DETAILED ACTION
The examiner prosecuting this application has changed.
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 .
Priority
Effective Filing Date is 01/30/2020 (see below Foreign Priority)
Foreign Priority:
Applicant’s claim for the benefit of a prior-filed application (EP20154496.2 whose filing date is 1/30/2020) under 35 U.S.C. 119(e) or under 35 U.S.C. 371 as a CONTINUATION of PCT/EP2021/052101 with the filing date 01/29/2021 is acknowledged. The priority date is 1/30/2020.
Status of Application/Amendment/Claims
This Office action is in response to the communications filed on September 30, 2025.
Currently, claims 1-13, 18-23, and 25 are pending in the instant application. Claims 1, 3, 4, 7, 10-12, and 18-23 have been amended to further specify certain features of the claims. Claim 24 has been canceled with no prejudice or disclaimer. New claim 25 has been added. The examiner acknowledges the applicant’s statement that “support for the claim amendments and new claim 25 can be found in the application as originally filed, for example, at paragraphs [0015], [0024], [0124], [0248], [0251], and [0269] of the application as published (US 2023/0151392 A1)” and that “no new matter has been introduced.” Accordingly, claims 1-13, 18-23, and 25 are under examination on the merits in the instant application.
The following rejections are either newly applied or are reiterated and are the only rejections and/or objections presently applied to the instant application.
Withdrawn Rejections
Any rejections/objections not repeated in this Office action are hereby withdrawn.
Maintained Rejections
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.
Claim(s) 1-3, 5, 11-13 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by WO2018148196A1 Natalie SEALOVER 2018-08-16 "Stable targeted integration.” The rejection has been modified to address claim amendment.
Claim 1 of Sealover recites “ integration of at least one exogenous sequence into genomic DNA of a cell, the method comprises integrating the at least one exogenous sequence into a site within a genomic sequence chosen from NCBI Reference Sequences NW_003614682.1 , NW_003617022.1 , NW_006880577.1 , NW_003613622.1 , NW_003615666.1 , NW_003615226.1 , NW_003617688.1 , NW_003613618.1 , NW_003613627.1 , NW_003613628.1 , or homolog thereof.” Sealover further teaches a representative number of specific genomic loci that can be suitable for stable integration within the recited NCBI reference sequences. In particular, paragraph [0014] teaches that “the genomic locus suitable for stable integration can be located within about 10 kb on either side of “ specifically identified nucleotide positions within the listed RefSeq sequences, for example nucleotide 83801 in RefSeq NW_003614682.1, which is within the CHO genome, and contains a very well known “safe harbor domain” called HIPP-11 or just H11. Hipp11 is an intergenic site flanked by Eif4enif1 and Drg1 (Fig. 1 ). It is described that a large human transgene was inserted into the Hipp11 intergenic region by CRISPR/CAS9 (pg.13 ¶[0035]). The Eif4enif1 gene codes for Eukaryotic Translation Initiation Factor 4E Transporter, Drg 1 for Developmentally Regulated GTP Binding Protein 1. (Pg 3 [¶ 0012])
According to the definition in the description the "essential gene" is interpreted as "to include those genes of a host cell coding for an essential polypeptide, and is preferably a gene that has not been shown to be nonessential in the host cell". The genes flanking Hipp11 site can be considered essential according to this definition. However, the distance between the TSS of the genes is about 57kb, this is too large for the limitations of claim one. Sealover further teaches in paragraph [0015] that integration may occur “into a site within a genomic sequence chosen from” the listed NCBI reference sequences, wherein “the integrated sequence does not adversely affect the cell and the function of the integrated sequence is predictable, consistent, and reproducible.” Therefore, NW_003614682.1 may very well contain additional “safe harbor domains,” for example between the Eif4enif1 gene and the Sfi1 spindle body protein gene. The TSSs of Eif4enif1 and Sfi1 are 6kb apart, which is greater than 80nt (claim 2) and less than 20,000nt (claim 3), and the two promoters are in opposite directions (claim 5).
PNG
media_image1.png
610
869
media_image1.png
Greyscale
(NW_003614682.1.(n.d.).https://www.ncbi.nlm.nih.gov/nuccore/NW_003614682.1?report=graph) Screen clipping taken: 6/26/2025 11:07 AM
PNG
media_image2.png
508
875
media_image2.png
Greyscale
(NW_003614682.1.(n.d.).https://www.ncbi.nlm.nih.gov/nuccore/NW_003614682.1?report=graph) Screen clipping taken: 6/26/2025 11:12 AM
Regarding the amended limitation requiring the two TSSs to be within 5,000 bases pairs of each other, Sealover teaches this limitation. Specifically, similar to the analysis above for reference sequence, NW_003614682.1, an analysis of the reference sequence, NW_003613618.1, revealed two essential genes, Pms2 and Aimp2, transcribed in opposite directions (claim 5) and separated by 87 base pairs, which is greater than 80nt (claim 2) and less than the amended 5,000nt limitation (claims 1 and 3). Pms2 and Aimp2 are essential genes, as evidenced by OMIM entries 600259 and 600859, respectively, which describe each as require for fundamental cellular processes (DNA mismatch repair and aminoacyl-tRNA synthetase complex function, respectively).
PNG
media_image3.png
626
1372
media_image3.png
Greyscale
(https://www.ncbi.nlm.nih.gov/nuccore/351517931?report=graph&tracks=)
Screen clipping taken: 5/16/2026 3:56 PM
The genomic loci, therefore, can be termed "safe harbors." The integrated sequence remains in the genomic locus and is not excised or altered in any manner. For example, the integrated sequence and adjacent sequences are not subject to gene silencing or position effects. Additionally, the integrated exogenous sequence does not affect the function of genes or other chromosomal sequences in the cell, i.e., global or local gene expression is not altered, there are no cell abnormalities or deficits, there is no position mutagenesis or other side effects, etc. Moreover, when the exogenous sequence encodes a protein or RNA molecule, expression of the exogenous sequence is stable, efficient, consistent, and predictable. (Pg 3 [¶ 0012])
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.
Claims 1-3, 5-13 and 22-24 remain rejected under 35 U.S.C. 103 as being unpatentable over WO2018148196A1 Natalie SEALOVER 2018-08-16 "Stable targeted integration” as applied to claims 1-3, and 5 above, and further in view of WO 2019/112899 VALAMEHR, 13 June 2019, IMMUNOTHERAPIES USING ENHANCED iPSC DERIVED EFFECTOR CELLS.
As described above, the genomic loci NW_003614682.1, can be termed a "safe harbor." The integrated sequence remains in the genomic locus and is not excised or altered in any manner, the integrated exogenous sequence does not affect the function of genes or other chromosomal sequences in the cell, i.e., global or local gene expression is not altered, there are no cell abnormalities or deficits, there is no position mutagenesis or other side effects, etc. and the TSSs of Eif4enif1 and Sfi1 are 6kb apart, which is greater than 80nt (claim 2) and less than 20,000nt (claim 3), and the two promoters are in opposite directions (claim 5).
Although Sealover describes integration ((b) Donor polynucleotide comprising exogenous sequence pg8 ¶[0025]), the insertion details that meet many of the key limitations of the instant application are not well described. Sealover does not describe the GOI is comprised in a heterologous expression cassette integrated at the target site in hematopoietic stem cell, an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, iPSCs, or an immune effector cell selected from a Natural Killer (NK) cell, a microglial cell, a macrophage, and a T cell, such as a cytotoxic T lymphocyte (CTL), a regulatory T cell, or a T helper cell, or where the GOI expression cassette encodes a chimeric antigen receptor (CAR).
With regard to Claims (6-10, 18, and 22-24) VALAMEHR teaches pg. 29 ¶[000113] As used herein, a "gene of interest" or "a polynucleotide sequence of interest” is a DNA sequence that is transcribed into RNA and in some instances translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. Pg. 34 ¶ [000124] As used herein, the term "targeting modality" refers to a molecule, e.g., a polypeptide, that is genetically incorporated into a cell to promote antigen and/or epitope specificity that includes but not limited to i) antigen specificity as it related to a unique chimeric antigen receptor (CAR) or T cell receptor (TCR), ii) engager specificity as it related to monoclonal antibodies or bi specific engager, iii) targeting of transformed cell, iv) targeting of cancer stem cell, and v) other targeting strategies in the absence of a specific antigen or surface molecule. Pg.61 ¶ [000178] When iPSCs and its functional derivative hematopoietic cells have a genotype comprising both CAR and IL, the CAR and IL are comprised in a bi-cistronic expression cassette comprising a 2A sequence. As comparison, in some other embodiments, CAR and IL are in separate expression cassettes comprised in iPSCs and its functional derivative hematopoietic cells.
It would have been obvious to a person having ordinary skill in the art before the effective filing date to modify the teachings of Sealover et. al. by using the cells and cassettes taught by VALAMEHR et. al., as a person of ordinary skill in the art would recognize that these claim elements were well suited for the stated goals of the instant application pg.1
Line 5 recombinant host cell and methods of expressing gene of interest (GOI) from a host cell. The invention relates particularly to methods of improving a host cell's capacity to stably express a GOI over a long period of time, and gene therapy employing the recombinant host cell. Line 21 Stable expression in eukaryotic cell lines is useful for numerous projects. For example, it supports recombinant protein production over a prolonged period in a cell culture, or it helps in transgenic eukaryotes for optimization of protein productions at high yields. Maintaining stable expression of transgenes is important to ensure functionality. Stable expression implies insertion into the genome. Randomly inserted genes are subject to position effects and silencing, making their expression unreliable and unpredictable. Targeted insertion in safe-harbor sites has made some progress. To this end, so called safe harbor loci can be targeted as well…
as those of VALAMEHR on pg. 1-2
¶[0003]…developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. The cell products developed under the present disclosure address critical limitations of patient-sourced cell therapies…. ¶[0005] There is a need for functionally improved effector cells that address issues ranging from response rate, cell exhaustion, loss of transfused cells (survival and/ or persistence), tumor escape through target loss or lineage switch, tumor targeting precision, off-target toxicity, off-tumor effect, to efficacy against solid tumors, i.e., tumor microenvironment and related immune suppression, recruiting, trafficking and infiltration. ¶[0006] It is an object of the present invention to provide methods and compositions to generate derivative non-pluripotent cells differentiated from a single cell derived iPSC (induced pluripotent stem cell) clonal line, which iPSC line comprises one or several genetic modifications in its genome.
One of skill in the art could have combined cells and cassettes taught by VALAMEHR et. al. with the “safe harbor Loci” of Sealover et. al by known methods with no change in their respective functions, and the combination would have yielded a predictable outcome of developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo while addressing the critical limitations of patient-sourced cell therapies.
Claim(s) 9-10 and 18-23 is/are rejected under 35 U.S.C. 103 as being unpatentable over WO 2019/112899 VALAMEHR, 13 June 2019, IMMUNOTHERAPIES USING ENHANCED iPSC DERIVED EFFECTOR CELLS in view of WO2019194751 PHAN, Toan Thang 10.10.2019 USE OF AN ESSENTIALLY PURE MESENCHYMAL STEM CELL POPULATION OF THE AMNIOTIC MEMBRANE OF UMBILICAL CORD FOR GENERATING A MAMMALIAN STEM CELL CARRYING A TRANSGENE.
VALAMEHR describes developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. The cell products developed under the present disclosure address critical limitations of patient-sourced cell therapies…. ¶[0005] There is a need for functionally improved effector cells, but is silent on cells differentiated to generate a neuron or cardiomyocyte. Phan teaches (pgs30-31)
¶[0096-0097] … stem cells can, for example, be differentiated into any type of mesenchymal cell such as, but not limited to, adipocytes, skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, mucin producing cells, cells derived from endocrine glands such as insulin producing cells (for example, b- islet cells) or neuro-ectodermal cells. The transgenic mesenchymal stem population can be differentiated in vitro in order to subsequently use the differentiated cell for medical purposes…Any disease can be treated using a mesenchymal stem cell population of the invention as long as by its recombinant expression, the transgene can, as described above, ameliorate, treat or prevent a disease in a host, typically a mammal including a human that suffers from a (genetic) dysfunction.
It would have been obvious to a person having ordinary skill in the art before the effective filing date to modify the teachings of VALAMEHR et. al. by using the cell differentiation and maturation taught by Phan et. al., as a person of ordinary skill in the art would recognize that these claim elements were well suited for the stated goals of the instant application. One of skill in the art could have combined cell fate procedures taught by Phan et. al. with the cassette and transfection strategies of VALAMEHR et. al by known methods with no change in their respective functions, and the combination would have yielded a predictable outcome of developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo while addressing the critical limitations of patient-sourced cell therapies within the variety of cells required (e.g., differentiated to generate a neuron or a cardiomyocyte)
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over WO2018148196A1 Natalie SEALOVER 2018-08-16 "STABLE TARGETED INTEGRATION.” in view of WO 2019/112899 VALAMEHR, 13 June 2019, "IMMUNOTHERAPIES USING ENHANCED iPSC DERIVED EFFECTOR CELLS" as applied to claims 1-3, 5-13 and 22-24 above, and further in view of in view of US-16610899. Yuting Liu 2021-08-19.
Although Sealover describes integration “Donor polynucleotide comprising exogenous sequence” (pg8 ¶[0025]), It does not use the SEQ ID NOs:1-21 sequences claimed in the instant application. US-16610899 has the sequences mentioned in the instant application. It would have been obvious to a person having ordinary skill in the art before the effective filing date to modify the teachings of SEALOVER et. al. by using the sequences Liu et. al., as a person of ordinary skill in the art would recognize that these claim elements required the design of gRNA. One of skill in the art could have combined sequences Liu et. al. with the cassette and transfection strategies of VALAMEHR et. al by known methods with no change in their respective functions, and the combination would have yielded a predictable outcome of developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo while addressing the critical limitations of patient-sourced cell therapies within the variety of cells required (e.g., differentiated to generate a neuron or a cardiomyocyte)
New Rejection(s) Necessitated by Amendment
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.
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over WO2018148196A1 (“Sealover”) as applied to claims 1-3 and 5 above, and further in view of US20030077812A1 (“McArthur”).
Sealover teaches an isolated mammalian host cell comprising a heterologous gene of interest (GOI) chromosomally integrated at a target site within an intergenic region between a pair of adjacent essential genes, as discussed above with respect to claim 1, including stable integration within a genomic safe harbor locus.
Sealover does not teach a method of treating hemophilia in a subject by administering a host cell comprising a GOI encoding a coagulation factor.
McArthur teaches gene therapy using host cells or vectors expressing a blood clotting factor, wherein the transgene is human factor IX (hFIX) or human factor VIII (hFVIII), for treatment of hemophilia, see abstract and claims.
It would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date to modify the host cell of Sealover by selecting a coagulation factor gene as the gene of interest, as taught by McArthur. A PHOSITA would have recognized that stable chromosomal integration at a safe harbor site locus would provide predictable and durable expression of the therapeutic coagulation factor, thereby predictably treating hemophilia.
Response to the Arguments
Applicant’s arguments, see pg. 6 paragraph 2, filed 09/30/2025, with respect to claims 11-13 and 18-23 have been fully considered and are persuasive. The REJECTION UNDER 35 USC § 112(a) of claims 11-13 and 18-23 has been withdrawn.
Applicant's arguments filed September 30, 2025 have been fully considered but they are not persuasive.
REGARDING THE REJECTION UNDER 35 USC § 102
Applicant argues that WO2018148196A1 (“Sealover”) fails to teach the amended limitation requiring that the pair of adjacent essential genes have transcription start sites (TSSs) separated by less than 5,000 nucleotides (nt).
As set forth in the maintained rejection, Sealover teaches integration into genomic sequence NW_003613618.1, which contains the genes Pms2 and Aimp2. These genes are arranged in opposite transcriptional orientation and are separated by 87 base pairs, which satisfies the amended limitation requiring the TSSs to be separated by less than 5,000 nt, as well as claim 2’s lower bound of at least 80 nt. Further, Pms2 and Aimp2 are essential genes, as evidences by OMIM entries 600259 and 600859, respectively, which describe these genes as involved in fundamental cellular processes including DNA mismatch repair and amino-tRNA synthetase complex function.
Applicant argues that Sealover merely discloses a list of NCBI reference sequences and fails to teach or suggest a representative set of sufficiently specific insertion sites “between a pair of adjacent essential genes” whose transcription start sites are separated by less than 5,000 nucleotides.
First, Sealover expressly provides a representative number of species that identify specific genomic loci suitable for stable integration. Paragraph [0014] teaches that suitable integration loci can be located within approximately 10kb of specifically identified nucleotide positions within the disclosed reference sequences. Paragraph [0015] further teaches integration into a site within the listed NCBI reference sequences where the integrated sequences exhibits stable, predictable, and reproducible expression, thereby directing a PHOSITA to non-limiting positions within the NCBI reference sequences so long as the insertion meets these criteria.
Second, the claims presently under examination are broad. Claim 1 does not require any particular named safe harbor locus, any particular coordinate, or any specific gene pair identity. Rather, claim 1 reads on any integration event occurring within an intergenic region between adjacent essential genes whose TSSs are less than 5,000 nt apart. As discussed in the rejection, the expressly disclosed Sealover reference sequence, NW_003613618.1, contains the adjacent essential genes Pms2 and Aimp2, which are arranged in opposite transcriptional orientation and whose TSSs are separated by 87bp, satisfying the amended claim limitations.
Furthermore, a PHOSITA, upon reviewing the expressly disclosed RefSeq sequences identified by Sealover, would readily understand how to analyze those sequences using routine genome annotation tools (e.g., NCBI genome viewer) to identify intergenic regions and neighboring genes. Such routine analysis does not render the reference non-anticipatory where the claimed structural limitation is expressly present within the disclosed genomic sequence.
Accordingly, Sealover teaches each and every limitation of claims 1-3, 5, and 11-13, and the rejection under 35 USC § 102(a)(1) is maintained.
REGARDING THE REJECTION UNDER 35 USC § 103
Applicant argues that the secondary references do not cure the deficiencies of Sealover.
This argument is not persuasive because the alleged deficiency in Sealover is not present for the reasons discussed above. Sealover teaches the claimed integration site limitations, including the amended “less than 5,000 nt” limitation.
The additional references were cited to teach cell type selection, therapeutic transgenes, differentiation, and therapeutic applications, not to remedy the genomic safe-harbor teachings already provided by Sealover.
Accordingly, the rejections under 35 USC § 103 are maintained.
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 COREY LANE BRETZ whose telephone number is (571)272-7299. The examiner can normally be reached M-F 7:30am - 6:30pm.
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, Ram Shukla can be reached at (571) 272-0735. 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.
/COREY LANE BRETZ/Patent Examiner, 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
Prosecution Insights