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
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 9-26-25 has been entered.
Claims 3-5, 14 have been canceled. Claims 1, 2, 6-13, 15-17 are pending.
Applicant's arguments filed 9-26-25 have been fully considered but they are not persuasive.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Specification
“G(N)19NGG” or (N20)NGG on pg 49, para 176; pg 102, para 328; claim 11, 12… include fewer than 4 specifically defined nucleotides and do not require SEQ ID NOs.
The amended copy of the specification filed 7-6-22 includes Table S1 on pg 102. The information in Table S1 is on pg 56 of provisional application 61/808594.
Priority
Provisional application 61/808594 did not teach the structure of the guide RNA or sequence within the B2M gene required to make an indel in a B2M gene of any somatic cell as required in claim 1. Therefore, the claims have priority to 4-4-14, the filing date of parent application PCT/US2014/033082.
Table S1 on pg 102 was added in the amendment filed 7-6-22. The information in Table S1 is on pg 56 of provisional application 61/808594 filed 4-4-13.
Claim Rejections - 35 USC § 112
Enablement
Claims 1, 2, 6-13, 15-17 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for contacting a population of isolated mammalian cells with a nucleic acid sequence encoding Cas9 and a guide RNA (gRNA) that targets an endogenous β2-microglobin (B2M) gene in the cell such that the gene is genetically modified, wherein efficiency of obtaining a genetic modification in the gene is in at least 4.5%---, does not reasonably provide enablement for those of skill to inactivate a B2M gene in at least about 9% of primary mammalian CD4+ T cells or primary mammalian hematopoietic stem cells as required in claim 1. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make/use the invention commensurate in scope with these claims.
Withdrawn rejection
The rejection regarding using isolated mammalian CD4+ T-cells or HSCs with an inactivated B2M gene provided in the method of claim 1 has been withdrawn because the cells can be used for in vitro research of the role of B2M in T-cell activation.
Pending rejections
A) The specification does not enable inactivating a B2M gene in at least about 9% of isolated primary mammalian CD4+T cells or hematopoietic stem cells using Cas9 and a pair of gRNAs that target offset sequences of the B2M gene as required in claim 1.
The claim encompasses inactivating the B2M gene in isolated primary mammalian CD4+ T cells or isolated primary mammalian hematopoietic stem cells and requires identifying a pair of gRNAs “that target offset sequences” of a B2M gene which are compatible with CRISPR technology (pg 2, ¶4).
Fig. 1 describes over 100 guide RNA sequences for targeting the human CCR5 gene (pg 36, para 129).
Fig. 2 describes over 100 guide RNA sequences for targeting the human CXCR4 gene (pg 36, para 130).
Fig. 4C shows a schematic of B2M CRISPR sites
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However, the target sequences of these “offset” gRNA pairs used for Fig. 6B, 8A and 8C required for achieving “about 9%” inactivation of a B2M gene cannot be found in the specification. Support has not been provided and none can be found. Specifically, the structure of guide combination “A+B”, “D+Q”, and “C+D” in Figure 6B or “L1+L2” in Fig. 8A or 8C cannot be found. The schematic in Fig. 4C does not teach the specific target sequences of “A+B”, “D+Q”, and “C+D” in Figure 6 or “L1+L2” in Fig. 8.
Moreover, the schematic in Fig. 4C does not teach the specific target sequences of “A+B”, “D+Q”, and “C+D” in Figure 6 or “L1+L2” in Fig. 8 are offset by 2.2 kb as required in claim 16.
Fig 4D shows the results of targeting the B2M locus with single guide RNAs in 293T cells. Fig. 4E shows the results of flow cytometry analysis using a single guide strategy targeting B2M in 293T cell, which demonstrates that B2M CRISPRs ablate B2M surface expression with high efficiency” (pg 37, lines 4-9).
Figure 5A-5C are limited to a double gRNA strategy in the CCR5 gene (pg 106, para 345; pg 37, para 134):
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Fig. 5D “is a schematic showing double B2M CRISPR combinations” (pg 37, last line):
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However, Fig. 5D and the rest of the specification do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that would inactivate the B2M gene are not disclosed in Fig. 5D or elsewhere in the specification.
The description of Fig. 6B (pg 38, para 135 - knockout efficiency in primary CD34+ hematopoietic stem cells (HSCs) with double gRNA strategy targeting a B2M gene) appears to say that the columns labeled “Null (%)” show at least 5 conditions that represent “about 9% efficiency” in inactivating the B2M gene in HSCs.
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However, Fig. 6B and the rest of the specification do not teach the specific structures of “A+B”, “D+Q” or “C+D” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “A+B”, “D+Q” or “C+D” gRNAs cannot be determined from the generic graphic of the approximate location of gRNAs within the B2M gene in Fig. 4C or the “L1”, “L2”, or “8” gRNAs within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that would inactivate the B2M gene are not disclosed in Fig. 4C, 5D or elsewhere in the specification.
The description of Fig. 8 (pg 38, para 137 - knockout efficiency in CD4+ T-cells with double gRNA strategy targeting a B2M gene), specifically Fig. 8A (L1+L2) and 8C (L1+8) is noted. The figures filed at the Patent Office are unreadable, but clearer drawings were provided in the response filed 9-26-25:
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However, Fig. 8A, 8C and the rest of the specification do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that would inactivate the B2M gene are not disclosed in Fig. 5D or elsewhere in the specification.
Pg 99, para 324, teaches the human codon-optimized Cas9 gene used in Example 1 was the one described by Mali (pg 99, line 5).
Pg 93, para 324, teaches the gRNA was introduced separately using a "20 nucleotide protospacer for each gRNA" (pg 93, lines 8-9).
Example 1 (pg 98) suggests inactivating a human gene in a human cell by transfecting an isolated cell with a plasmid encoding a human codon-optimized Cas9 gene subcloned with a C-terminal nuclear localization signal as described by Mali and a reporter protein operably linked to a CAG promoter and 2A peptides and a plasmid encoding guide RNA operably linked to the human U6 polymerase III promoter, does not reasonably provide enablement for the actual structure of the Cas9 gene, or B2M target sequence, or the gRNAs required to inactivate the B2M gene using a Cas protein as claimed. Pg 99, para 325, (AKT2, cCELSR2, CIITA, GLUT4, LINC00116, SORT1, LDLR) and 326 (AKT2) in particular does not contemplate designing gRNA for targeting a B2M gene.
Pg 99, para 325, states gRNAs were designed matching G(N)19NGG in 7 genes and compared to TALENs targeting the same genes. Table S1 is mentioned on pg 99 in para 325 and was added to pg 101 of the specification filed 7-6-22. Table S1 is in 61/808594 (4-4-13) but not in parent application PCT/US2014/033082 (4-4-14). Table S1 describes efficiencies of "mutants" using TALEN vs CRISPRs. Furthermore, para 325 fails to describe modifying the B2M gene as claimed, teach using pairs of gRNAs as claimed, or that a deletion occurred as claimed.
Example 2 (pg 103) describes targeting “clinically relevant genes” in primary somatic cells including B2M using a pair of gRNA (para 336). The codon-optimized Cas9 of Cong, Jinek, Mali, Ding, Wang, Li, and Niu was used (Pg 103, para 333). Example 2 and Fig. 4C do not teach the specific structure of the target sequence within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. Example 2 does not teach any deletion occurred in the B2M gene as claimed.
Pg 105, para 339, discusses transfection of HEK293T cells with Cas9 and gRNA that target a B2M gene but do not teach the target, the structure of any gRNA pairs, that the target sequences for gRNAs were “offset”, that the final mutation was a deletion, or that any “deletion” that occurred had any effect of B2M expression. Pg 104, para 339, states B2M expression was “abrogated in up to 60% of transfected HEK293T cells (Fig. 4)”; however, Fig. 4C-4E are the only part of Fig. 4 that relates to B2M, but they are limited to single gRNA. Fig. 4C does not teach the specific structure of the offset B2M target sequences (see above). Fig. 4D shows the results of targeting the B2M locus but is limited to single guide RNAs in 293T cells (pg 37, para 133).
Fig. 5, 6, and 8 mentioned in the Examples are discussed above.
Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to inactivate a B2M gene in at least about 9% of mammalian CD4+ T cells or HSCs using Cas9 and two different gRNAs as required in claim 1 because the specification does not teach the specific structure of the two offset target sequences for any B2M gene in Fig. 4C or elsewhere in the specification required to obtain the results in Fig. 6B, 8A, or 8C.
Response to arguments
Applicants argue Fig. 8A and 8C show gRNA pairs “L1+L2” and “L1+8” show at least 9% inactivation of a B2M gene and Fig. 5D shows a schematic of the B2M gene and the generic positions of “L1”, “L2” and “8”. Fig. 8A and 8C taken with the schematic in Fig. 5D are not persuasive the specification does not teach the specific structures of “L1”, “L2” or “8” within the B2M gene in Fig. 5D or elsewhere in the specification. The specification does not teach the specific structure of any target sequences for gRNA pairs that are “offset” as claimed.
Applicants argue Fig. 6A and 6B demonstrate inactivating a B2M gene in HSCs, Fig. 8A & 8C demonstrate inactivating a B2M gene in CD4+ T-cells, and Fig. 5D shows a schematic of the B2M gene including the location of gRNAs “L1”, “L2”, and “8”. Applicants’ argument is not persuasive. Fig. 6A, 6B, 8A, 8C and the rest of the specification taken with Fig. 5D do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that would inactivate the B2M gene are not disclosed in Fig. 4C, 5D, or elsewhere in the specification.
B) The specification does not enable any “offset” target sequences for gRNA pairs “to which the first gRNA and second gRNA bind are offset by approximately 2.2 kB” as required in claim 16. Pg 47, praa 170, in the middle, says the B2M gene has four exons that span about 8 kb. Fig. 5D says gRNAs “L1” and “8” are 2.2 kb apart; however, Fig. 5D and the specification in general do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that are at least 2.2 kb apart capable of inactivating the B2M gene are not disclosed in Fig. 4C, 5D, or elsewhere in the specification.
Even assuming “L1” and “8” are 2.2 kb apart from each other (as suggested at the left side of Fig. 5D), there is no description of them being “offset” by 2.2 kb or being “approximately” 2.2 kb apart.
Even assuming “L1” and “8” are 2.2 kb apart from each other (as suggested at the left side of Fig. 5D), applicants do not describe any other gRNA target sequences that are 2.2 kb apart.
Given the complete lack of guidance or suggestion in the specification for any pair of “offset” gRNA target sequences within a B2M gene that are at least 2.2 kb apart, taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to arrive at any pair of gRNAs “offset” by 2.2 kb capable of causing at least 9% of the cells to have a deletion.
Response to arguments
Applicants argue Fig. 5D supports the concept because “L1” and “8” are 2.2 kb apart. Applicants’ argument is not persuasive. Fig. 5D says gRNAs “L1” and “8” are 2.2 kb apart; however, Fig. 5D and the specification in general do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that are at least 2.2 kb apart capable of inactivating the B2M gene are not disclosed in Fig. 4C, 5D, or elsewhere in the specification. The specification does not teach any gRNA pairs that are greater than 2.2 kb apart as broadly encompassed by claim 16. Therefore, the concept is not enabled.
Written Description
Claims 1, 2, 6-13, 15-17 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
Withdrawn rejection
The rejection regarding using isolated mammalian CD4+ T-cells or HSCs with an inactivated B2M gene provided in the method of claim 1 has been withdrawn because the cells can be used for in vitro research of the role of B2M in T-cell activation.
Pending rejections
A) The specification lacks written description for inactivating a B2M gene in at least about 9% of isolated primary mammalian CD4+T cells or hematopoietic stem cells using Cas9 and a pair of gRNAs that target offset sequences of the B2M gene as required in claim 1.
The claim encompasses inactivating the B2M gene in isolated primary mammalian CD4+ T cells or isolated primary mammalian hematopoietic stem cells and requires identifying a pair of gRNAs “that target offset sequences” of a B2M gene which are compatible with CRISPR technology (pg 2, ¶4).
Fig. 1 describes over 100 guide RNA sequences for targeting the human CCR5 gene (pg 36, para 129).
Fig. 2 describes over 100 guide RNA sequences for targeting the human CXCR4 gene (pg 36, para 130).
Fig. 4C shows a schematic of B2M CRISPR sites (See above). However, the target sequences of these “offset” gRNA pairs used for Fig. 6B, 8A and 8C required for achieving “about 9%” inactivation of a B2M gene cannot be found in the specification. Support has not been provided and none can be found. Specifically, the structure of guide combination “A+B”, “D+Q”, and “C+D” in Figure 6B or “L1+L2” in Fig. 8A or 8C cannot be found. The schematic in Fig. 4C does not teach the specific target sequences of “A+B”, “D+Q”, and “C+D” in Figure 6 or “L1+L2” in Fig. 8. Moreover, the schematic in Fig. 4C does not teach the specific target sequences of “A+B”, “D+Q”, and “C+D” in Figure 6 or “L1+L2” in Fig. 8 are offset by 2.2 kb as required in claim 16.
Fig 4D shows the results of targeting the B2M locus with single guide RNAs in 293T cells. Fig. 4E shows the results of flow cytometry analysis using a single guide strategy targeting B2M in 293T cell, which demonstrates that B2M CRISPRs ablate B2M surface expression with high efficiency” (pg 37, lines 4-9).
Figure 5A-5C are limited to a double gRNA strategy in the CCR5 gene (pg 106, para 345; pg 37, para 134) (see above).
Fig. 5D “is a schematic showing double B2M CRISPR combinations” (pg 37, last line) (see above). However, Fig. 5D and the rest of the specification do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that would inactivate the B2M gene are not disclosed in Fig. 5D or elsewhere in the specification.
The description of Fig. 6B (pg 38, para 135 - knockout efficiency in primary CD34+ hematopoietic stem cells (HSCs) with double gRNA strategy targeting a B2M gene) appears to say that the columns labeled “Null (%)” show at least 5 conditions that represent “about 9% efficiency” in inactivating the B2M gene in HSCs (See Fig. 6B above). However, Fig. 6B and the rest of the specification do not teach the specific structures of “A+B”, “D+Q” or “C+D” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “A+B”, “D+Q” or “C+D” gRNAs cannot be determined from the generic graphic of the approximate location of gRNAs within the B2M gene in Fig. 4C or the “L1”, “L2”, or “8” gRNAs within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that would inactivate the B2M gene are not disclosed in Fig. 4C, 5D or elsewhere in the specification.
The description of Fig. 8 (pg 38, para 137 - knockout efficiency in CD4+ T-cells with double gRNA strategy targeting a B2M gene), specifically Fig. 8A (L1+L2) and 8C (L1+8) is noted. The figures filed at the Patent Office are unreadable, but clearer drawings were provided in the response filed 9-26-25 (See above). However, Fig. 8A, 8C and the rest of the specification do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that would inactivate the B2M gene are not disclosed in Fig. 5D or elsewhere in the specification.
Pg 99, para 324, teaches the human codon-optimized Cas9 gene used in Example 1 was the one described by Mali (pg 99, line 5).
Pg 93, para 324, teaches the gRNA was introduced separately using a "20 nucleotide protospacer for each gRNA" (pg 93, lines 8-9).
Example 1 (pg 98) suggests inactivating a human gene in a human cell by transfecting an isolated cell with a plasmid encoding a human codon-optimized Cas9 gene subcloned with a C-terminal nuclear localization signal as described by Mali and a reporter protein operably linked to a CAG promoter and 2A peptides and a plasmid encoding guide RNA operably linked to the human U6 polymerase III promoter, does not reasonably provide enablement for the actual structure of the Cas9 gene, or B2M target sequence, or the gRNAs required to inactivate the B2M gene using a Cas protein as claimed. Pg 99, para 325, (AKT2, cCELSR2, CIITA, GLUT4, LINC00116, SORT1, LDLR) and 326 (AKT2) in particular does not contemplate designing gRNA for targeting a B2M gene.
Pg 99, para 325, states gRNAs were designed matching G(N)19NGG in 7 genes and compared to TALENs targeting the same genes. Table S1 is mentioned on pg 99 in para 325 and was added to pg 101 of the specification filed 7-6-22. Table S1 is in 61/808594 (4-4-13) but not in parent application PCT/US2014/033082 (4-4-14). Table S1 describes efficiencies of "mutants" using TALEN vs CRISPRs. Furthermore, para 325 fails to describe modifying the B2M gene as claimed, teach using pairs of gRNAs as claimed, or that a deletion occurred as claimed.
Example 2 (pg 103) describes targeting “clinically relevant genes” in primary somatic cells including B2M using a pair of gRNA (para 336). The codon-optimized Cas9 of Cong, Jinek, Mali, Ding, Wang, Li, and Niu was used (Pg 103, para 333). Example 2 and Fig. 4C do not teach the specific structure of the target sequence within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. Example 2 does not teach any deletion occurred in the B2M gene as claimed.
Pg 105, para 339, discusses transfection of HEK293T cells with Cas9 and gRNA that target a B2M gene but do not teach the target, the structure of any gRNA pairs, that the target sequences for gRNAs were “offset”, that the final mutation was a deletion, or that any “deletion” that occurred had any effect of B2M expression. Pg 104, para 339, states B2M expression was “abrogated in up to 60% of transfected HEK293T cells (Fig. 4)”; however, Fig. 4C-4E are the only part of Fig. 4 that relates to B2M, but they are limited to single gRNA. Fig. 4C does not teach the specific structure of the offset B2M target sequences (see above). Fig. 4D shows the results of targeting the B2M locus but is limited to single guide RNAs in 293T cells (pg 37, para 133).
Fig. 5, 6, and 8 mentioned in the Examples are discussed above.
Accordingly, the specification lacks written description for determining how to inactivate a B2M gene in at least about 9% of mammalian CD4+ T cells or HSCs using Cas9 and two different gRNAs as required in claim 1 because the specification does not describe two specific offset target sequences for any B2M gene in Fig. 4C or elsewhere in the specification required to obtain the results in Fig. 6B, 8A, or 8C.
Response to arguments
Applicants argue Fig. 8A and 8C show gRNA pairs “L1+L2” and “L1+8” show at least 9% inactivation of a B2M gene and Fig. 5D shows a schematic of the B2M gene and the generic positions of “L1”, “L2” and “8”. Fig. 8A and 8C taken with the schematic in Fig. 5D are not persuasive the specification does not teach the specific structures of “L1”, “L2” or “8” within the B2M gene in Fig. 5D or elsewhere in the specification. The specification does not teach the specific structure of any target sequences for gRNA pairs that are “offset” as claimed.
Applicants argue Fig. 6A and 6B demonstrate inactivating a B2M gene in HSCs, Fig. 8A & 8C demonstrate inactivating a B2M gene in CD4+ T-cells, and Fig. 5D shows a schematic of the B2M gene including the location of gRNAs “L1”, “L2”, and “8”. Applicants’ argument is not persuasive. Fig. 6A, 6B, 8A, 8C and the rest of the specification taken with Fig. 5D do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that would inactivate the B2M gene are not disclosed in Fig. 4C, 5D, or elsewhere in the specification.
B) The specification lacks written description for any “offset” target sequences for gRNA pairs “to which the first gRNA and second gRNA bind are offset by approximately 2.2 kB” as required in claim 16. Pg 47, praa 170, in the middle, says the B2M gene has four exons that span about 8 kb. Fig. 5D says gRNAs “L1” and “8” are 2.2 kb apart; however, Fig. 5D and the specification in general do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that are at least 2.2 kb apart capable of inactivating the B2M gene are not disclosed in Fig. 4C, 5D, or elsewhere in the specification.
Even assuming “L1” and “8” are 2.2 kb apart from each other (as suggested at the left side of Fig. 5D), there is no description of them being “offset” by 2.2 kb or being “approximately” 2.2 kb apart.
Even assuming “L1” and “8” are 2.2 kb apart from each other (as suggested at the left side of Fig. 5D), applicants do not describe any other gRNA target sequences that are 2.2 kb apart.
Accordingly, the specification lacks written description for any pair of “offset” gRNA target sequences within a B2M gene that are at least 2.2 kb apart capable of causing inactivation in at least 9% of the cells.
Response to arguments
Applicants argue Fig. 5D supports the concept because “L1” and “8” are 2.2 kb apart. Applicants’ argument is not persuasive. Fig. 5D says gRNAs “L1” and “8” are 2.2 kb apart; however, Fig. 5D and the specification in general do not teach the specific structures of “L1”, “L2” or “8” within the B2M gene or the specific structure of any target sequences for gRNA pairs that are “offset” as claimed. The specific sequence of “L1”, “L2” or “8” gRNAs cannot be determined from the generic graphic of the approximate location of “L1”, “L2” or “8” within the B2M gene in Fig. 5D. The specific sequence of any other “offset” gRNA pairs that are at least 2.2 kb apart capable of inactivating the B2M gene are not disclosed in Fig. 4C, 5D, or elsewhere in the specification. The specification does not teach any gRNA pairs that are greater than 2.2 kb apart as broadly encompassed by claim 16. Therefore, the concept lacks written description.
Indefiniteness
Claims 1, 2, 6-13, 15-17 remain rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A) The metes and bounds of a “primary” cell in claim 1 cannot be determined. It cannot be determined if the phrase is limited to “fresh” cells taken directly from an organism. If so, how fresh must they be to be considered “primary” cells. It cannot be determined if the phrase encompasses any cell taken from tissue of any organism and maintained for growth in culture medium. The line at which “fresh” cells are no longer “primary” cells is not defined in the specification or the art at the time of filing. The number of passages that define when cells are/are not “primary” is not described by applicants or the art. The time in culture that defines when cells are/are not “primary” is not described by applicants or the art.
It is unclear whether “primary” cells are limited to cells isolated directly from tissue before being placed in culture or if they encompass cells removed from tissue and placed into a culture dish. If the phrase encompasses cells removed from tissue and placed into a culture dish, it is unclear whether the phrase is limited to cells placed into culture for a limited number of seconds, or if it encompasses cells placed into culture for minutes, hours, days, weeks, months, etc. If the phrase encompasses cells removed from tissue and placed into a culture dish for a limited number of minutes, hours, days, weeks, months, etc., it is unclear when the cells are/are not “primary”. It is unclear whether “primary” cells are limited to cells that have undergone no changes in media in culture or if the phrase encompasses any number of changes in media in culture. If the phrase encompasses any number of changes in media in culture, it is unclear whether “primary” cells are limited to cells that have undergone a particular number of changes in media or if the phrase encompasses an unlimited number of changes in media. If the phrase encompasses an unlimited number of changes in media, then it is unclear how the “primary” further limits the term “cells” at all.
Accordingly, it cannot be determined if the term “primary” is limiting in any way, and if so how. The metes and bounds of “primary cells” are not defined in the specification or the art at the time of filing. It is unclear when cells are “primary” and when they are no longer “primary” and become a “cell line”. Therefore, those of skill would not be able to determine when they were infringing on the claim.
Response to arguments
Applicants argue the specification makes clear in paragraphs 133 and 336 that “primary cells” are not cell lines. Applicants’ arguments are not persuasive. Para 133 simply compares cell line 293T, cell line K562, and primary CD34+ HSPCs without defining the metes and bounds of “primary” cells. Par 336 contemplates using CRISPR technology in CD34+ HPCs without defining the metes and bounds of “primary” cells. Even assuming “cell lines” are not “primary” cells, applicants fail to define when cells taken “directly” from living tissue are no longer “primary”.
Applicants argue Haflick (1965) taught primary cells are subject to “replicative senescence”. Applicants’ arguments are not persuasive. Haflick, the specification, and the art at the time of filing fail to teach the amount of “replicative senescence” that defines “primary” cells.
Applicants argue Oh (2007) resemble cells in vivo. Applicants’ argument is not persuasive because Oh, the art at the time of filing, and the specification do not define when cells are within the realm of being primary or the amount of “resemblance” required to be considered “primary”.
Applicants point to Lozzio who taught K562 cells were derived from a pleural effusion, subcultured weekly for 175 passages. Applicants somehow conclude K562 were not isolated “directly” from tissues or organs. Applicants’ argument is not persuasive because the pleural effusion clearly contained pleural tissue. The argument is also not persuasive because Lozzio, the specification and the art at the time of filing do not teach the line at which cells from the pleural effusion were primary vs. the K562 cell line by providing the structures, functions, number of minutes (hours, days, weeks, months), or number of passages that define when “primary” cells became the K562 cell line.
B) The metes and bounds of when target sequences are “offset” in any gene cannot be determined as required in claim 1. It is unclear whether the phrase encompasses any difference in length, if the sequences can have any overlap, or if the sequences must be mutually exclusive. For example, it is unclear whether the 1st target sequence is ATAGCTACATGGA can the 2nd target sequence be ATAGCTACATGGAG. it is unclear whether the 1st target sequence is ATAGCTACATGGA can the 2nd target sequence be TACATGGAGCATACTT. The specification uses the term “offset” frequently without defining it. The art at the time of filing does not define when target sequences are “offset”. Therefore, those of skill would not be able to determine when they were infringing on the claim.
Response to arguments
Applicants argue the term is clear to those of skill in the art and in view of the depiction in Fig. 5D. Applicants’ argument is not persuasive for reasons set forth in the rejection.
C) The phrase “wherein the population of cells are primary hematopoietic stem cells that are isolated from a mammalian subject’s peripheral blood” in claim 15 makes the claim indefinite. It is unclear whether applicants are attempting to limit the cells of claim 1 to “hematopoietic stem cells” (in which case just say ---wherein the isolated mammalian primary CD4+ T-cells or hematopoietic stem cells are hematopoietic stem cells---) or if applicants are simply trying to further limit the source of the HSCs (i.e. from the peripheral blood). If applicants are trying to further limit the source of the HSCs to those obtained from the peripheral blood, then the limitation does not further limit the structure or function of the HSCs and does not make sense. HSCs are specialized cells found in the bone marrow that give rise to all types of blood cells; they are not isolated from peripheral blood as claimed. It is unclear how HSCs isolated from the peripheral blood are distinguished structurally or functionally from those isolated from bone marrow. Essentially, claim 15 has buried a product-by-process limitation (i.e. HSCs isolated from peripheral blood) into a method claim, but the process by which the HSCs are isolated does not make sense and does not distinguish them structurally or functionally from those isolated from bone marrow. This makes claim 15 indefinite.
D) The phrase “the offset sequences to which the two gRNAs bind are offset by approximately 2.2 kb” in claim 16 makes the claim indefinite. Claim 1 requires to “offset sequences of an endogenous B2M gene”. It does not refer to “the offset sequences to which the two gRNAs bind” as in claim 16. Therefore, the phrase lacks antecedent basis.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
E) The phrase “wherein the population of cells are primary CD4+ T-cells that are isolated from a mammalian subject’s peripheral blood” in claim 15 makes the claim indefinite. It is unclear whether applicants are attempting to limit the cells of claim 1 to “CD4+ T-cells” (in which case just say ---wherein the isolated mammalian primary CD4+ T-cells or hematopoietic stem cells are CD4+ T-cells---) or if applicants are simply trying to further limit the source of the CD4+ T-cells (i.e. from the peripheral blood). If applicants are trying to further limit the source of the CD4+ T-cells to those obtained from the peripheral blood, then the limitation does not further limit the structure or function of the CD4+ T-cells. It is unclear how CD4+ T-cells isolated from the peripheral blood are distinguished structurally or functionally from those isolated from lymph nodes or other organs. Essentially, claim 17 has buried a product-by-process limitation (i.e. CD4+ T-cells isolated from peripheral blood) into a method claim, but the process by which the CD4+ T-cells are isolated does not distinguish them structurally or functionally from those isolated from lymph nodes or other organs. This makes claim 17 indefinite. Claim 17 does not require an active step of isolating CD4+ T-cells from peripheral blood.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
Claim Rejections - 35 USC § 103
The rejection of claims 1, 2, 6, 8-13, 15, 16 under 35 U.S.C. 103 as being unpatentable over Cho (Nature Biotech., published online on January 29, 2013, Vol. 31(3), pp. 230-232, plus Supplemental Materials) in view of Arnould (WO 2008/102274) and Mali (Science, Feb. 15, 2013, Vol. 339, No. 6121, pg 823-826) has been withdrawn because the combined teachings of Cho, Arnould, and Mali did not teach the cells were CD4+ T-cells or HSCs as required in claim 1.
Pending rejections
A) Claims 1, 2, 6, 8-13, 15, 16 remain rejected under 35 U.S.C. 103 as being unpatentable over Cho (Nature Biotech., published online on January 29, 2013, Vol. 31(3), pp. 230-232, plus Supplemental Materials) in view of Arnould (WO 2008/102274), Mali (Science, Feb. 15, 2013, Vol. 339, No. 6121, pg 823-826), Carroll (Mol. Ther., Sept. 2012, Vol. 20, No. 9, pg 1658-1660) and Ando (7951925).
Cho contacted a population of isolated human K562 cells with a nucleic acid sequence encoding Cas9 codon-optimized for humans (pg 1 of Supplementary methods, “Construction of Cas9-encoding plasmids”) and a gRNA that targets an endogenous CCR5 gene in the cell such that a deletion in the CCR5 gene occurs, wherein efficiency of obtaining the genetic modification in the CCR5 gene is up to 33% (see 2nd para on pg 231; pg 230, col. 2, 2nd para).
Cho did not target the B2M gene as required in claim 1. However, targeting the B2M gene of human cells was well-known in the art as described by Arnould (“Meganuclease variants cleaving a DNA target sequence from the beta-2-microglobulin gene and uses thereof”). Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to use CRISPR technology to make deletions in an endogenous gene of isolated human cells as described by Cho wherein the gene was B2M described by Arnould. Those of ordinary skill in the art at the time of filing would have been motivated to target the B2M gene to “suppress MHC complexes” in cells (Arnould, 2nd paragraph).
Cho did not teach using a pair of gRNAs that target “offset” sequences of a B2M gene as required in claim 1. However, Mali taught using single or double gRNA guides [T1/T2] that “achieved NHEJ rates of 10 and 25% in 293Ts, 13 and 38% in K562s, and 2 and 4% in PGP1-iPS cells (Fig. 2B). Simultaneous introduction of both T1 T2 gRNAs resulted in high-efficiency deletion of the intervening 19-bp fragment (Fig. S8) (see also pg 824, col. 2). Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to use gRNA and Cas9 to make deletions in an endogenous B2M gene in isolated human cells as described by Cho and Arnould wherein gRNA is a pair of gRNAs that target “offset” sequences of a gene described by Mali. Those of ordinary skill in the art at the time of filing would have been motivated to use a pair of gRNAs that target “offset” sequences of a gene to ensure deletion occurs.
Cho did not teach applying the technology to CD4+ T-cells or hematopoietic stem cells as required in claim 1. However, Carroll described “A CRISPR approach to gene targeting (Title) and stated: “Cells of the hematopoietic lineages are obvious targets, and as more pluripotent cell types are identified or generated, the applications will expand” (pg 1660, col. 2). Ando confirms it was well-known to contact isolated human primary CD34+ hematopoietic stem cells with endonucleases to cleave a target gene (col. 25, Example 4, claim 14, 16, 19). And Arnould targeted the B2M gene of human cells and suggested doing so in pancreatic, renal, or muscular tissues (3rd paragraph), all of which are “primary cells” as required in claim 1. Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to use CRISPR technology to make deletions in an endogenous gene of isolated human cells as described by Cho wherein the cells were primary hematopoietic, pancreatic, renal, or muscular tissues as described by Carroll and Arnould. Those of ordinary skill in the art at the time of filing would have been motivated to use primary hematopoietic, pancreatic, renal, or muscular tissues for ex vivo gene therapy.
Cho used a Cas9 from Streptococus pyogenes as required in claim 2 (abstract).
Cho used a sequence encoding Cas9 that is codon-optimized for expression in human cells which is a “modified nucleic acid” as required in claim 6.
Cho used 20 base targets (“X20”) as required in claim 8 (pg 135, col. 1, last two lines and throughout).
Cho used 20 base targets (“X20”) as required in claim 8 (pg 135, col. 1, last two lines and throughout) immediately followed by NGG (pg 132, col. 2, 2nd para) which is equivalent to claims 9-11.
The formula G(N)19NGG in claim 12 has been included because the phrase is an obvious variant of (N)20NGG described by Cho. Those of ordinary skill in the art at the time of filing would have been motivated to delete a random “N” and make it a reliable “G” to make the target sequence more specific.
Cho used a plasmid as required in claim 13 (pg 138, col. 2).
Cho taught a number of deletions that knocked out gene function (pg 232, Fig. 2A) which is equivalent to a deletion that “knocks-out” as required in claim 15.
The combined teachings of Cho, Arnould, Mali, Carroll, and Ando did not teach the “offset” sequences to which the gRNA pair bind are separated by 2.2 kb as required in claim 17. However, it was well-within the purview of the ordinary artisan to choose any length of separation between targets that would result in a deletion. Those of ordinary skill in the art at the time of filing would have been motivated to choose a separation of 2.2 kb to ensure inactivation of the B2M gene.
Response to arguments
Applicants argue Arnauld teaches away from the invention because it “led a skilled artisan to modify the B2M gene only in pancreatic, renal, and muscular tissues with meganuclease [ ] which are excluded from the claims (pg 14). Applicants’ argument is not persuasive. Arnauld need not teach every limitation as claimed. Applicants’ argument that the absence of teachings in Arnauld somehow equates to “teaching away” is scientifically and legally illogical. Arnauld has been relied upon for targeting the B2M gene of human cells as well as evidence that those of skill would have had a reasonable expectation of doing so in a number of mammalian tissues.
Applicants argue Mali is limited to using two different gRNAs that target AAVS1 “which is excluded from the claims”. Applicants’ argument is not persuasive. Mali need not teach every limitation as claimed. Mali has been relied upon for targeting a desired gene in mammalian cells using two different gRNAs.
B) Claim 7 remains rejected under 35 U.S.C. 103 as being unpatentable over Cho (Nature Biotech., published online on January 29, 2013, Vol. 31(3), pp. 230-232, plus Supplemental Materials) in view of Arnould (WO 2008/102274), Mali (Science, Feb. 15, 2013, Vol. 339, No. 6121, pg 823-826), Carroll (Mol. Ther., Sept. 2012, Vol. 20, No. 9, pg 1658-1660) and Ando (7951925) as applied to claims 1, 2, 6, 8-13, 15, 16 and further in view of Hoerr (WO 2008/052770).
The combined teachings of Cho, Arnould, Mali, Carroll, and Ando taught contacting a population of isolated primary hematopoietic, pancreatic, renal, or muscular cells with a nucleic acid sequence encoding Cas9 and a pair of gRNAs that target “offset” sequences of an endogenous B2M gene such that a deletion in the B2M gene occurs, wherein efficiency is 33%.
The combined teachings of Cho, Arnould, Mali, Carroll, and Ando did not modify the gRNA as required in claim 7.
However, modifying RNA for increased expression using pseudouridine or 5 methylcytosine was well known as described by Hoerr (title; pg 80-83 Examples; pg 83, lines 15-26).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to use Cas9 and a pair of gRNAs that target “offset” sequences of an endogenous B2M gene to make a deletion in the B2M gene of isolated human cells as described by Cho, Arnould, Mali, Carroll, and Ando wherein the pair of gRNAs were modified with pseudouridine or 5 methylcytosine. Those of ordinary skill in the art at the time of filing would have been motivated to do so to improve gRNA expression as described by Hoerr.
Dependent claims have been included for reasons set forth above.
Response to arguments
Applicants argue Arnauld teaches away from the invention because it “led a skilled artisan to modify the B2M gene only in pancreatic, renal, and muscular tissues with meganuclease [ ] which are excluded from the claims (pg 14). Applicants’ argument is not persuasive. Arnauld need not teach every limitation as claimed. Applicants’ argument that the absence of teachings in Arnauld somehow equates to “teaching away” is scientifically and legally illogical. Arnauld has been relied upon for targeting the B2M gene of human cells as well as evidence that those of skill would have had a reasonable expectation of doing so in a number of mammalian tissues.
Applicants argue Mali is limited to using two different gRNAs that target AAVS1 “which is excluded from the claims”. Applicants’ argument is not persuasive. Mali need not teach every limitation as claimed. Mali has been relied upon for targeting a desired gene in mammalian cells using two different gRNAs.
C) Claims 1, 2, 6, 8-13, 15-17 remain rejected under 35 U.S.C. 103 as being unpatentable over Cho (Nature Biotech., published online on January 29, 2013, Vol. 31(3), pp. 230-232, plus Supplemental Materials) in view of Arnould (WO 2008/102274), Mali (Science, Feb. 15, 2013, Vol. 339, No. 6121, pg 823-826), Carroll (Mol. Ther., Sept. 2012, Vol. 20, No. 9, pg 1658-1660), Ando (7951925) and Cannon (Curr. Opin. HIV AIDS, Jan. 2012, Vol. 6, No. 1, pg 74-79).
The combined teachings of Cho, Arnould, Mali, Carroll, and Ando taught contacting a population of isolated primary hematopoietic, pancreatic, renal, or muscular cells with a nucleic acid sequence encoding Cas9 and a pair of gRNAs that target “offset” sequences of an endogenous B2M gene such that a deletion in the B2M gene occurs, wherein efficiency is 33%.
The combined teachings of Cho, Arnould, Mali, Carroll, and Ando did not teach the cells were CD4+ T-cells as required in claim 17.
However, knocking out genes in CD4+ T-cells was well known as described by Cannon (pg 2, 2nd full paragraph).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to use Cas9 and a pair of gRNAs that target “offset” sequences of an endogenous B2M gene to make a deletion in the B2M gene of isolated human cells as described by Cho, Arnould, Mali, Carroll, and Ando wherein the cells were CD4+ T-cells described by Cannon. Those of ordinary skill in the art at the time of filing would have been motivated to replace the cells of Cho with CD4+ T-cells for in vitro research of the role of B2M in T-cell activation.
Dependent claims have been included for reasons set forth above.
Response to arguments
Applicants argue Arnauld teaches away from the invention because it “led a skilled artisan to modify the B2M gene only in pancreatic, renal, and muscular tissues with meganuclease [ ] which are excluded from the claims (pg 14). Applicants’ argument is not persuasive. Arnauld need not teach every limitation as claimed. Applicants’ argument that the absence of teachings in Arnauld somehow equates to “teaching away” is scientifically and legally illogical. Arnauld has been relied upon for targeting the B2M gene of human cells as well as evidence that those of skill would have had a reasonable expectation of doing so in a number of mammalian tissues.
Applicants argue Mali is limited to using two different gRNAs that target AAVS1 “which is excluded from the claims”. Applicants’ argument is not persuasive. Mali need not teach every limitation as claimed. Mali has been relied upon for targeting a desired gene in mammalian cells using two different gRNAs.
D) Claims 1, 2, 6, 8-13, 16, 17 remain rejected under 35 U.S.C. 103 as being unpatentable over Cho (Nature Biotech., published online on January 29, 2013, Vol. 31(3), pp. 230-232, 2013, plus Supplemental Materials) in view of Arnould (WO 2008/102274), Mali (Science, Feb. 15, 2013, Vol. 339, No. 6121, pg 823-826), and Cannon (Curr. Opin. HIV AIDS, Jan. 2012, Vol. 6, No. 1, pg 74-79).
Cho contacted a population of isolated human K562 cells with a nucleic acid sequence encoding Cas9 codon-optimized for humans (pg 1 of Supplementary methods, “Construction of Cas9-encoding plasmids”) and a gRNA that targets an endogenous CCR5 gene in the cell such that the CCR5 gene has a genetic modification that is a deletion, wherein efficiency of obtaining the genetic modification in the CCR5 gene is up to 33% (see 2nd para on pg 231; pg 230, col. 2, 2nd para; pg 232, Fig. 2A). The phrase “primary cells” in claim 1 encompasses any cells that are isolated/harvested directly from living tissue/organ. K562 cells are “primary cells” because they were isolated directly from human tissue and maintained for growth in culture medium.
Cho did not target the B2M gene as required in claim 1. However, targeting the B2M gene of human cells was well-known in the art as described by Arnould (“Meganuclease variants cleaving a DNA target sequence from the beta-2-microglobulin gene and uses thereof”) and doing so in pancreatic, renal, or muscular tissues (3rd paragraph). Thus, it would have be