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 10/027/2025. Claims 382-397 and 399-412 are currently pending and are examined herein. Claim 412 is new. The election of species requirement mailed November 29, 2023 is still deemed proper. Applicant's elected a) Cas 12b as the one genome-modifying protein; b) B2M as the one or more target genes; and c) CD47 as the exogenous polynucleotide. without traverse in the reply filed January 26, 2024.
The genome modifying protein in claim 400; the target genes in claim 408; and an exogenous polynucleotide in claim 393 that were not elected remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 1/26/24.
Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 120 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63/232161, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. There is no mention of re-clustering, dissociating, or maintaining suspensions of modified cells in Provisional Application No. 63/232161 (filing date August 11, 2021). Support for re-clustering, dissociating, or maintaining suspensions of modified cells first appears in Provisional Application No. 63/297694 (filing date January 7, 2022). However, there is no support for motion or movement during incubation of modified cells in Provisional Application No. 63/232161 (filing date August 11, 2021), Provisional Application No. 63/297694 (filing date January 7, 2022), or Provisional Application No. 63344502 (filing date May 20, 2022). Therefore, the earliest priority date is June 17, 2022 receiving benefit from Provisional Application No. 63353531.
Claim Rejections - 35 USC § 103 – new necessitate by amendment
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.
Claims 382-397, 399-411 remain and claim 412 is rejected under 35 U.S.C. 103 as being unpatentable over Hering (Hering and Burlack. US20160165861A1; published June 16, 2016), in view of Bevacqua (Bevacqua et al. CRISPR-based genome editing in primary human pancreatic islet cells. Nat Commun; published April 23, 2021) and Walker (Walker et al. Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells. JCI Insight 5, e137017; published April 30, 2020). This rejection has been modified to address newly added claim 412.
Hering’s disclosure is directed to methods of making genetically modified cells, tissues, and organs for treating or preventing diseases (see abstract).
Regarding claim 382, Hering teaches a method of modifying primary islet cells (see paras 0019, 0022, 0077, 0083, 0088, 0167-0175, 0184, and 0347). Hering teaches dissociating one or more primary islet clusters into a suspension of primary islet cells by standard enzymatic digestion of minced pancreas from a subject or in a Ricordi chamber by enzymatic and mechanical forces (see paras 0331-0334). Hering further teaches contacting the primary islet cells with a gene editing system comprising a genome-modifying protein, including proteins from the CRISPR/cas system and incubating the cells to produce modified cells (see paras 0242-0261; and Example 9). Hering teaches introducing exogenous polynucleotides into modified cells (see paras 0009 and 0019; claims 1 and 17). Hering teaches that modified cells are incubated with gentle sloshing and in a shaking incubator after gene editing reagents are applied and small aggregates might be observed (see Example 8).
However, while Hering suggests that small aggregates of modified cells may form, Hering does not specifically teach that modified cells are re-clustered into modified primary islet clusters and that the incubating with motion is carried out prior to a step of selecting for islet cells that have modified gene expression relative to the primary islet cells before the contacting.
Bevacqua’s disclosure is directed to CRISPR-based genome editing in primary human pancreatic islet cells (see abstract). Bevacqua teaches that prior genetic approaches in human pseudoislets had not allowed direct modification of pancreatic beta cell DNA and that their techniques of delivery and expression of sgRNAs and Cas9 with a dispersion and reaggregation method permitted efficient genome editing (see p. 2, left column, paras 3-4).
Regarding claim 382, Bevacqua teaches genome editing of primary islet cells using a strategy based on the dispersion and reaggregation of the cells into primary organoids or pseudoislets (see p. 2, left column, paras 3-4; Fig. 1). Bevacqua further teaches dissociating primary human islet clusters into a solution, contacting with lentivirus for transduction and re-clustering to form modified primary islet clusters (see Fig. 1).
Walker’s disclosure is directed to studying primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters and teach human islets dispersed into single cells and then reaggregated into pseudoislets (see abstract; and Fig. 1A; and p. 2, para 4).
Regarding claim 382, Walker teaches that the 3D structure of intact islets makes virally mediated manipulation of human islet cells challenging due to poor viral penetration into the center of the islet and that they adopted the pseudoislet system to overcome this challenge by transducing the dispersed single islet cells before reaggregation (see Fig. 3A). Walker further teaches that while they focused on virally mediated gene expression to alter signaling pathways, this system could also be adapted to accommodate CRISPR technologies (p. 11, para 2). Walker further teaches that after islet dispersion into single cells, techniques to purify live-cell populations such as FACS with cell surface antibodies could be incorporated to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation (p. 11, para 2). Walker teaches that combined with accurate cell-specific targeting, this approach would allow the measurement of intracellular dynamics at the individual cell level and distinguish intracellular responses of islet endocrine cells to stimuli (p. 11, para 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Hering’s method of modifying primary islet cells using the CRISPR/Cas system with the dispersion and reaggregation strategies of Bevacqua and Walker by incubating the unmodified primary islet cells and gene editing reagents with motion prior to a step of selecting for islet cells that have modified gene expression to produce modified islet cells. Hering teaches the dissociation of islets, applying gene editing reagents, and then incubating cells with motion (see paras 0331-0334, paras 0242-0261, and Example 8). Bevacqua teaches that prior genetic approaches in human pseudoislets had not allowed direct modification of pancreatic beta cell DNA and that their dispersion and reaggregation method permitted efficient genome editing (see p. 2, left column, paras 3-4). One of ordinary skill in the art would have been motivated to modify Hering’s method of modifying primary islet cells with Bevacqua and Walker’s techniques of dispersion and reclustering the islet cells because Walker provides the motivation to try the technique in CRISPR and suggests sorting prior to pseudoislet formation would incorporated to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation (p. 11, para 2). One of ordinary skill in the art would have had a reasonable expectation of success because Hering, Bevacqua, and Walker teach modifying pancreatic islet cells by dissociating the cells and treating with a Cas nuclease and guide RNA to achieve modified primary islet cells. Thus, the claimed invention as a whole is prima facie obvious.
Regarding claim 383, the combined teachings Hering, Bevacqua, and Walker render obvious the method of modifying primary islet cells, comprising the steps of modifying primary islet cells by dissociating islet clusters into a suspension of primary islet cells, contacting the cells with Cas9 nuclease, guide RNA, and or an agent comprising an exogenous polynucleotide encoding a protein, incubating the cells, and reclustering the cells (see above regarding claim 382). Hering further teaches that isolated cells can be genetically modified, analyzed, sorted and that these isolated cells can be further genetically modified, which would require the steps of dissociating the first modified primary islet clusters into a suspension of primary islet cells, contacting the cells with Cas9 nuclease, guide RNA, and or an agent comprising an exogenous polynucleotide encoding a protein, incubating the cells, and reclustering the one or more second modified primary islet cells (see paras 0167-0174). Walker further teaches that after islet dispersion into single cells, purifying live-cell populations by FACS with cell surface antibodies could be incorporated to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation (p. 11, para 2).
Regarding claim 384, Walker teaches dispersion of islets into single cells and techniques to purify live-cell populations, such as FACS, with cell surface antibodies to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation (p. 11, para 2).
Regarding claim 385, Hering teaches the step of reclustering the cells and incubating with motion (see paras 0425-0426).
Regarding claims 386-387, Hering teaches that isolated cells can be genetically modified, analyzed, sorted and that these isolated cells can be further genetically modified, which would require the steps of dissociating the second modified primary islet clusters into a suspension of primary islet cells, contacting the cells with Cas9 nuclease, guide RNA, and/or an agent comprising an exogenous polynucleotide encoding a protein, incubating the cells, and reclustering the one or more further modified primary islet cells (see paras 0167-0174). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to repeat the steps of dissociating islet clusters and incubating the gene editing reagents, such as multiple gRNAs targeted towards different genes or delivering an exogenous polynucleotide encoding a polypeptide until the desired phenotype is achieved. It would have been a matter of combining known techniques to yield predictable results with a reasonable expectation of success. Thus, the claimed invention as a whole is prima facie obvious.
Regarding claims 388-389, Hering teaches reagents for reducing cell surface expression of MHC class I and MHC II class molecules (see paras 0007-0008 and 0016).
Regarding claims 390-391, Hering teaches gene editing systems comprising a Cas nuclease and one or more guide RNAs (see paras 0242-0261; and Examples 9 and 11).
Regarding claims 392 and 408, Hering teaches that target genes comprise CIITA and B2M, and that the reagents disrupt the genes, respectively (see paras 0008, 0116, 0121-0122, 0139-0144, and 0172).
Regarding claims 393-394, Hering teaches reagents comprising an exogenous polynucleotides encoding CD47, CD46, CD55, CD59, CTLA4-Ig, FasL, PD-L1HLA-E, and HLA-G (see paras 0016-0019, 0232, and 0452; and Fig. 22A).
Regarding claim 395, Hering teaches that the exogenous polynucleotide is integrated by targeted insertion (see paras 0010 and 0140).
Regarding claim 396, Hering teaches that the exogenous polynucleotide is integrated by non-targeted insertion (see paras 0193 and 0269).
Regarding claim 397, Walker teaches incubation with adenovirus for 2 hours (see p. 4, para 3, and p. 11, para 5).
Regarding claim 399, Hering teaches orbital motion at a speed of 125 rpm (see Example 8).
Regarding claim 400, Hering teaches the genome-modifying protein Cas9 (see para 0246).
Regarding claim 401, Hering teaches a method of modifying primary islet cells (see paras 0019, 0022, 0077, 0083, 0088, 0167-0175, 0184, and 0347). Hering teaches steps i) and iv) of dissociating one or more primary islet clusters into a suspension of primary islet cells by standard enzymatic digestion of minced pancreas from a subject or in a Ricordi chamber by enzymatic and mechanical forces (see paras 0331-0334). Hering further teaches steps ii) and v) of contacting the primary islet cells with a gene editing system comprising a genome-modifying protein, including proteins from the CRISPR/cas system and guide RNAs and incubating the cells to produce modified and further modified cells (see paras 0242-0261; and Example 9). Hering teaches that a combination of transgenes and gene disruptions can be used (paras 0143). Hering teaches that target genes comprise CIITA and B2M (see paras 0008, 0116, 0121-0122, 0139-0144, and 0172). Hering teaches that modified cells in steps iii) and vi) are incubated with gentle sloshing and in a shaking incubator after gene editing reagents are applied and small aggregates might be observed (see Example 8). Hering further teaches introducing exogenous polynucleotides into modified cells (see paras 0009 and 0019; claims 1 and 17).
However, while Hering suggests that small aggregates of modified cells may form, Hering does not specifically teach that modified cells are re-clustered into modified primary islet clusters and that the incubating with motion is carried out prior to a step of selecting for islet cells that have modified gene expression relative to the primary islet cells before the contacting.
Regarding claim 401, Bevacqua teaches genome editing of primary islet cells using a strategy based on the dispersion and reaggregation of the cells into primary organoids or pseudoislets (see p. 2, left column, paras 3-4; Fig. 1). Bevacqua further teaches dissociating primary human islet clusters into a solution, contacting with lentivirus for transduction and re-clustering to form modified primary islet clusters (see Fig. 1).
Regarding claim 401, Walker teaches that the 3D structure of intact islets makes virally mediated manipulation of human islet cells challenging due to poor viral penetration into the center of the islet and that they adopted the pseudoislet system to overcome this challenge by transducing the dispersed single islet cells before reaggregation (see Fig. 3A). Walker further teaches that while they focused on virally mediated gene expression to alter signaling pathways, this system could also be adapted to accommodate CRISPR technologies (p. 11, para 2). Walker further teaches that after islet dispersion into single cells, techniques to purify live-cell populations such as FACS with cell surface antibodies could be incorporated to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation (p. 11, para 2). Walker teaches that combined with accurate cell-specific targeting, this approach would allow the measurement of intracellular dynamics at the individual cell level and distinguish intracellular responses of islet endocrine cells to stimuli (p. 11, para 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Hering’s method of modifying primary islet cells with a CRISPR/Cas system using gRNAs targeting CIITA and B2M and an exogenous polynucleotide encoding CD47 with Bevaqua’s strategy of dispersion and reaggregation and contacting with a lentiviral vector. Hering teaches the dissociation of islets, applying gene editing reagents, and then incubating cells with motion (see paras 0331-0334, paras 0242-0261, and Example 8). Bevacqua teaches that prior genetic approaches in human pseudoislets had not allowed direct modification of pancreatic beta cell DNA and that their dispersion and reaggregation method permitted efficient genome editing (see p. 2, left column, paras 3-4). Walker teaches that sorting modified dispersed islet cells prior to pseudoislet formation to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation (p. 11, para 2). One of ordinary skill in the art would have been motivated to modify Hering’s method of modifying primary islet cells with Bevacqua and Walker’s techniques of dispersion and reclustering the islet cells to improve the genome editing of the primary islet cells because Walker suggests sorting prior to pseudoislet formation to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation (p. 11, para 2). One of ordinary skill in the art would have had a reasonable expectation of success because Hering, Bevacqua, and Walker teach modifying pancreatic islet cells by dissociating the cells and treating with a Cas nuclease and guide RNA to achieve modified primary islet cells. Thus, the claimed invention as a whole is prima facie obvious.
Regarding claim 402, Hering teaches SEQ ID NO: 59 encoding is human CD47 (NP_001768.1) with 100% sequence identity to Applicant’s SEQ ID NO: 2 (see attached OA Appendix of sequence alignment).
Regarding claim 403 and 407, Hering teaches selecting cells comprising modified gene expression by performing flow cytometry (see paras 0067, 0120, 0156, and 0170).
Regarding claim 404, Hering teaches after selecting the islet cells, the step of reclustering the cells and incubating with motion (see paras 0425-0426).
Regarding claims 405, Hering teaches that isolated cells can be genetically modified, analyzed, sorted and that these isolated cells can be further genetically modified, which would require the steps of dissociating the second modified primary islet clusters into a suspension of primary islet cells, contacting the cells with Cas9 nuclease, guide RNAs targeting CIITA and B2M, and/or a lentivirus comprising an exogenous polynucleotide encoding CD47, incubating the cells, and reclustering the one or more further modified primary islet cells (see paras 0167-0174). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to repeat the steps of dissociating islet clusters and incubating the gene editing reagents, such as multiple gRNAs targeted towards different genes or delivering an exogenous polynucleotide encoding a polypeptide until the desired phenotype is achieved. It would have been a matter of combining known techniques to yield predictable results with a reasonable expectation of success. Thus, the claimed invention as a whole is prima facie obvious.
Regarding claim 406, neither Hering nor Bevacqua specifically teach the 1 minute to 60 minutes range of time for the contacting to be carried out. However, it would have been obvious to one of ordinary skill in the art to arrive at the 1 minute to 60 minutes. One of ordinary skill in the art could have arrived at the range of time through routine optimization with a reasonable expectation of success because both Hering and Bevacqua teach methods of transduction, transfection, infection, electroporation, etc. to introduce disruptions to the genome of the cell.
Regarding claim 408, Hering teaches the target genes B2M, TAP1, NLRC5, and CIITA (see paras 0014-0015, 0044, 0121-0124; and Example 1).
Regarding claim 409, Hering teaches a population of engineered primary islets produced by the method of claim 382 (see para 0040).
Regarding claim 410, Hering teaches treating diabetes in a patient in need thereof (see para 0346).
Regarding claim 411, Hering teaches that modified cells are incubated with motion, which includes orbital and undulating motion (see Example 8 and Example 23).
Regarding claim 412, Walker teaches allowing the cell to reaggregate for 6 days before harvesting (see p. 11, para 5).
Therefore the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Response to Arguments
Applicant's arguments filed 10/27/2025 have been fully considered but they are not persuasive. Applicant argues that the motion of gentle sloshing in Hering is part of the step of selecting for edited cells and does not teach or suggest using the motion to re-cluster modified islet cells into one or more modified primary islet cell clusters recited in claims 382 and 401 and further that "gentle sloshing" by Hering does not teach or suggest a method "wherein the incubating with motion is carried out prior to a step of selection for islet cells that have modified gene expression relative to the primary islet cells before the contacting" as recited in claims 382 and 401. Applicant further argues that Bevacqua never teaches or suggests using motion (e.g., an orbital motion) to re-cluster modified primary cells, and that Bevacqua teaches using ultra-low attachment cell plates for promoting re-clustering of the modified primary cells. Page 9, right column of Bevacqua. Applicant further argues that Bevacqua does not teach the recited claim element of "wherein the incubating with motion is carried out prior to a step of selecting for islet cells that have modified gene expression relative to the primary islet cells before the contacting." Applicant argues that Walker mainly focuses on establishing methods for studying the function of human primary islet cells using genetically modified pseudoislets that resemble native islets and does not focus on manipulating the primary cells and then transplanting the manipulated primary cells to a recipient. Applicant argues that the Office posits that Walker suggests sorting modified primary cells prior to pseudoislet formation and that it would [be] incorporated to allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation. Page 9 of the Office Action, however, that Walker clearly suggests that after islet dispersion into single cells and prior to re-clustering into one or more pseudoislets, sorting by FACS with cell surface antibodies could be incorporated. In contrast, claims 382 and 401 recite that the step of selecting/sorting modified islet cells is carried out after re-clustering by motion of the modified primary cells. Applicant argues that Walker uses non-attachment cell plates for promoting re-clustering of primary cells and does not teach "wherein at least a portion of the incubating is carried out with motion and wherein the modified islet cells are re-clustered into one or more first modified primary islet cell clusters."
The Office disagrees. Regarding motion during incubation, the office is relying on Hering, not Maxwell to teach motion during methods of culturing primary islet cells.
In response to applicant's argument that Hering, Bevaqua, and Walker individually and in combination do not teach claims 382 and 401, 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).
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). In this case, the rejection of amended claims above is under 35 U.S.C. 103 relying on related and relevant prior art references rather than a single reference under 35 U.S.C. 102. One of ordinary skill in the art would have been motivated to combine Hering’s method of modifying primary islet cells with Bevacqua and Walker’s techniques of dispersion and reclustering the islet cells because Walker provides the motivation to try the technique in CRISPR and suggests sorting prior to pseudoislet formation would allow manipulation of the pseudoislet cellular composition as well as cell-specific gene manipulation (p. 11, para 2). Thus, the claimed invention as a whole is prima facie obvious.
Conclusion
No claim 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 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 KHALEDA B HASAN whose telephone number is (571)272-0239. The examiner can normally be reached IFP, Monday - Friday 7:30am-5pm.
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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.
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/KHALEDA B HASAN/Examiner, Art Unit 1636
/BRIAN WHITEMAN/Primary Examiner, Art Unit 1636