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 .
DETAILED ACTION
Status of the Claims
Claims 1-2, 5-6, 10, 13, 15-16, 18, 28, 36, 41-42, 45-46, 49, 53, 61 and 67-68 are pending. Claims 1-2, 5-6, 13, 15-16, 18, 28, 36, 41-42, 45-46, 49, 53, 61 and 67-68 are the subject of this NON-FINAL Office Action. This is the first action on the merits.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-2, 5-6, 13, 15-16, 18, 28, 36, 41-42, 45-46, 49, 53, 61 and 67-68) in the reply filed on 06/22/2026 is acknowledged. Claim 10 is withdrawn.
Note on Claims
Applicants claims read on IEX chromatography performed routinely in the art of gene editing to determine HbA and HbF. See e.g. US 20200155606 (“The functional effects of alterations caused or facilitated by the genome editing systems and methods of the present disclosure can be assessed in any number of suitable ways. . . . Fetal hemoglobin protein may be assessed by high pressure liquid chromatography (HPLC), for example, according to the methods discussed on pp. 143-44 of Chang 2017, incorporated by reference herein, or fast protein liquid chromatography (FPLC) using ion-exchange and/or reverse phase columns to resolve HbF, HbB and HbA and/or γA and γG globin chains as is known in the art.”)
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. § 102 that form the basis for the rejections under this section made in this Office action:
(A) A person shall be entitled to a patent unless –
(1)the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention; or
(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-2, 5-6, 13, 15-16, 18, 28, 36, 42, 45-46, 49, 53 and 67-68 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by WO2019209914.
As to claim 1, WO2019209914 teaches methods and compositions for modifying hemoglobin loci, such as hemoglobin-related mutations including sickle cell mutations, and relates to a nucleic acid for homology directed repair (HOR) of an HBB gene which codes for the hemoglobin subunit beta. Reference is made to a vector for promoting HOR of HBB protein expression in a cell, wherein the vector includes one or more of: a first sequence encoding a HBB gene; a second sequence encoding one or more guide RNA cleavage sites; and a third sequence encoding one or more nuclease binding sites. The nuclease can be TALEN nuclease. The cells can be K562 cells. The 13-globin can be 13Ts7o_ The polypeptides and polynucleotides can be deliverd into cultured cells in vitro using for example, electroporation or transfection, and may include the use of polybrene. Further the document teaches erythroid cell lysis and the detection of globin subtypes using RP-HPLC on UFLC chromatograph, and using ion exchange chromatography (IEC) and detecting peaks at 418nm for HbA and HbF, the cells being treated with viral vector rAAV6 alone and RNP plus GTC (E6V) rAAV6 , see 01 para [0003], [0006], [0017]-[0026], [0052], [0132]-[0138], [0151], [0154], [0155], [0162]-[0173], [0194], [0221 ]-[0023]; figure 11J, K.
As to claim 2, WO2019209914 teaches vector encoding alpha-globin gene into the population of cells (e.g. para. 0011).
As to claims 5-6, WO2019209914 teaches an endonuclease or polynucleotide encoding an endonuclease, and a donor repair template encoding alpha-globin (e.g. para. 0117).
As to claims 13 and 15-16, WO2019209914 teaches K562 cells (e.g. para. 0156).
As to claim 18, WO2019209914 teaches cell density between 5×105 to 1×106 cells/ml (e.g. para. 0214).
As to claim 28, WO2019209914 teaches flasks, or 12 or 24-well plates (para. 0214).
As to claim 36, WO2019209914 teaches cells cultured 48-96 hours after modification/transduction (2 days; paras. 0052, 0065, 0104, 0194-0200).
As to claim 42, WO2019209914 teaches HbA comprises alpha and beta globin chain dimers or tetramers (paras. 0053, 0095-98, 0224, 0230).
As to claim 45, WO2019209914 teaches βA-T87Q globin, βA-G16D/E22A/T87Q_ globin, or a βA-T87Q/K95E/K120E_ globin (Table 2).
As to claim 45, WO2019209914 teaches vector is a lentiviral vector (paras. 0114, 0148).
As to claim 49, WO2019209914 teaches transducing comprises transduction of vector at a multiplicity of infection (MOI) of about 5 to about 40, about 5 to about 30, about 10 to about 40, or about 10 to about 30 (para. 0214).
As to claim 53, WO2019209914 teaches transducing comprises transduction with vector at one or more MOIs in different wells or plates (id.).
As to claim 67, WO2019209914 teaches chromatography separates HbF multimers from HbA multimers (paras. 0053, 0096-98). In addition, this is a function/result of the IEX chromatography recited in claim 1, which fails to distinguish the IEX chromatography of claim 1 because the claim fails to recite any specific structure or composition.
As to claim 68, WO2019209914 teaches chromatographic identification of HbA and HbF is made based on the matched retention time of the analyte peaks relative to a hemoglobin standard (paras. 0053, 0096-98).
Claims 1-2, 5-6, 13, 15-16, 28, 36, 41-42, 45-46, 49, 53 and 67-68 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by WO2016044416.
As to claim 1, WO2016044416 teaches targeted modification of the genome of a hematopoietic stem cell and discloses methods and compositions for altering the expression or for correcting one or more genes encoding proteins involved in a genetic disease. Especially the document refers to a method of altering human beta-hemoglobin (Hbb) expression in a cell by introducing into the cell by transduction using for example lentivirus one or more polynucleotides leading to the alteration of the Hbb gene expression. The document refers to the optimization of the ZFN (zinc finger nuclease) and IDLV (integrase defective lentivirus) doses used for transfection. The amount of Hba was analyzed in supernatants of lysed cells, the supernatant frozen after lysis, thawed and diluted for analysis using HPLC with cation exchange column (polyCAT). The relative percentage of HbA produced was calculated based on the sum of total areas under to curve for each of the hemoglobin peaks including HbF, HbA and HbS, see WO2016044416 the whole document, especially para [0015], [0018]-[0023], [0029], [0043], [0045], [0198], [0212]; figures 2 and 16C. WO2016044416 does not explicitly mention the used wavelength for detecting the heme groups, but as WO2016044416 equally applies an ion exchange chromatography column, it is necessary that the detection is at around 418nm.
As to claim 2, WO2016044416 teaches vector encoding alpha-globin gene into the population of cells (e.g. para. 0029).
As to claims 5-6, WO2016044416 teaches an endonuclease or polynucleotide encoding an endonuclease, and a donor repair template encoding alpha-globin (e.g. para. 0029).
As to claims 13 and 15-16, WO2016044416 teaches K562 cells (e.g. para. 0034).
As to claim 28, WO2016044416 teaches flasks, or 12 or 24-well plates (e.g. para. 0194).
As to claim 36, WO2016044416 teaches cells cultured 48-96 hours after modification/transduction (e.g. paras. 0028-29).
As to claim 41, WO2016044416 teaches cells are frozen after lysis and prior to analyzing the cell lysates with ion exchange (IEX) chromatography (paras. 0197-98, 0201).
As to claim 42, WO2016044416 teaches HbA comprises alpha and beta globin chain dimers or tetramers (e.g. para. 0211).
As to claim 45, WO2016044416 teaches βA-T87Q globin, βA-G16D/E22A/T87Q_ globin, or a βA-T87Q/K95E/K120E_ globin (paras. 0012, 0017-18, 0028, 0031).
As to claim 45, WO2016044416 teaches vector is a lentiviral vector (e.g. para. 0022).
As to claim 49, WO2016044416 teaches transducing comprises transduction of vector at a multiplicity of infection (MOI) of about 5 to about 40, about 5 to about 30, about 10 to about 40, or about 10 to about 30 (e.g. para. 0191, 0201).
As to claim 53, WO2016044416 teaches transducing comprises transduction with vector at one or more MOIs in different wells or plates (id.).
As to claim 67, WO2016044416 teaches chromatography separates HbF multimers from HbA multimers (paras. 0010, 0043, 0197-98, 0212).
As to claim 68, WO2016044416 teaches chromatographic identification of HbA and HbF is made based on the matched retention time of the analyte peaks relative to a hemoglobin standard (id.).
Claims 1-2, 5-6, 13, 15-16, 28, 36, 41-42, 45-46, 49, 53 and 67-68 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by US20200109399.
US20200109399 teaches
Transduction of CD34+ Cells
Human (h) CD34+ cells were isolated from normal donors or from subjects have sickle cell disease and prestimulated at 1×106 cells/mL for 48 h in CellGro® Serum-free Media (CellGenix) supplemented with hSCF, hTPO, and hFlt-3L in a standard humidified tissue culture incubator (5% CO2). Then cells were enumerated, distributed into 21 wells (3 replicates per condition) and transduced at 4×106 cells/mL for 24 h according to the experimental design summarized in Table 2.
TABLE 2
Experimental Design Summary
Transduction Conditions Wells #
Normal hCD34+ cells, MOCK transduction 1, 2, 3
Normal hCD34+ cells transduced with BB694 (MOI 25) + 4, 5, 6
protamine sulfate
Normal hCD34+ cells transduced with BB694 (MOI 25) + 7, 8, 9
F108 + PGE2
Normal hCD34+ cells transduced with BB694 (MOI 50) + 10, 11, 12
protamine sulfate
Normal hCD34+ cells transduced with BB694 (MOI 50) + 13, 14, 15
F108 + PGE2
Normal hCD34+ cells transduced with D12G5 (MOI 25) + 16, 17, 18
protamine sulfate
Normal hCD34+ cells transduced with D12G5 (MOI 25) + 19, 20, 21
F108 + PGE2
SCD hCD34+ cells, MOCK transduction 22, 23, 24
SCD hCD34+ cells transduced with BB694 (MOI 25) + 25, 26, 27
protamine sulfate
SCD hCD34+ cells transduced with BB694 (MOI 25) + 28, 29, 30
F108 + PGE2
SCD hCD34+ cells transduced with BB694 (MOI 50) + 31, 32, 33
protamine sulfate
SCD hCD34+ cells transduced with BB694 (MOI 50) + 34, 35, 36
F108 + PGE2
SCD hCD34+ cells transduced with D12G5 (MOI 25) + 37, 38, 39
protamine sulfate
SCD hCD34+ cells transduced with D12G5 (MOI 25) + 40, 41, 42
F108 + PGE2
Protamine sulfate was used at 8 μg/mL, F108 at 200 μg/mL, PGE2 at10 μM
After the transduction, cells were washed with phosphate buffered saline (PBS). 500 cells per condition were used for clonogenic culture (MethoCult, H4434, StemCell Technologies) and the remaining cells were divided equally between liquid culture in SCGM for day 6 (D6) VCN assessment and erythroid differentiation in liquid culture for hemoglobin analysis.
Liquid Culture in SCGM for D6 VCN Assessment
Transduced hCD34+ cells were cultured in SCGM for VCN assessment in CellGro® Serum-free Media (CellGenix) supplemented with hSCF, hTPO, hFlt-3L, and IL-3 for 6 days in a standard humidified tissue culture incubator (5% CO2). The cells were harvested, genomic DNA extraction was extracted, and the average vector copy number per diploid genome was determined by qPCR. The D6 VCNs for the transduction conditions in Table 2 are shown in FIG. 2.
Clonogenic Assay
500 cells from each transduction condition were washed and transferred to 3 mL aliquots of cytokine-supplemented methylcellulose (for example, Methocult M4434 Classic). 1.1 mL was then transferred to parallel 35-mm tissue culture dishes using a blunt 16-gauge needle. Dishes were maintained in a standard humidified tissue culture incubator for 14-16 days at 37° C. and 5% CO2 and colonies were scored for size, morphology, and cellular composition. The transduction conditions did not lead to unexpected differences in clonogenic frequency or increase toxicity. FIGS. 3A-3B.
Individual colonies were pooled and subjected to VCN analysis. FIG. 4.
Erythroid Differentiation in Liquid Culture
About half of the transduced cells were cultured in erythroid differentiation media in a standard humidified tissue culture incubator for 14-16 days at 37° C. and 5% CO2. The erythroid differentiation media (HF media) comprises IMDM supplemented with Pen/Strep, hSCF, hIL-3, erythropoietin (R&D #287-TC), and 20% heat-inactivated FBS (Lot 1658396). After 14 days, cells were centrifuged (˜300 g 10 min), washed in PBS and lysed in HPLC grade water. After high speed centrifugation (20 000 g 30 min 4° C.), hemoglobin content in the supernatant was analyzed by ion-exchange high-performance liquid chromatography (HPLC).
Hemoglobin Analysis by HPLC
Hemoglobins were analyzed with a Prominence chromatograph (Shimadzu): DGU-20A 3R degassing unit, two LC-20AD mobile phase delivery units (pumps), in series with a CBM-20A system controller, a SIL-20AC HT autosampler, a CTO-20AC column oven and a SPO-20A dual wavelength UV-vis detector. Automated sample injections were performed with SIL-20AC HT autosampler.
One to thirty microliters of the supernatant was injected onto a 100×2.1 mm, 5 μm diameter particle size with 1000-angstrom pores, PolyCAT A column (PolyLC, Columbia, Md.). Hemoglobins were eluted with a gradient of two Tris buffers (buffer A: Tris 40 mM, KCN 3 mM, and adjusted at pH 6.5 with acetic acid; buffer B: Tris 40 mM, KCN 3 mM, NaCl 200 mM, adjusted at pH 6.5 with acetic acid) of different ionic strength at a flow rate of 0.3 ml/minute. The gradients used were 0-2 minutes, 2% B; 2-6 minutes, 20% B; 8-12 minutes, 60% B; 12-12:30 minutes 100% B; and 13 minutes, 2% B. The column oven was set at 30° C. The detection wavelength was 418 nm. Data acquisition and date analysis were performed with the software LC Solution from Shimadzu. Hemoglobins were identified thanks to their retention time and a reference standard run in the same batch. The proportion of the different hemoglobins was assessed with the peak area of each peak at 418 nm.
FIG. 5 shows the relative fetal hemoglobin, normal hemoglobin, and sickled hemoglobin levels produced by erythroid cells derived from the healthy (left panel) and SCD (right panel) CD34+ donor cells transduced under the conditions in Table 2.
[ . . . ]
Conclusion
The bb694 lentiviral vector was superior to the D12G5 vector under all conditions tested. bb694 lentiviral vector was produced at high titer (>1.108 TU/mL), it was able to transduce around 40% of erythroid progenitors at MOI 25 and more than 80% of erythroid progenitors at MOI of 25 in the presence of F108 and PGE2. Under the latter conditions, the percentage of HbF was higher than 70%
(paras. 0312-20 & 0324).
Claim Rejection - 35 USC § 103
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.
Claim 61 is rejected under 35 U.S.C. § 103 as being unpatentable over WO2016044416 or WO2019209914, in view of US 20200393472.
It would have been prima facie obvious to a person of ordinary skill in the hemoglobin cation IEX chromatography art before effective filing to apply familiar cation exchange sulfonic ligands to the columns in order to achieve familiar cation exchange results with a reasonable expectation of success.
Neither WO2016044416 nor WO2019209914 explicitly teach sulfonic cation exchange ligands on IEX columns.
However, as explained above, both WO2016044416 or WO2019209914 teach to use cation exchange IEX columns; and sulfonic cation exchange groups on IEX columns were routine in the art. For example, US 20200393472 teaches
The hemoglobin analysis method of the present invention comprises separating hemoglobins in a sample by ion exchange chromatography. As a sample to be used in the method of the present invention, a blood sample containing hemoglobins used for normal hemoglobin analysis can be used. For example, a blood sample obtained by hemolyzing or diluting blood collected from a human can be used.
The chromatography performed in the method of the present invention is preferably liquid chromatography, more preferably high performance liquid chromatography (HPLC). The ion exchange chromatography in the present invention can be performed using a known liquid chromatography system, that is, an ion exchange column connected to a system including a pump for eluent feeding, a sampler, a detector, and the like.
Preferably, the ion exchange chromatography performed in the method of the present invention is cation exchange chromatography using a cation exchange column. The cation exchange column used in the method of the present invention may be a column packed with a stationary phase having a cation exchange group. Examples of the cation exchange group include a carboxyl group, a phosphoric acid group, and a sulfonic acid group, among which a sulfonic acid group is preferable.
(paras. 0021-23; emphasis added). Thus, sulfonic cation exchange groups in IEX for hemoglobin analysis was preferred.
In sum, a skilled artisan would have been motivated to apply familiar sulfonic cation exchange groups in IEX as it is “preferred.”
Claims 49 and 53 are rejected under 35 U.S.C. § 103 as being unpatentable over WO2016044416 or WO2019209914, in view of US20200109399.
It would have been prima facie obvious to a person of ordinary skill in the hemoglobinopathies art to use MOIs of 5-40 in various wells to achieve better results with a reasonable expectation of success.
For example, as explained above, US20200109399 teaches that
The bb694 lentiviral vector was superior to the D12G5 vector under all conditions tested. bb694 lentiviral vector was produced at high titer (>1.108 TU/mL), it was able to transduce around 40% of erythroid progenitors at MOI 25 and more than 80% of erythroid progenitors at MOI of 25 in the presence of F108 and PGE2. Under the latter conditions, the percentage of HbF was higher than 70%
(para. 0324). Thus, a skilled artisan would have been motivated to optimize MOI for other beta-globin vectors along similar amounts to achieve similar results.
Potential Double Patenting
US Application 19/215,081 has the same inventors, and contains claims to assessing HbA formation after lysis of cells using IEX (claim 26). However, the claims do not explicitly recite a lysis step. It is also noted that the specification discloses MOI 5-40.
Prior Art
The following prior art, among many, also teaches IEX chromatography to assess HbA heme groups in beta-globin infected cells: Ouyang Wenjie ET AL: "Restoration of [beta]-globin expression with optimally designed lentiviral vector for [beta]-thalassemia treatment in Chinese patients", bioRxiv, 21 July 2020 (2020-07-21), XP093264565, DOI: 10.1101/2020.07.18.209759; WO 2020/198691; US 20200155606 (“The functional effects of alterations caused or facilitated by the genome editing systems and methods of the present disclosure can be assessed in any number of suitable ways. . . . Fetal hemoglobin protein may be assessed by high pressure liquid chromatography (HPLC), for example, according to the methods discussed on pp. 143-44 of Chang 2017, incorporated by reference herein, or fast protein liquid chromatography (FPLC) using ion-exchange and/or reverse phase columns to resolve HbF, HbB and HbA and/or γA and γG globin chains as is known in the art.”); US 20190184035; US 20190284542; US 20210309995; US 20190358347; US 20240360414.
The following prior art demonstrate the routine optimization of conditions for lentiviral infection: Broad Institute: "Protocol: Optimization of lentiviral transduction using spinfection", 20 October 2018 (2018-10-20), XP093264622.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MELODY TSUI whose telephone number is (571)272-1846. The examiner can normally be reached Monday - Friday, 9am - 5pm.
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/YUNG-SHENG M TSUI/ Primary Examiner, Art Unit 1684
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