DETAILED ACTION
The finality of the last Office action is withdrawn.
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 .
Claim Status
The after final amendment, filed 4/14/20206, has been entered.
Claim 1 is amended. Claim 7 is cancelled.
Claims 1, 4-6, 9-10, 12, 16-17, 19, 23-25, 35 and 36 are examined on the merits.
Priority
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in parent Application No. PCT/EP2021/061999, filed on 05/06/2021, claiming a priority date 05/06/2020.
Withdrawn Rejections
The rejection of claims 1, 4-6, 9-10, 12, 16-17, 19, 23-25, 35-36, under 35 U.S 103, has been withdrawn in view of Applicant’s amendments and arguments to the claims in the reply in the reply filed 04/14/2026.
The rejection of claim 7 under 35 U.S 103, is moot in view of Applicant’s cancellation of the claim in the reply in the reply filed 04/14/2026.
Claim Rejections - 35 USC § 102
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 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 person shall be entitled to a patent unless –
(a)(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, 4-6, 9, 12, 19, 23-25 and 36 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Webber et al. (“Webber”, US 2022/0282285 A1, with priority date Sep. 23, 2019, Provisional application 62904299 [‘299]), as evidenced by Zhang et al. (“Zhang”, Molecular Therapy, 2019).
Regarding claims 1 and 19, Webber teaches a method comprising cleaving a target site in the genome of a plurality of mammalian immune cells with an endonuclease to produce at least one double stranded break in the genome, and introducing an exogenous transgene flanked by homology arms comprising at most 5-100 base pairs, where the homology arms comprise sequence homologous to the locus with the double stranded break (e.g., paragraphs [0057]-[0069]; '299 at paragraphs [0051]-[0062]). Webber teaches the method where the cells are first electroporated with an mRNA encoding the endonuclease, which is a zinc finger nuclease, TALEN or meganuclease (e.g., paragraphs [0317], [0349]-[0350], [0497] and [0628]; '299 at paragraphs [0309], [0323], [0338]-[0352], [0528] and [0573]). Webber teaches the first electroporation step results in the functional presence (cleavage activity) of the nuclease (e.g., paragraph [0628]; '299 at [0573]). Webber teaches the method where the first electroporation step is followed by a second electroporation step to introduce an exogenous polynucleic acid (e.g., paragraph [0624]; '299 at paragraph [0569]). Webber teaches the exogenous polynucleic acid comprises a transgene, such as a T cell receptor (TCR) or chimeric antigen receptor (CAR), flanked homology arms of 50 to 200 nucleotides (e.g., paragraphs [0472]-[0473], [0485], [0499]-[0502], [0630]; '299 at paragraphs [0425]-[0431], [0436], [0450]-[0453] and [0575]). Webber teaches the interval between the first and second electroporation steps are from about 5 hours to about 28 hours, such as 6, 7, 8, 9, 10, 11, 1,2 1, 14, 15, 16, 17, 18, 19 or 20 hours (e.g., paragraph [0625]; '299 at paragraph [0570]). Webber teaches that cleavage of the genome of the immune cell to be edited creates a double-stranded break that is repaired via a process that results in insertion of the exogenous polynucleic acid at the locus containing the break (e.g., paragraphs [00421]-[0422] and [0472]; '299 at paragraphs [0390]-[0391] and [0425]). Webber teaches the exogenous polynucleic acid in the form of double-stranded DNA (e.g., paragraph [0058]; '299 at paragraph [0052]).
Zhang is cited to show that nuclease delivered as mRNA is expressed as protein at 4-6 hours after transfection (e.g., page 738, Table 2). Thus, the interval of Webber allows for a duration between electroporation steps where the nuclease is functional in the cell for at least 5 hours prior to the introduction of the exogenous polynucleic acid.
Regarding claim 4, Webber teaches the method where the zinc finger nuclease, TALEN or meganuclease is delivered as mRNA in a first electroporation to result in a functional presence (cleavage activity) in the cell, and the exogenous polynucleic acid is delivered 5 to 28 hours, or more specifically 16 hours, 17 hours, 18 hours, 19 hours and 20 hours, after the mRNA encoding the nuclease (e.g., paragraph [0625]; '299 at paragraph [0570]).
Zhang is cited to show that nuclease delivered as mRNA is expressed as protein at 4-6 hours after transfection (e.g., page 738, Table 2). Further, Zhang is cited to show that the nuclease is expressed as protein for about 24-48 hours (e.g., page 738, Table 2). Thus, the interval of Webber allows for a duration between electroporation steps where the nuclease is functional in the cell for at least 15 hours prior to the introduction of the exogenous polynucleic acid.
Regarding claim 5, Webber teaches the method where the exogenous polynucleic acid is introduced at 6, 7, 8, 9 or 10 hours after transfection of the mRNA encoding the nuclease, where the exogenous polynucleic acid encodes a reporter gene, such as GFP, and where the integration efficiency is determined by examining cultured cells for the presence of the reporter by flow cytometry, which can count the cells expressing the fluorescent protein (e.g., paragraphs [0512] and [0625]; '299 at paragraphs [0463] and [0570]).
Regarding claim 6, Webber teaches the method where the exogenous polynucleic acid is introduced at 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours after transfection of the mRNA encoding the nuclease, where the exogenous polynucleic acid encodes a reporter gene, such as GFP, and where the integration efficiency is determined by examining cultured cells for the presence of the reporter by flow cytometry, which can count the cells expressing the fluorescent protein (e.g., paragraphs [0512] and [0625]; '299 at paragraphs [0463] and [0570]).
Regarding claim 9, Webber teaches the method where the nuclease is a TALE-nuclease (e.g., paragraphs [0317], [0349]-[0350], [0497] and [0628]; '299 at paragraphs [0309], [0323], [0338]-[0352], [0528] and [0573]).
Regarding claim 12, Weber teaches the method where integration is by a process of homologous recombination (e.g., paragraph [0472]; '299 at paragraph [0425]).
Regarding claims 23-25, Weber teaches the method where the cell is a primary cells, specifically immune cells that are differentiated progeny of HSC, such as T cells (e.g., paragraph [0065]; '299 at paragraph [0027]).
Regarding claim 36, Weber teaches the introduction of the nuclease as mRNA, which is not a viral vector, and introduction of the exogenous polynucleic acid as a plasmid or minicircle, which is not a viral vector (e.g., paragraph [0058]; '299 at paragraph [0052]).
Claim Rejections - 35 USC § 103
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.
Claims 10 and 35 are rejected under 35 U.S.C. 103 as being unpatentable over Webber et al. (“Webber”, US 2022/0282285 A1, with priority date Sep. 23, 2019) as applied to claims 1, 4-6, 9, 12, 19, 23-25 and 36 above, and further in view of Cabaniols et al. (“Cabaniols”, WO 2018/007263 A1, cited as reference 1 on IDS filed 10/31/2022).
The teaching of Webber et al. are described above and applied as before.
Webber does not teach transfecting Cas9 and sgRNA as a first step, as required by the instant claim. Webber does not teach a method for producing therapeutic cells, as required by the instant claim. However, this is cured by Cabaniols.
Regarding claim 10, Cabaniols teaches a method of sequential gene
editing aiming to improve the genetic modification of primary human cells, especially immune cells originating from individual donors or patients
(e.g., paragraph 1st, page 4; Fig. 1). Cabaniols teaches first and/or second sequence-specific reagent is a conjugate of RNA guide and a Cas9 or Cpf1
polypeptide (e.g., paragraph 8th, page 6).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the TALEN by Cas9/gRNA during the first step of the sequential gene-editing of primary cells, because 1) Webber teaches the method where the cells are first electroporated with an mRNA encoding the endonuclease, TALEN, and 2) Cabaniols teaches the use of Cas9/gRNA during the first electroporation of the sequential gene editing of primary immune cells, therefore, it is within the ordinary skill in the art to substitute TALEN by Cas9/gRNA. A person of ordinary skill in the art would have been motivated to make such a modification in order to receive the expected benefit to improve the genetic modification of primary human cells as taught by Cabaniols.
Regarding claim 35, Cabaniols teaches sequential steps to produce batches of engineered primary immune cells of therapeutic grade (e.g., line 27, page 15). Cabaniols teaches the method that allows producing engineered primary immune cells within a limited time frame of about 15 to 30 days, so that they keep their full immune therapeutic potential, especially with respect to their cytotoxic activity. These populations of cells can be expanded under closed culture recipients to comply with highest manufacturing practices requirements and can be frozen prior to infusion into a patient, thereby providing "off the shelf' or "ready to use" therapeutic compositions (e.g., line 20, page 33).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to incorporate the sequential targeted insertion method of Webber, into the therapeutic cell manufacturing process taught by Cabaniols, because Cabaniols expressly teaches using genetically modified immune cells as therapeutic products and teaches purification and freezing as conventional downstream manufacturing steps for clinical administration. A person of ordinary skill in the art would have been motivated to combine the genome editing method of Webber with the therapeutic cell production workflow of Cabaniols to obtain engineered immune cells suitable for storage and subsequent therapeutic use with reasonable expectation of success.
Claims 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Webber et al. (“Webber”, US 2022/0282285 A1, with priority date Sep. 23, 2019) as applied to claims 1, 4-6, 9, 12, 19, 23-25 and 36 above, and further in view of Fahrenkrug et al. (“Fahrenkrug”, US 2019/0323031 A1, cited as reference 2 on IDS filed 10/31/2022).
The teachings of Webber et al. are described above and applied as before.
Webber teaches that a transgene can be flanked by homology arms
where the degree of homology between the arm and its complementary sequence is sufficient to allow homologous recombination between the two. Two homologous non-identical sequences can be any length and their degree of nonhomology can be as small as a single nucleotide (e.g., for
correction of a genomic point mutation by targeted homologous
recombination) (e.g., paragraph [0549]). Webber teaches that sequence
in the genome can be replaced by another sequence, for example, to replace a disease-associated sequence (e.g., SNP or mutation) with a normal sequence, or to alter the function of a gene product ( e.g., binding affinity for an antigen, ligand, agonist, antagonist etc.) (e.g., paragraph [0631]). Webber teaches homology arms of the disclosure can be single
stranded DNA (ssDNA) (e.g., paragraph [0473]).
Webber does not teach that the DNA template is a single stranded polynucleotide. Webber does not teach that the DNA template is a short single-stranded oligodeoxynucleotide. However, this is cured by Fahrenkrug.
Fahrenkrug teaches that the method comprising contacting the animal cell with a homology-dependent repair (HDR) template such that the HDR template is incorporated into genomic DNA of the animal cell, thereby altering the genome of the animal cell. the HDR template can be single-stranded DNA (e.g., paragraph 0014). Fahrenkrug teaches the use of single stranded oligonucleotides (ssOligos) as a template for homologous recombination at the bovine GDFS locus. TALENs (btGDF83.1, arrow) and two ssODNs were designed to introduce an 11 bp deletion into exon-3 of the bovine GDFS gene (Belgium Blue mutation) by homologous recombination. TALENs and subsequent transfection of ssODNs using Lipofectamine L TX 24 hours later (e.g., paragraph [0086]).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to employ a single-stranded DNA or a short single-stranded oligodeoxynucleotide (ssODN) as the DNA template as taught by Fahrenkrug in the sequential transfection of an endonuclease and DNA template to replace a disease-associated sequence (e.g., SNP or mutation) taught by Webber, because as demonstrated by Fahrenkrug single stranded DNA or ssODN are well-known formats for DNA templates used in targeted genome editing via homology-direct repair with reasonable expectation of success achieving targeted insertion at the endonuclease cleavage site.
One of ordinary skill in the art before the effective filing date of the
invention would have been motivated to do so in order to replace a disease-associated sequence (e.g., SNP or mutation) in primary immune cells using site-specific endonucleases and short single-stranded oligodeoxynucleotides.
Conclusion
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/JULIO WASHINGTON GOMEZ RODRIGUEZ/Examiner, Art Unit 1637
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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