the plasma membrane. The rejection is not based on ESyts, and thus, the arguments regarding ESyts are not material to the rejection.
The rejection is based on the capability of Syts to insert into the plasma membrane and on extrapolating Tremblay’s teaching of SNAP tag labeling to other Syts (such as Syt3) capable of inserting into the plasma membrane. The combination of the cited prior art suggests attaching the SNAP tag to Syt3 and further use it to follow insulin secretory granule trafficking in single β-cells. Tremblay does not teach or suggests that Syt3-SNAP cannot be used to follow the trafficking of the insulin secretory granules.
The arguments addressing Brown are not found persuasive. Brown clearly teaches that Syt3 is involved in insulin exocytosis from beta-cells. Brown and Ohara-Imaizumi provide the motivation to use Syt3-SNAP for monitoring and measuring insulin secretion.
The applicant refers Tremblay’s teachings of SNAP permeable labels. However, these teachings are not material to the rejection because they only pertain to ESyts which are not capable of inserting into the plasma membrane; they are localized intracellularly. The rejection is based on Syts and Tremblay teaches detecting Syt1 on the cell surface in the absence of permeabilization (see Fig. 1; p. 16145, column 1).
The arguments addressing Tremblay, Ohara-Imaizumi, and Ward individually are not found persuasive. 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).
The applicant argues that Ward teaches SNAP-tagged GPCR proteins, not SNAP-tagged proteins as stated in the rejection.
However, Ward teaches that SNAP tags could be used to monitor the localization of any membrane protein of interest (see p. 1440, paragraph bridging columns 1 and 2; p. 1451, paragraph bridging columns 1 and 2). One of skill in the art would have reasonably concluded that Ward’s teachings could be extrapolated to monitor other proteins of interest, including Syt2. MPEP 2141.03 states:
"A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.
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The "hypothetical ‘person having ordinary skill in the art’ to which the claimed subject matter pertains would, of necessity have the capability of understanding the scientific and engineering principles applicable to the pertinent art." Ex parte Hiyamizu, 10 USPQ2d 1393, 1394 (Bd. Pat. App. & Inter. 1988).
Conclusion
8. 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 ILEANA POPA whose telephone number is (571)272-5546. The examiner can normally be reached 8:00 am to 4:30 pm.
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/ILEANA POPA/Primary Examiner, Art Unit 1633