KDETAILED ACTION
Final Rejection
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of Claims
2. Applicant cancelled claims 3 and 5-6 on 12/17/2025.
3. Claims 1-2, 8-10, 13-22, 38-39, and 58-60 as filed on 12/17/2025 are pending.
4. Claims 1-2, 8-10, 13-22, 38-39, and 58-60 as filed on 12/17/2025 are under examination.
Priority
5. This application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/US2020/049627, filed September 7, 2020, which claims the benefit of U.S. Provisional Application Serial No. 62/896,376, filed September 5, 2019. International Application No. PCT/US2020/049627 was published under PCT Article 21(2) in English. A certified copy of foreign priority application filed on 03/07/2022 is acknowledged.
Information Disclosure Statement
6. The information disclosure statement (IDS) submitted on 10/17/2022 is in compliance with the provisions of 37 CFR 1.97 and is being considered by the examiner.
Claim Rejections - 35 USC § 103 (Modified)
On 12/17/2025, applicant amended claims, cancelled claims 3, and 5-6 and added new claims 58-60. Applicant’s amendment changed the scope of the claimed inventions requiring additional searching and modification of the office action by applying new prior art for rejection of claims 58-60 under 35 U.S.C. 103 of as recited below. The prior art by Kohn et al 2017 (disclosed in IDS filed on 10/17/2022 and cited in prior non-final rejection office action (06/17/2025), US20170157270A1 published 06/08/2017) was used for clarity on the PBMC claim limitation for dependent claim 2 and other claims.
7. 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. 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.
8. Claims 1, 8, 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), and further in view of Nicholas et al 2007 (Stem Cells and Development 16:109–117) and Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250).
Claim 1: A transduction efficiency assay comprising:
a) transducing a population of cells from a sample with a lentiviral vector comprising a polynucleotide encoding a therapeutic gene;
b) culturing the transduced cells for a period of at least three days;
c) assaying the cultured transduced cells using single cell PCR;
d) measuring presence of genomic and viral DNA sequences in the cells in the sample;
e) quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and viral DNA sequences;
f) quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences; and
g) calculating efficiency of lentiviral vector transduction (percentage of transduced cells), wherein the efficiency of the transduction is measured as:
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Charrier et al 2011 discloses a transduction efficiency assay, quantification of lentiviral copy numbers in individual hematopoeitic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction, comprising: a) transducing a population or cells from a sample with a lentiviral vector comprising a polynucleotide encoding a therapeutic gene (See, Charrier et al 2011, p.479 abstract, p. 479, a human cell population transduced with a rHIV LV, col 1 , second paragraph, and P. 480, col 1, top of first paragraph; HIV vectors are actively developed, including for instance a treatment of Wiskott-Aldrich syndrome (WAS). ALV encoding the WAS protein (WASP) is being developed to treat this life-threatening X-linked primary deficiency, P. 479, col 1, first paragraph); b) culturing the transduced cells for a period of at least three days (when cells were re-infected, the second hit of vector was added overnight. At the end of the transduction step, cells were washed ...cultures were incubated at 37 degrees Celsius, 5% CO2 for 14 days, P. 486, third paragraph); c) assaying the cultured transduced cells using single cell PCR (quantitative PCR (Q-PCR) has been used to provide more precise measures and the technique can be calibrated on dilutions of a plasmid bearing both vector and genomic sequences to determine average vector copy number (VCN) in a human cell transduced with a rHIV LV, P. 479, col 2, second paragraph; and P. 480, col 1, top of first paragraph; The transduction of hematopoietic progenitor cells can therefore be assessed at the clonal level by measuring VCN in a single unit of CFC (P. 481, col 2, top of first paragraph); d) measuring presence of genomic and viral DNA sequences in the cells in the sample (genomic DNA of the cells can be extracted from these colony-forming cells (CFC) with proteinase K and phenol/chloroform to determine the presence or absence of vector by PCR, P. 480, col 1, first paragraph); e) quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and viral DNA sequences (determine the presence or absence of vector by PCR and agarose gels, thus, determining the frequency of transduced hematopoietic progenitor cells in the initially-infected population of cells, P. 480, col 1, first paragraph); f) quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences (determine the presence or absence of vector by PCR and agarose gels, thus, determining the frequency of transduced hematopoietic progenitor cells in the initially-infected population of cells, P. 480, col 1, first paragraph); and g) calculating efficiency of lentiviral vector transduction (percentage of transduced cells). wherein the efficiency of the transduction is measured as: Transduction efficiency(%) =( ∑ transduced cells/ ∑ tranduced and untransduced cells) x 100 (CD34+ cells were infected with the GFP-LV at 2x10A8 IG per ml giving mean average VCN of 1.4±0.4 in the whole population (n¼3 experiments) and in this set of experiments, the presence of vector was measured by Q-PCR on CFC (Table 2 and Figure 5a). This showed 70% vector-positive individual CFC (Table 2 and Figure 5a), which is close to the expected transduction efficiency of 65% calculated from the Poisson distribution as per Ref. 18, P. 482, col 2, bottom of third paragraph). Thus, Charrier et al 2011 teaches quantitative analysis of vector copy number in individual colony-forming cell (CFC) unit with Q-PCR and determines the frequency and the distribution of vector copies in the population of hematopoietic progenitor cells that were initially targeted by the vector.
Charrier et al 2011 does not teach assaying the cultured transduced cells using single cell PCR.
Nicholas et al 2007 teaches a method for single cell sorting and expansion of genetically modified human embryonic stem cells, a method for producing and expanding hES cell lines from single cells that have been isolated by fluorescence-activated cell sorting (FACS) following genetic modification by lentivirus vectors (See, Nicholas et al 2007, abstract, entire article).
Penter et al 2018 teaches fluorescence activated cell sorting (FACS) single cell index sorting is highly reliable and determines immune phenotypes of clonally expanded T cells (See, Penter et al 2018, Figs 1-2, abstract, entire article).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the prior art teachings of Charrier et al 2011 on quantitative analysis of vector copy number in individual colony-forming cell (CFC) unit by incorporating teachings of Nicholas et al 2007 and Penter et al 2018 on FACS based single cell sorting to arrive at the invention of claim 1. One of ordinary skills in the art would have been motivated to incorporate the teachings of Nicholas et al 2007 and Penter et al 2018 on FACS based single cell sorting to for rapid turnover time for quantitative analysis of transduction efficiency assay for determining both genomic and viral DNA sequences, applying to cell types that do not form colonies in soft agarose or methylcellulose medium overlay and for commercial success. Moreover, high infection efficiency is not necessary when using FACS for isolation of individual transduced cells (See, Nicholas et al 2007, P.114 col 1, para 1). One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 1 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 as applied to claim 1 and as recited supra.
Claims 8, 16-17: The combined teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 teaches the assay of claim 1 as recited supra.
Claim 8: Charrier et al 2011 further teaches added limitation of claim 8, wherein the cells are hematopoietic stem or progenitor cells by disclosing an assay for quantification of lentiviral vector copy numbers in individual hematopoietic stem cells (HSC) colony forming units (See, Charrier et al 2011, Title and abstract).
Claim 16: Charrier et al 2011 further teaches added limitation of claim 16, wherein the culturing of the transduced cells occurs for a period of 3 to 10 days by disclosing transduction and incubation of cultures for 14 days (See, P.486, col 2, para 3 on transduction and culture).
Claim 17: Charrier et al 2011 further teaches added limitation of claim 17, wherein a cell is considered as transduced when it is measured as having a Threshold Cycle (Ct) value of < 32 for both genomic and viral DNA sequences by disclosing the lowest amount of genomic DNA that could provide an interpretable signal with a Ct of 31.2± 0.5 (n=94) …, thus confirming the range of predicted sensitivity of the Q-PCR.
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to further modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 with teachings of Charrier et al 2011 to arrive at the inventions of claims 8, and 16-17. One of ordinary skills in the art would have been motivated to modify the applied combined teachings to claim 1 to optimize the assay for inventions of claims 8 and 16-17 and for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claims 8 and 16-17 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 as applied to claims 8 and 16-17 and as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claims 8 and 16-17. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
9. Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250) and further in view of Kohn et al 2017 (Disclosed in IDS filed on 10/17/2022, US20170157270A1 published 06/08/2017), and Harrop 2018 (WO2018167486A1 published 09/20/2018).
Claim 2: The assay of claim 1, wherein the cells are peripheral blood mononuclear cells (PBMCs).
The combined teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 teaches the assay of claim 1 as recited supra. Charrier fails to explicitly disclose the added limitation of claim 2, wherein the cells are peripheral blood mononuclear cells.
Kohn et al 2017 (US20170157270A1) is in the art and a recombinant lentiviral vector is provided comprising an expression cassette comprising a nucleic acid construct comprising an anti-sickling human beta globin gene encoding an anti-sickling-beta globin polypeptide comprising the mutations Gly16Asp, Glu22Ala and Thr87Gln, where the lentiviral vector is a TAT-independent and self-inactivating (SIN). In certain embodiments the vector additionally contains one or more insulator elements are useful in gene therapy for the treatment of sickle cell disease (See, abstract). Kohn et al 2017 teaches the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cell, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ cells (See, para [0125], [0127]- [0128], [0132]-[0133], claims 1, 18-19, and 23).
Harrop 2018 (WO2018167486A1) is in the field of treating haematological cancers (See, abstract) and teaches cells are peripheral blood mononuclear cells, transduction of 5T4 CAR construct into PBMCs from ovarian cancer patients, P. 3, lines. 23-24, entire prior art). Harrop et al 2018 teaches cells are PBMCs isolated from a subject that has cancer, transduction of 5T4 CAR construct into PBMCs from ovarian cancer patients (See, Harrop 2018, WO2018167486A1, P. 3, lines 23-24). Harrop 2018 further teaches the lentiviral vector comprises an engineered antigen receptor by disclosing chimeric antigen receptor (CAR) and uses thereof in treating cancers (See, Harrop 2018 WO2018167486A1, abstract, claim 9).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to further modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018 as applied to the assay of the claim 1 with the additional teachings of Kohn et al 2017 (US20170157270A1) transduction of peripheral blood stem cells, that involve obtaining PBMCs from a subject or a human, with the lentiviral vector encoding globulin gene sequence, and Harrop 2018 (WO2018167486A1) on PBMCs in chimeric antigen receptor (CAR) and uses thereof in treating cancers (See, Harrop 2018 WO2018167486A1, abstract, claim 9) and incorporate the teachings on PBMCs to arrive at the invention of claim 2. One of ordinary skills in the art would have been motivated to further modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018 with additional teachings of Kohn et al 2017 and Harrop 2018 for measuring transduction efficiency (VCN copy number) and expression of the therapeutic gene in the PBMC for gene therapy approaches of SCD approaches for improved and specific treating hematological cancers and for commercial success (See, Kohn et al 2017 US20170157270A1 entire prior art, and Harrop 2018, WO2018167486A1, abstract, figures 1-19 with legends and entire prior art). One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 2 given the combined prior teachings of prior arts as applied to claim 2 and as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 2. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
10. Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250) and further in view of Kohn et al 2017 (US20170157270A1 published 06/08/2017).
Claim 9: The assay of claim 8, further comprising obtaining the hematopoietic stem or progenitor cells from a subject that has sickle cell disease or β-thalassemia.
The combined prior teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 teaches claim 8 as recited supra; however, fails to explicitly disclose further comprising obtaining the hematopoietic stem or progenitor cells from a subject that has sickle cell disease or β-thalassemia.
Kohn et al 2017 (US20170157270A1) is in the field of lentiviral vector for stem cell gene therapy of sickle cell disease (Title) and teaches obtaining the hematopoietic stem or progenitor cells from a subject that has sickle cell disease (human hematopoietic progenitor cells were re-isolated from the marrow, para [0012); hematopoietic stem and progenitor cells) that can then be transplanted into a subject in need thereof (e.g., a subject that has the sickle cell mutation), para [0072); the cell can be autologous to the subject (i.e., from the subject), para [0124).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 with teachings of Kohn et al 2017 to arrive at the invention of claim 9. One of ordinary skills in the art would have been motivated to modify the applied combined teachings for the purpose of producing sufficient levels of an anti-sickling Hb protein to improve the physiological parameters of the RBCs that can be utilized for clinical gene therapy of SCD (Kohn et al 2017 US20170157270A1, para [00121) and for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 9 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018, Kohn et al 2017 as applied to claim 9 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 9. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
11. Claims 10 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250) and further in view of Bonner et al 2019 (US20190078059A1, published 03/14/2019).
Claim 10: The assay of claim 8, wherein the hematopoietic stem or progenitor cells comprise CD34+ cells, CD133+ cells, and/or CD34+CD38LOCD90+CD45RA- cells.
The combined prior teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 teaches claim 8 as recited supra; however, fails to explicitly disclose the added limitation of claim 10 wherein the hematopoietic stem or progenitor cells comprise CD34+ cells, CD133+ cells, and/or CD34+CD38LOCD90+CD45RA- cells.
Charrier et al 2011 further discloses, wherein the hematopoietic stem or progenitor cells comprise CD34+ cells (following transduction of CD34+ cells, P. 485, col 2, second paragraph).
Bonner et al 2019 (US20190078059A1) is in the art of increasing transduction efficiency and vector copy number (VCN) of human hematopoietic stem and progenitor cells (HSPCs) to yield improved gene therapy compositions. Bonner et al 2019 teaches lentiviral transducing a population of CD34+ hematopoietic stem or progenitor cells (See, Bonner et al 2019, US20190078059A1, claim 1). The population of hematopoietic cells of claim 1, wherein the HSPCs comprise CD34+ cells or CD133+ cells (See, Bonner et al 2019, US20190078059A1, claim 6). The population of hematopoietic cells of any one of claims 1-6, wherein the HSPCs comprise CD34+CD38LoCD90+CD45RA− cells (See, Bonner et al 2019, US20190078059A1, claim 7).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 with teachings of Bonner et al 2019 to arrive at the invention of claim 10. One of ordinary skills in the art would have been motivated to modify the combined teachings for the purpose of the assay quantification of the vector genome copies of hematopoietic stem or progenitor cells comprise CD34+ cells, CD133+ cells, and/or CD34+CD38LOCD90+CD45RA- cells used for cancer treatment and for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 10 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 and Bonner et al 2019 as applied to claim 10 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 10. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
Claim 13: The assay of claim 8, wherein the hematopoietic stem or progenitor cells comprise a pair of β-globin alleles selected from the group consisting of: βE/β0, βC/β0, β0/β0, βC/βC, βE/βE, βE/β+, βC/βE, βC/β+, β0/β+, or β+/β+.
Bonner et al 2019 teaches gene therapies comprise hematopoietic stem and progenitor cell compositions with increased therapeutic efficacy (See, abstract) and discloses the population of hematopoietic cells the β-globin alleles of the subject are βE/β0, βC/β0, β0/β0, βC/βC, βE/βE, βE/β+, βC/βE, βC/β+, β0/β+, or β+/β+ (See, Bonner et al 2019, claim 85).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 with teachings of Bonner et al 2019 to arrive at the invention of claim 13. One of ordinary skills in the art would have been motivated to modify the combined teachings for the purpose of the assay quantification of the vector genome copies of hematopoietic stem or progenitor cells for treatment of the claimed β-globin alleles defects or deficiencies and for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 13 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 and Bonner et al 2019 as applied to claim 13 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 13. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
Claim 14: The assay of claim 8, wherein the polynucleotide encodes a globin selected from the group consisting of a human β-globin, a human δ-globin, an anti-sickling globin, a human y-globin, a human βA-TS7Q-globin, a human βA-G16D/E22A/T87Q-globin, and a human βA-T87Q/K95E/K20E-globin protein.
Bonner et al 2019 teaches gene therapies comprise hematopoietic stem and progenitor cell compositions with increased therapeutic efficacy (See, abstract). Bonner et al 2019 discloses hematopoietic cells wherein the gene of interest encodes an anti-sickling protein or a globin gene (See, claim 82); a human β-globin protein, a human δ-globin protein, a human γ-globin protein, a human βA-T87Q-globin protein, a human βA-G16D/E22A/T87Q-globin protein, or a human βA-T87Q/K95E/K120E-globin protein (See, Bonner et al 2019, claim 83).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 with teachings of Bonner et al 2019 to arrive at the invention of claim 14. One of ordinary skills in the art would have been motivated to modify the combined teachings for the purpose of the assay quantification of the vector genome copies of hematopoietic stem or progenitor cells for treatment of the claimed globin alleles defects related disease and for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 13 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 and Bonner et al 2019 as applied to claim 14 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 14. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
Claim 15: Bonner et al 2019 teaches the added limitation of the claim 15, wherein the lentiviral vector is an AnkT9W vector, a T9Ank2W vector, a TNS9 vector, a lentiglobin HPV569 vector, a lentiglobin BB305 vector, a BG-1 vector, a BGM-1 vector, a d432βAγ vector, a mLARβΔγV5 vector, a GLOBE vector, a G-GLOBE vector, a βAS3-FB vector, a V5 vector, a V5m3 vector, a V5m3-400 vector, a G9 vector, or a BCL11A shmir vector (See, Bonner et al 2019, claim 117).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to further modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 with additional teachings of Bonner et al 2019 on the claimed lentiviral vector(s) as disclosed by Bonner et al 2019 in claim 117 to arrive at the invention of claim 15. One of ordinary skills in the art would have been motivated to further modify the combined teachings for the purpose of using the lentiviral vector that has a fully characterized genetic and sequence elements (to avoid undesirable lentiviral genome recombination in the transduced cells) for insertion of a desired therapeutic transgene in desirable cells or hematopoietic stem or progenitor cells for treatment of the target genetic disease or cancer for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 15 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 and Bonner et al 2019 as applied to claim 15 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 15. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
12. Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250) and further in view of Kuate et al 2012 (RNA Viral Vectors, vol 20, Suppl 1S137) and Sastry et al 2003 (Molecular Therapy vol. 8, No. 5, November 2003).
Claim 18: The assay of claim 1, wherein the viral DNA sequences is a lentiviral vector psi-gag DNA sequence.
The combined teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 teaches the assay of claim 1 as recited supra, however, fails to teach the added limitation of the claim 18 wherein the viral DNA sequences is a lentiviral vector psi-gag DNA sequence.
Kuate et al 2012 teaches a cell-based assay to detect rare psi-gag recombinants in lentiviral vector preparations (See, Kuate et al 2012, abstract).
Sastry et al 2003 teaches a psi-gag assay that detects recombination between gag regions in the transfer vector and the packaging construct, psi–gag recombinants (See, Sastry et al 2003, abstract, entire article).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 and incorporate the teachings of Kuate et al 2012 and Sastry et al 2003 to arrive at the invention of claim 18. One of ordinary skills in the art would have been motivated to develop an assay for detection of psi-gag recombinant vectors that can arise, where sequences from the packaging construct (gag) are incorporated into the vector genome. These recombinants can potentially lead to replication-competent lentivirus (RCL), which is undesirable in clinical applications (See, Kuate et al 2012 and Sastry et al 2003). One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 18 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Kuate et al 2012 and Sastry et al 2003 as applied to claim 18 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 18. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
13. Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250) and further in view of McNees et al 2005 (Journal of Clinical Virology 34, p. 52–62).
Claim 19: The assay of any one of claim 1, wherein the genomic DNA sequence is a RNAseP DNA sequence.
The combined teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 teaches the assay of claim 1 as recited supra, however, fails to teach the added limitation of the claim 19 wherein the genomic DNA sequence is a RNAseP DNA sequence.
McNees et al 2005 teaches a development and evaluation of a quantitative real time PCR assay to measure the single copy human RNAse P gene in order to normalize viral gene copy numbers to cell numbers (See, abstract, p. 56 fig 3-D, and entire article).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 and incorporate the teachings of McNees et al 2005 to arrive at the invention of claim 19. One of ordinary skills in the art would have been motivated to develop a human RNAse P gene quantification assay for normalizing or relative quantification of lentiviral delivered target disease transgene integration following transduction of cell. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 19 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Kuate et al 2012 and McNees et al 2005 as applied to claim 19 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 19. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
14. Claims 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250) and further in view of Superti-Furga et al 2017 (US20170356911A1 published 12/14/2017), Kohn et al 2017 (Disclosed in IDS filed on 10/17/2022, US20170157270A1 published 06/08/2017).
Claim 20: A transduction efficiency assay comprising: a) obtaining a peripheral blood or bone marrow sample from a subject; b) isolating nucleated cells from the peripheral blood by density gradient centrifugation; c) assaying the isolated cells using single cell PCR; d) measuring presence of genomic and lentiviral vector DNA sequences in the cells in the sample; e) quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and lentiviral vector DNA sequences; f) quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences; and g) calculating efficiency of lentiviral vector transduction, wherein the efficiency of the transduction is measured as:
The combined teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 as recited supra rendering obvious claim 1 are incorporated here in its entirety, and the combined prior arts teaches a transduction efficiency assay, inter alia, comprising transducing a population of cells (hematopoeitic stem cells) from a sample with a lentiviral vector comprising a polynucleotide encoding a therapeutic gene; culturing the transduced cells, assay of the the cultured transduced cells using single cell PCR, measuring presence of genomic and viral DNA sequences in the cells in the sample, quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and viral DNA sequences, quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences; and calculating efficiency of lentiviral vector transduction (percentage of transduced cells), wherein the efficiency of the transduction is measured as:
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The combined teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 fails to explicitly disclose: a) obtaining a peripheral blood or bone marrow sample from a subject; b) isolating nucleated cells from the peripheral blood by density gradient centrifugation.
Superti-Furga et al 2017 is in the field of peripheral blood mononuclear cell (PBMC) monolayers (Abstract) and teaches obtaining a peripheral blood or bone marrow sample from a subject (the sample comprising PBMCs, para [00111]; b) isolating nucleated cells from the peripheral blood by density gradient centrifugation ([t]he polymorphonuclear cells can be further isolated by lysing the red blood cells, i.e. non-nucleated cells. Common density gradients useful for such centrifugation include, but are not limited to, Ficoll, para [0053].
Kohn et al 2017 (US20170157270A1) is in the art and a recombinant lentiviral vector is provided comprising an expression cassette comprising a nucleic acid construct comprising an anti-sickling human beta globin gene encoding an anti-sickling-beta globin polypeptide comprising the mutations Gly16Asp, Glu22Ala and Thr87Gln, where the lentiviral vector is a TAT-independent and self-inactivating (SIN). In certain embodiments the vector additionally contains one or more insulator elements are useful in gene therapy for the treatment of sickle cell disease (See, abstract). Kohn et al 2017 teaches the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cell, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ cells (See, para [0125], [0127]- [0128], [0132]-[0133], claims 1, 18-19, and 23).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the combined teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 by incorporating teachings of Superti-Furga et al 2017 and Kohn et al 2017 on PBMCs and peripheral blood stem cells to arrive at the invention of the claim 20. One of ordinary skills in the art would have been motivated to modify the applied prior art teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 for the purpose of efficiently separating whole blood into components separated by layers: a top layer of plasma, followed by a layer of PBMCs (Superti-Furga et al 2017, para [0053] for efficient transduction of purified PBMCs and as per teachings of Kohn et al 2017 peripheral blood stem cells obtained from PBMCs. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 20 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Superti-Furga et al 2017 and Lohn et al 2017 as applied to claim 20 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 20. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
Claim 21: The assay of claim 20, wherein the nucleated cells are peripheral blood mononuclear cells.
The combined teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Superti-Furga et al 2017 and Kohn et al 2017 renders obvious claim 20 as recited supra
Superti-Furga et al 2017 further teaches nucleated cells are peripheral blood mononuclear cells, the polymorphonuclear cells can be further isolated by lysing the red blood cells, i.e. non-nucleated cells. Common density gradients useful for such centrifugation include, but are not limited to, Ficoll, (See, para. [00531].
Kohn et al 2017 (US20170157270A1) is in the art and a recombinant lentiviral vector is provided comprising an expression cassette comprising a nucleic acid construct comprising an anti-sickling human beta globin gene encoding an anti-sickling-beta globin polypeptide comprising the mutations Gly16Asp, Glu22Ala and Thr87Gln, where the lentiviral vector is a TAT-independent and self-inactivating (SIN). In certain embodiments the vector additionally contains one or more insulator elements are useful in gene therapy for the treatment of sickle cell disease (See, abstract). Kohn et al 2017 teaches the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cell, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ cells (See, para [0125], [0127]- [0128], [0132]-[0133], claims 1, 18-19, and 23).
It would have been obvious to one of ordinary skill in the art at the time of the invention to further modify the combined teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 by incorporating teachings of Superti-Furga et al 2017 on nucleated PBMCs and Kohn et al 2017 teachings on PBMC derived peripheral blood stem cells to arrive at the invention of the claim 21. One of ordinary skills in the art would have been motivated to modify the applied prior art teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 for the purpose of efficiently separating whole blood into components separated by layers: a top layer of plasma, followed by a layer of nucleated PBMCs (Superti-Furga et al 2017, para [0053]) and Kohn et al 2017 PBMC comprising peripheral blood stem cells for efficient transduction of purified nucleated PBMCs and integration in the nucleated PBMC chromosomes of the lentiviral genome comprising a desirable transgene for engineered immune cell therapeutic purpose. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 21 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Superti-Furga et al 2017, and Kohn et al 2017 as applied to claim 21 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 21. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
15. Claims 22 and 38 are rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250), Superti-Furga et al 2017 (US20170356911A1 published 12/14/2017), Kohn et al 2017 (Disclosed in IDS filed on 10/17/2022, US20170157270A1 published 06/08/2017),
and further in view of Bonner et al 2019 (US20190078059A1, published 03/14/2019).
Claim 22: The assay of claim 20, wherein the nucleated cells are hematopoietic stem or progenitor cells.
The combined teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Superti-Furga et al 2017 and Kohn et al 2017 renders obvious claim 20 as recited supra
Bonner et al 2019 teaches gene therapies comprise hematopoietic stem and progenitor cell compositions with increased therapeutic efficacy. Bonner et al 2019 discloses the invention contemplates compositions and methods for increasing the engineered lentiviral transduction efficiency and vector copy number (VCN) of human hematopoietic stem and progenitor cells (HSPCs) to yield improved gene therapy compositions, a population of HSPCs transduced with a lentiviral vector, a method of treating sickle cell disease in a subject comprising administering the subject an effective amount of the population of hematopoietic cells (See, Bonner et al 2019, abstract, claim 1). It is evident that the lentiviral genome integrates in chromosomal DNA of the hematopoietic stem and progenitor cells and thus renders obvious the instant claim 22 added limitation. It would have been obvious to one of ordinary skill in the art at the time of the invention to further modify the combined teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018, Superti-Furga et al 2017 by incorporating teachings of Bonner et al 2019 on the nucleated cells are hematopoietic stem or progenitor cells to arrive at the invention of the claim 22. One of ordinary skills in the art would have been motivated to modify the applied prior art teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018 for the purpose of engineering human hematopoietic stem and progenitor cells with lentiviral vector comprising a desirable transgene for treatment of genetic disease e.g. treating sickle cell disease. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 22 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Superti-Furga et al 2017 and Bonner et al 2019 as applied to claim 22 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 22. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
Claim 38: The assay of any one of claim 20, wherein the subject has sickle cell disease or β-thalassemia.
The combined teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Superti-Furga et al 2017 and Kohn et al 2017 renders obvious claim 20 as recited supra, however, fails to teach the added limitation of instant claim 38, wherein the subject has sickle cell disease or β-thalassemia.
Bonner et al 2019 teaches gene therapies comprise hematopoietic stem and progenitor cell compositions with increased therapeutic efficacy. Bonner et al 2019 discloses the invention contemplates, in part, a method of treating a β-thalassemia in a subject comprising administering the subject an effective amount of the population of hematopoietic cells (See, para [0186], [0009], [0495], claims 170, 174, abstract]).
It would have been obvious to one of ordinary skill in the art at the time of the invention to further modify the combined teachings of Charrier et al 2011, Nicholas et al 2007, Penter et al 2018, Superti-Furga et al 2017, and Kohn et al 2017 by incorporating teachings of Bonner et al 2019 on a subject having sickle cell disease or β-thalassemia comprising administering the subject an effective amount of the population of hematopoietic cells to arrive at the invention of the claim 38. One of ordinary skills in the art would have been motivated to modify the applied prior art teachings for the purpose of treating a subject having sickle cell disease or β-thalassemia comprising administering the subject an effective amount of the population of hematopoietic cells. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 38 given the combined prior teachings as applied as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 38. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
16. Claim 39 is rejected under 35 U.S.C. 103 as being unpatentable over Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), and further in view of Nicholas et al 2007 (Stem Cells and Development 16:109–117) and Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250).
Claim 39: A method for measuring transduction efficiency comprising: a. assaying a population of cells using single cell PCR, wherein the population of cells are transduced with a lentiviral vector comprising a polynucleotide encoding a therapeutic gene; b. measuring presence of genomic and viral DNA sequences in the cells; c. quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and viral DNA sequences; d. quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences; and e. calculating efficiency of the lentiviral vector transduction, wherein the efficiency of the transduction is measured as:
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Charrier et al 2011 discloses a method for measuring transduction efficiency assay, quantification of lentiviral copy numbers in individual hematopoeitic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction, comprising: a) transducing a population or cells from a sample with a lentiviral vector comprising a polynucleotide encoding a therapeutic gene (See, Charrier et al 2011, p.479 abstract, p. 479, a human cell population transduced with a rHIV LV, col 1 , second paragraph, and P. 480, col 1, top of first paragraph; HIV vectors are actively developed, including for instance a treatment of Wiskott-Aldrich syndrome (WAS). ALV encoding the WAS protein (WASP) is being developed to treat this life-threatening X-linked primary deficiency, P. 479, col 1, first paragraph); b) culturing the transduced cells for a period of at least three days (when cells were re-infected, the second hit of vector was added overnight. At the end of the transduction step, cells were washed ...cultures were incubated at 37 degrees Celsius, 5% CO2 for 14 days, P. 486, third paragraph); c) assaying the cultured transduced cells using single cell PCR (quantitative PCR (Q-PCR) has been used to provide more precise measures and the technique can be calibrated on dilutions of a plasmid bearing both vector and genomic sequences to determine average vector copy number (VCN) in a human cell transduced with a rHIV LV, P. 479, col 2, second paragraph; and P. 480, col 1, top of first paragraph; The transduction of hematopoietic progenitor cells can therefore be assessed at the clonal level by measuring VCN in a single unit of CFC, P. 481, col 2, top of first paragraph); d) measuring presence of genomic and viral DNA sequences in the cells in the sample (genomic DNA of the cells can be extracted from these colony-forming cells (CFC) with proteinase K and phenol/chloroform to determine the presence or absence of vector by PCR, P. 480, col 1, first paragraph); e) quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and viral DNA sequences (determine the presence or absence of vector by PCR and agarose gels, thus, determining the frequency of transduced hematopoietic progenitor cells in the initially-infected population of cells, P. 480, col 1, first paragraph); f) quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences (determine the presence or absence of vector by PCR and agarose gels, thus, determining the frequency of transduced hematopoietic progenitor cells in the initially-infected population of cells, P. 480, col 1, first paragraph); and g) calculating efficiency of lentiviral vector transduction (percentage of transduced cells). wherein the efficiency of the transduction is measured as: Transduction efficiency(%) =( ∑ transduced cells/ ∑ tranduced and untransduced cells) x 100 (CD34+ cells were infected with the GFP-LV at 2x10A8 IG per ml giving mean average VCN of 1.4±0.4 in the whole population (n¼3 experiments) and in this set of experiments, the presence of vector was measured by Q-PCR on CFC (Table 2 and Figure 5a). This showed 70% vector-positive individual CFC (Table 2 and Figure 5a), which is close to the expected transduction efficiency of 65% calculated from the Poisson distribution as per Ref. 18, P. 482, col 2, bottom of third paragraph). Thus, Charrier et al 2011 teaches a method for quantitative analysis of vector copy number in individual colony-forming cell (CFC) unit with Q-PCR and determines the frequency and the distribution of vector copies in the population of hematopoietic progenitor cells that were initially targeted by the vector.
Charrier et al 2011 do not explicitly teach assaying a population of cells using single cell PCR in the recited method supra.
Nicholas et al 2007 teaches a method for single cell sorting and expansion of genetically modified human embryonic stem cells, a method for producing and expanding hES cell lines from single cells that have been isolated by fluorescence-activated cell sorting (FACS) following genetic modification by lentivirus vectors (See, Nicholas et al 2007, abstract, entire article).
Penter et al 2018 teaches a method for fluorescence activated cell sorting (FACS) single cell index sorting is highly reliable and determines immune phenotypes of clonally expanded T cells (See, Penter et al 2018, Figs 1-2, abstract, entire article).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the prior art teachings of Charrier et al 2011 on a method for quantitative analysis of vector copy number in individual colony-forming cell (CFC) unit by incorporating teachings of Nicholas et al 2007 and Penter et al 2018 on methods of FACS based single cell sorting to arrive at the invention of claim 39. One of ordinary skills in the art would have been motivated to incorporate the teachings of Nicholas et al 2007 and Penter et al 2018 on methods of FACS based single cell sorting to for rapid turnover time for quantitative analysis of transduction efficiency assay for determining both genomic and viral DNA sequences, applying to cell types that do not form colonies in soft agarose or methylcellulose medium overlay and for commercial success. Moreover, high infection efficiency is not necessary when using FACS for isolation of individual transduced cells (See, Nicholas et al 2007, P.114 col 1, para 1). One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 39 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 as applied to claim 39 and as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 39. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
17. Claim 58 is rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250), Superti-Furga et al 2017 (US20170356911A1 published 12/14/2017), Bonner et al 2019 (US20190078059A1, published 03/14/2019) as applied to claim 22 above and further additional teachings of Bonner et al 2019 (US20190078059A1, published 03/14/2019) teaches added limitation of instant claim 58.
Claim 58: The assay of claim 22, wherein the hematopoietic stem or progenitor cells comprise one or more of CD34+ cells, CD133+ cells, and CD34+CD38LoCD90+CD45RA- cells.
The combined prior teachings of Charrier et al 2011, Nicholas et al 2007, and Penter et al 2018, Superti-Furga et al 2017 and Bonner et al 2019 teaches claim 22 as recited supra.
The added limitation of instant claim 58, wherein the hematopoietic stem or progenitor cells comprise one or more of CD34+ cells, CD133+ cells, and CD34+CD38LoCD90+CD45RA- cells is taught by Charrier et al 2011 and Bonner et al 2019 as recited below:
Charrier et al 2011 is in the art and discloses, wherein the hematopoietic stem or progenitor cells comprise CD34+ cells (following transduction of CD34+ cells, P. 485, col 2, second paragraph).
Bonner et al 2019 (US20190078059A1) is in the art and discloses increasing transduction efficiency and vector copy number (VCN) of human hematopoietic stem and progenitor cells (HSPCs) to yield improved gene therapy compositions. Bonner et al 2019 teaches lentiviral transducing a population of CD34+ hematopoietic stem or progenitor cells (See, Bonner et al 2019, US20190078059A1, claim 1). The population of hematopoietic cells of claim 1, wherein the HSPCs comprise CD34+ cells or CD133+ cells (See, Bonner et al 2019, US20190078059A1, claim 6). The population of hematopoietic cells of any one of claims 1-6, wherein the HSPCs comprise CD34+CD38LoCD90+CD45RA− cells (See, Bonner et al 2019, US20190078059A1, claim 7).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018, Bonner et al 2019 with additional teachings of Charrier et al 2011 and Bonner et al 2019 to arrive at the invention of claim 58. One of ordinary skills in the art would have been motivated to modify the combined teachings for the purpose of the assay quantification of the vector genome copies of hematopoietic stem or progenitor cells comprise CD34+ cells, CD133+ cells, and/or CD34+CD38LOCD90+CD45RA- cells used for cancer treatment and for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 58 given the combined prior teachings applied to claim 58 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 58. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
18. Claim 59 is rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250) and Superti-Furga et al 2017 (US20170356911A1 published 12/14/2017) as applied to claim 20, and further in view of Kohn et al 2014 (Clinical Trial ID No. NCT02247843, First Posted Date 09/25/2014), Kohn et al 2017 (Disclosed in IDS filed on 10/17/2022, US20170157270A1 published 06/08/2017), Ribeil et al 2017 (N Engl J Med 2017; 376:848-855), Romero et al 2013 (J Clin Invest. 2013 Jul 1;123(8):3317–30), and Weber et al 2018 (Molecular Therapy: Methods & Clinical Development Vol. 10 September 2018).
Claim 59: The assay of claim 20, wherein the peripheral blood is obtained from a subject treated with a drug product comprising a lentiviral vector comprising a polynucleotide encoding a globin.
The combined teachings of Charrier et al 2011 (teaches the cells are PBMCs isolated from a subject that has cancer), Nicholas et al 2007, Penter et al 2018 and Superti-Furga et al 2017 teaches claim 20 as recited supra. However, does not explicitly disclose the added limitation of the instant claim 60, wherein the peripheral blood is obtained from a subject treated with a drug product comprising a lentiviral vector comprising a polynucleotide encoding a globin.
Kohn et al 2014 (Clinical Trial ID: NCT02247843) is in the art and teaches Clinical Research Study of Autologous Stem Cell Transplantation for Sickle Cell Disease (SCD) Using Peripheral Blood CD34+ Cells Modified With the Lenti/G-βAS3-FB Lentiviral Vector. Kohn et al 2014 teaches a phase I clinical trial to assess the safety and initial evidence for efficacy of an autologous transplant of lentiviral vector modified peripheral blood for adults with severe sickle cell disease. Autologous HSCT using a patient's own peripheral blood stem cells that have been corrected by transfer of a modified human beta-globin gene that inhibits polymerization of the HbS (stem cell gene therapy) may provide a better therapeutic alternative, as it would avoid the immunologic complications and donor limitations of allogeneic HSCT. CD34+ from the peripheral blood of patients with sickle cell disease (SCD) are transduced ex-vivo with the Lenti/βAS3-FB lentiviral vector. The transduced cells are then infused into the patient (See, Kohn et al 2014 (Clinical Trial ID: NCT02247843, PDF print out - attached).
Kohn et al 2017 (US20170157270A1) is in the art and a recombinant lentiviral vector is provided comprising an expression cassette comprising a nucleic acid construct comprising an anti-sickling human beta globin gene encoding an anti-sickling-beta globin polypeptide comprising the mutations Gly16Asp, Glu22Ala and Thr87Gln, where the lentiviral vector is a TAT-independent and self-inactivating (SIN). In certain embodiments the vector additionally contains one or more insulator elements are useful in gene therapy for the treatment of sickle cell disease (See, abstract). Kohn et al 2017 teaches the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cell, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ cells (See, para [0125], [0127]- [0128], [0132]-[0133], claims 1, 18-19, and 23).
Ribeil et al 2017 is in the art of treatment of sickle cell disease (SCD). Ribeil et al 2017 discloses a clinical trial NCT02151526 conducted for treatment of SCD and the treatment of a first patient with lentiviral vector-mediated addition of an antisickling β-globin gene into autologous hematopoietic stem cells. Fifteen months after treatment, the level of therapeutic antisickling β-globin remained high (approximately 50% of β-like–globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease (See, abstract, Table 1, entire article).
Romero et al 2013 is in the art of autologous hematopoietic stem cells (HSCs) gene therapy to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. Romero et al 2013 teaches treatment of sickle cell disease by disclosing β-globin gene transfer to human bone marrow by using a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells (See, abstract, entire article). One of the ordinary skills is apprised of Peripheral blood mononuclear cells (PBMCs) are a variety of specialized immune cells produced in the bone marrow from hematopoietic stem cells (HSCs).
Weber et al 2018 teaches treatment of an SCD by disclosing an optimized Lentiviral vector efficiently corrects the human sickle cell disease phenotype. The expression of the anti-sickling HBB and the reduced incorporation of the βS-chain in hemoglobin tetramers allowed up to 50% reduction in the frequency of RBC sickling under hypoxic conditions. Together, these results demonstrate the ability of a high-titer LV to express elevated levels of a potent anti-sickling HBB transgene ameliorating the SCD cell phenotype (See, abstract). Weber et al 2018 teaches analysis of VCN analysis for VCN per diploid genome was determined by duplex TaqMan qPCR that can be reasonably used to evaluate the efficacy of the treatment by using the DNA extracted from peripheral blood cells (nucleated cells) for VCN analysis (See, page 277, clol 2 VCN analysis, page 278 col 1 para 1).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to further modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018, Superti-Furga et al 2017with additional teachings of Kohn et al 2014, Kohn et al 2017, Ribeil et al 2017, Romero et al 2013 and Weber et al 2018 to obtain the peripheral blood from subjects or humans treated with a drug product comprising a lentiviral vector comprising a polynucleotide encoding a globin to monitor and assess the efficacy of the treatment to detect and quantify the globin transgene lentiviral vector copy number (globin VCN) in the peripheral blood cells (nucleated cells) to arrive at the inventions of claim 59. One of ordinary skills in the art would have been motivated to modify the prior art teachings as applied to the claim 20 above to perform the VCN assay using peripheral blood cells of the invention of instant claim 59 to study the efficacy of the treatment and for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 59 given the combined prior teachings applied to the claim 59 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 59. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
19. Claim 60 is rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487), Nicholas et al 2007 (Stem Cells and Development 16:109–117), Penter et al 2018 (Eur. J. Immunol. 2018. 48: 1248–1250) and additional teachings of Charrier et al 2011 (Gene Therapy, vol 18, p. 479–487).
Claim 60: The method of claim 39, wherein the cells are hematopoietic stem or progenitor cells.
Charrier et al 2011 further teaches added limitation of instant claim 60, wherein the cells are hematopoietic stem or progenitor cells by disclosing an assay for quantification of lentiviral vector copy numbers in individual hematopoietic stem cells (HSC) colony forming units (See, Charrier et al 2011, Title and abstract).
It would have been obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to further modify the combined prior art teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 with additional teachings of Charrier et al 2011 to arrive at the inventions of claim 60. One of ordinary skills in the art would have been motivated to modify the prior art teachings as applied to claim 39 to optimize the assay for inventions of claims 60 for hematopoietic stem or progenitor cells and for commercial success. One of the ordinary skills in the art would have been apprised of a reasonable expectation of success to arrive at the invention of claim 60 given the combined prior teachings of Charrier et al 2011, Nicholas et al 2007 and Penter et al 2018 as recited supra. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed inventions of claim 60. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, A-G).
Response to Arguments
20. Applicant’s arguments with respect to claims 1-2, 8-10, 13-22, 38-39, and 58-60 amended and filed on 12/17/2025 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
In response to applicant's argument that the examiner has combined an excessive number of references, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991).
The response to the applicant’s arguments is given below:
Applicant’s argument 1:
Rejections under 35 U.S.C. §103:
Claims 1, 8, 16-17 and 39 stand rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. (Gene Therapy, 18: 479-487 (2011)) in view of Nicholas et al. (Stem Cells and Development 16:109-117 (2007)) and Penter et al. (Eur. J. Immunol. 48: 1248-1250 (2018)).
Charrier allegedly discloses evaluating "the transduction of hematopoietic progenitor cells at the single-cell level" by measuring "VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method." Charrier at Abstract. The Office recognizes that Charrier does "not teach assaying the cultured transduced cells using single cell PCR," and cites to Nicholas and Penter as allegedly teaching FACS-based single cell sorting to solve the deficiencies of Charrier. See Action at page 6. However, the combination of Charrier, Nicholas and Penter fails to teach or suggest "assaying the cultured transduced cells using single cell PCR," as required by the pending claims. For example, the subject application recognized that "[m]easurement of % LVV+ cells can be complicated by the lack of expression of the transgenic protein in the assayed cells, absence of fluorescent reporters in clinical vectors, and/or lack of suitable methods for detection of transgene expression." US 2022/0325336 at paragraph [0002]. The single cell PCR (scPCR) assay of the subject application was developed to detect individual cells with one or more integrations of LVV sequences, thereby enabling the quantification of the % LVV cells in a population. Id. at paragraph [0003]. "Prior to the development of [the claimed assay], transduction efficiency of LentiGlobin could only be measured by single colony PCR or estimated from bulk VCN using the Poisson equation," and estimating TE from VCN using a Poisson distribution gives a large over-estimation of the true TE in the sample. Id. at paragraph [0100]. Thus, there is a need for an assay that measures TE that does not rely on VCN. In fact, the subject application explicitly states that "[s]ingle cell PCR is an alternative to FACS or single colony qPCR for quantifying transduction efficiency without the need for transgene expression" (paragraph [0077]).
Accordingly, Charrier, Nicholas and Penter, whether considered alone or in combination,
fail to teach or suggest all of the elements of the pending claims. Specifically, the references fail to disclose assaying the cells transduced with a lentiviral vector using single cell PCR. Reconsideration and withdrawal of the rejection are respectfully requested.
In Response 1: Applicant’s arguments have been considered and are not persuasive because the claims 1, 8, 16-17 and 39 stand rejected under 35 U.S.C. § 103 based on the combined teachings of the prior arts and obviousness analysis and motivations as recited supra in the rejection. The prior arts teach single cell isolation and quantification of the VCN as recited supra.
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).
Applicant’s argument 2:
Claims 2-3 and 5 stand rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. in view of Nicholas et al. and Penter et al., and further in view of Harrop (WO 2018/167486).
As an initial matter, without acceding to the rejection, claims 3 and 5 are cancelled, and the rejection of these claims is now moot. Regarding claim 2, as discussed above, the combination of Charrier, Nicholas and Penter does not teach or suggested a transduction efficiency assay that includes assaying cultured transduced cells using single cell PCR, as required by claim 1. Harrop fails to cure the deficiencies of the combination of references. Harrop is merely cited as allegedly teaching peripheral blood mononuclear cells. However, Harrop fails to disclose or suggest a transduction efficiency assay that includes assaying cultured transduced cells using single cell PCR. Accordingly, reconsideration and withdrawal of the rejection are respectfully requested.
In Response 2: Applicant’s arguments have been considered and are not persuasive because the claims 2-3 and 5 stand rejected under 35 U.S.C. § 103 based on the combined teachings of the prior arts and obviousness analysis and motivations as recited supra in the rejection. The prior arts teach single cell isolation and quantification of the VCN as recited supra. Harrop is merely cited as allegedly teaching peripheral blood mononuclear cells. The rejection of claim 2 as recited supra teaches the added claim limitation on PBMCs. It is obvious that the PBMCs are obtained from a subject to purify the peripheral blood stem cells for transduction with lentiviral vector encoding globin for treatment of SCD in a subject.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, 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 this case, Harrop teaches PBMC purification. For clarity Kohn et al 2017 (cited in IDS and previously cited in the non-final rejection office action) was cited for claim 2 on teachings of obtaining peripheral blood stem cells from PBMCs for transduction with lentiviral vector encoding globin. The rejection is based on 35 U.S.C. 103 obviousness analysis.
Applicant’s argument regarding the rejection of claims 3 and 5 is moot because the applicant has cancelled claims 3 and 5 in claim version filed on 12/17/2025.
Therefore, the rejection of the claim 2 is maintained.
Applicant’s argument 3:
Claim 6 stands rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. in view of Nicholas et al., Penter et al., and Harrop, and further in view of Leung et al. (JCI Insight 2019; 4(11): e124430). Without acceding to the rejection, claim 6 is cancelled, and the rejection is now moot.
In Response 3: Applicant’s argument regarding the rejection of claim 6 is moot because the applicant has cancelled claim 6 in claim version filed on 12/17/2025.
Applicant’s argument 4:
Claim 9 stands rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. in view of Nicholas et al. and Penter et al., and further in view of Kohn et al. (US 2017/0157270). As discussed above, the combination of Charrier, Nicholas and Penter does not teach or suggested a transduction efficiency assay that includes assaying cultured transduced cells using single cell PCR, as required by claim 1. Kohn fails to cure the deficiencies of the combination of references. Kohn is merely cited as allegedly teaching obtaining hematopoietic stem or progenitor cells from a subject that has sickle cell disease. However, Kohn fails to disclose or suggest a transduction efficiency assay that includes assaying cultured transduced cells using single cell PCR.
Accordingly, reconsideration and withdrawal of the rejection are respectfully requested.
In Response 4: Applicant’s arguments have been considered and are not persuasive because the claim 9 stand rejected under 35 U.S.C. § 103 based on the combined teachings of the prior arts and obviousness analysis and motivations as recited supra in the rejection. The prior arts teach single cell isolation and quantification of the VCN as recited supra. In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, 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 this case, the combined prior art as recited supra in the office action renders obvious the claim 9.
Therefore, the rejection of the claim 9 is maintained.
Applicant’s argument 5:
Claims 10 and 13-15 stand rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. in view of Nicholas et al. and Penter et al., and further in view of Bonner et al. (US 2019/0078059).
As discussed above, the combination of Charrier, Nicholas and Penter does not teach or suggested a transduction efficiency assay that includes assaying cultured transduced cells using
single cell PCR, as required by claim 1. Bonner fails to cure the deficiencies of the combination of references. Bonner is merely cited as allegedly teaching lentiviral transduction of a population of hematopoietic stem or progenitor cells, including CD34+ cells or cells comprising a pair of β-globin alleles, or a polynucleotide encodes a globin. However, Bonner fails to disclose or suggest a transduction efficiency assay that includes assaying cultured transduced cells using single cell PCR.
Accordingly, reconsideration and withdrawal of the rejection are respectfully requested.
In Response 5: Applicant’s arguments have been considered and are not persuasive because the claims 10 and 13-15 stand rejected under 35 U.S.C. § 103 based on the combined teachings of the prior arts and obviousness analysis and motivations as recited supra in the rejection. The prior arts teach single cell isolation and quantification of the VCN as recited supra. In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, 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 this case, the combined prior art as recited supra in the office action renders obvious the claims 10 and 13-15.
Therefore, the rejection of the claims 10 and 13-15 is maintained.
Applicant’s argument 6:
Claim 18 stands rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. in view of Nicholas et al. and Penter et al., and further in view of Kuate et al. (RNA Viral Vectors, 20: Suppl 1S137) and Sastry et al. (Molecular Therapy, 8(5): November 2003).
As discussed above, the combination of Charrier, Nicholas and Penter does not teach or suggested a transduction efficiency assay that includes assaying cultured transduced cells using
single cell PCR, as required by claim 1. Kuate and Sastry fail to cure the deficiencies of the combination of references. Kuate and Sastry are merely cited as allegedly teaching a lentiviral vector psi-gag DNA sequence. However, Kuate and Sastry fail to disclose or suggest a transduction efficiency assay that includes assaying cultured transduced cells using single cell PCR. Accordingly, reconsideration and withdrawal of the rejection are respectfully requested.
In Response 6: Applicant’s arguments have been considered and are not persuasive because the claim 18 stand rejected under 35 U.S.C. § 103 based on the combined teachings of the prior arts and obviousness analysis and motivations as recited supra in the rejection. The prior arts teach single cell isolation and quantification of the VCN as recited supra.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, 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 this case, the combined prior art as recited supra in the office action renders obvious the claim 18.
Therefore, the rejection of the claim 18 is maintained.
Applicant’s argument 7:
Claim 19 stands rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. in view of Nicholas et al. and Penter et al., and further in view of McNees et al. (Journal of Clinical Virology 34: 52-62 (2005)).
As discussed above, the combination of Charrier, Nicholas and Penter does not teach or suggested a transduction efficiency assay that includes assaying cultured transduced cells using
single cell PCR, as required by claim 1. McNees fails to cure the deficiencies of the combination of references. McNees is merely cited as allegedly teaching that a genomic DNA sequence is a RNAseP DNA sequence. However, McNees fails to disclose or suggest a transduction efficiency assay that includes assaying cultured transduced cells using single cell PCR. Accordingly, reconsideration and withdrawal of the rejection are respectfully requested.
In Response 7: Applicant’s arguments have been considered and are not persuasive because the claim 19 stand rejected under 35 U.S.C. § 103 based on the combined teachings of the prior arts and obviousness analysis and motivations as recited supra in the rejection. The combined prior arts teachings teach single cell isolation and quantification of the VCN as recited supra.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, 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 this case, the combined prior art as recited supra in the office action renders obvious the claim 19.
Therefore, the rejection of the claim 19 is maintained.
Applicant’s argument 8:
Claims 20-21 stand rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. in view of Nicholas et al. and Penter et al., and further in view of Superti-Furga et al. (US 2017/0356911).
The Office has reiterated the arguments regarding the transduction efficiency assay of claim 1 and has cited Superti-Furga as allegedly disclosing the process of obtaining a peripheral blood or bone marrow sample from a subject and isolating nucleated cells from the peripheral blood by density gradient centrifugation. However, as discussed above with respect to claim 1, the combination of Charrier, Nicholas and Penter fails to disclose a transduction efficiency assay that includes the utilization of single cell PCR. Superti-Furga fails to cure the deficiencies of Charrier, Nicholas and Penter, as Superti-Furga fails to disclose or suggest a transduction efficiency assay that includes using single cell PCR. Accordingly, reconsideration and withdrawal of the rejection are respectfully requested.
In Response 8: Applicant’s arguments have been considered and are not persuasive because the claims 20-21 stand rejected under 35 U.S.C. § 103 based on the combined teachings of the prior arts and obviousness analysis and motivations as recited supra in the rejection. The prior arts teach single cell isolation and quantification of the VCN as recited supra.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, 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 this case, the combined prior art as recited supra in the office action renders obvious the claims 20-21.
Therefore, the rejection of the claims 20-21 is maintained.
Applicant’s argument 9:
Claims 22 and 38 stand rejected under 35 U.S.C. § 103 as allegedly unpatentable over Charrier et al. in view of Nicholas et al., Penter et al., Superti-Furga and Bonner.
As discussed above, the combination of Charrier, Nicholas, Penter and Superti-Furga does not teach or suggested a transduction efficiency assay that includes assaying assaying isolated cells using single cell PCR, as required by claim 20. Bonner fails to cure the deficiencies of the combination of references. Bonner is merely cited as allegedly teaching lentiviral transduction of a population of hematopoietic stem or progenitor cells. However, Bonner fails to disclose or suggest a transduction efficiency assay that includes assaying isolated cells using single cell PCR.
Accordingly, reconsideration and withdrawal of the rejection are respectfully requested.
In Response 9 Applicant’s arguments have been considered and are not persuasive because the claims 22 and 38 stand rejected under 35 U.S.C. § 103 based on the combined teachings of the prior arts and obviousness analysis and motivations as recited supra in the rejection. The prior arts teach single cell isolation and quantification of the VCN as recited supra. In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, 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 this case, the combined prior art as recited supra in the office action renders obvious the claims 22 and 38.
Therefore, the rejection of the claims 22 and 38 is maintained.
Rejection of New Claims 58-60:
The newly added claims 58-60 (amended and filed claim listing 12/17/2025) are rejected under 35 U.S.C. § 103 based on the combined prior art teachings including new prior arts as recited supra.
Relevant Prior Arts:
Urbinati et al 2018. Gene Therapy for Sickle Cell Disease: A Lentiviral Vector Comparison Study. Human Gene Therapy, vol 29 (10), 1153-1166.
Conclusion
21. No claim is allowed.
22. 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).
23. 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 SAMADHAN J JADHAO whose telephone number is (703)756-1223. The examiner can normally be reached M-F 8:00-5:00.
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/SAMADHAN JAISING JADHAO/Examiner, Art Unit 1672
/BENNETT M CELSA/Primary Examiner, Art Unit 1600