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
Claims 3-5, 14, 15 have been canceled. Claims 1, 2, 6-13, 16, 17 are pending.
Applicant's arguments filed 5-4-26 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, 16, 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Withdrawn rejection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using isolated mammalian CD4+ T-cells or HSCs with an inactivated B2M gene provided in the method of claim 1 has been withdrawn because the cells can be used for in vitro research of the role of B2M in T-cell activation.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, a 1st gRNAs that targets a coding region of exon 1 of the B2M gene, and a 2nd gRNAs that targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1.
The claim encompasses inactivating the B2M gene in isolated primary human CD4+ T cells. The specification contemplates identifying a pair of gRNAs “that target offset sequences” of a B2M gene which are compatible with CRISPR technology (pg 2, ¶4). Claim 1 requires a 1st gRNAs that targets a coding region of exon 1 of the B2M gene, and a 2nd gRNAs that targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene.
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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Fig. 5D:
However, the target sequences of these gRNA pairs used for Fig. 4C, 5D required for achieving “about 9%” inactivation of a B2M gene” cannot be found in the specification. There is nothing in Fig. 4C that says a 1st gRNA targets a coding region of exon 1 of the B2M gene or that a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1. Support has not been provided and none can be found.
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 have the pattern 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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Applicants arguments filed 5-4-26 say gRNA L1 targets a coding sequence in exon 1; gRNA L2 targets a 5’ UTR region of exon 1, gRNA 8 targets intron 1, and gRNA 10 targets exon 2 (pg 6-7 of the response). However, Fig. 5D and the rest of the specification do not teach the specific target sequences of “L1”, “L2”, “8”, “10 within the human B2M gene. 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. Fig. 5D does not say a 1st gRNA targets a coding region of exon 1 of the B2M gene was used in combination with a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1. Fig. 5D does not infer the gRNA pairs that bind the targets are capable of inactivating a B2M gene or with the efficiency required in claim 1.
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 There is nothing in Fig. 4C that says the “guide combination” is a 1st gRNA targets a coding region of exon 1 of the B2M gene and a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1. 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 gRNA pairs having the structures claimed 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 a 1st gRNA targets a coding region of exon 1 of the B2M gene and a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1. 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. 8. The specific sequence of any 1st gRNA targets a coding region of exon 1 of the B2M gene and a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene that would inactivate the B2M gene are not disclosed in Fig. 8 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 target the sequences 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 are those claimed, 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 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 target sequences for the B2M gene in Fig. 4C were the target sequences claimed or elsewhere in the specification required to obtain the results in Fig. 6B, 8A, or 8C.
Response to arguments
Applicants argue Fig. 5D shows that gRNA L1 targets a coding sequence in exon 1; gRNA L2 targets a 5’ UTR region of exon 1, gRNA 8 targets intron 1, and gRNA 10 targets exon 2 (pg 6-7 of the response). Applicants’ argument is not persuasive. Fig. 5D and the rest of the specification do not teach the specific structures of “L1”, “L2”, “8”, “10 within the B2M gene or the specific structure of any target sequences for gRNA pairs that target the sequences 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 gRNA pairs that target the sequences claimed and would inactivate the B2M gene are not disclosed in Fig. 5D or elsewhere in the specification. There is nothing in Fig. 5D that says a 1st gRNA targets a coding region of exon 1 of the B2M gene was used in combination with a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1.
B) The specification does not enable any 1st gRNA that targets a coding sequence in exon 1 and a 2nd gRNA that targets a 5’ UTR region of exon 1, intron 1, or exon 2 and which is approximately 2.2 kb away from the target of the 1st gRNA as required in claim 16. Pg 47, para 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 that they target sequences for gRNA pairs 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 gRNA pairs that bind the locations claimed, are at least 2.2 kb apart, and 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 by applicants in the left side of Fig. 5D), there is no description of their structure, and 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 gRNAs 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 having the target sequences claimed that are at least 2.2 kb apart and capable of inactivating the B2M gene in at least 9% of the cells as required in claim 1.
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 have the targets 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 gRNA pairs that target the sequences claimed and 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.
Applicants argue the assembly of chromosome 37 (GRCh37) mRNA reveals the coding region of exon 1 has 4 NGG target motifs, the 5’UTR of exon 1 has 2 NGG target motifs, exon 2 has 9 NGG target motifs, and intron 1 has a handful of NGG target motifs (pg 8 of response). Applicants’ argument is not persuasive because the “B2M mRNA track of the GRCh37 genome assembly (released in February 2009)” cannot be found. There is nothing in the specification that shows the sequence of the B2M gene or the delineation of the exon 1 UTR, the exon 1 coding region, intron 1, or exon 2 was known at the time of filing. The IDSs and prior art listings in the application have been reviewed for a clue about the “genome assembly” to which applicants refer, but none can be found. More importantly, the specification does not teach the specific structure of any gRNA that immediately precedes either of the 5’UTR of exon 1 NGG target motifs, any of the 4 exon 1 NGG target motifs, the 9 exon 2 NGG target motifs, or the handful of intron 1 NGG target motifs.
Written Description
Claims 1, 2, 6-13, 16, 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, a 1st gRNAs that targets a coding region of exon 1 of the B2M gene, and a 2nd gRNAs that targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1.
The claim encompasses inactivating the B2M gene in isolated primary human CD4+ T cells. The specification contemplates identifying a pair of gRNAs “that target offset sequences” of a B2M gene which are compatible with CRISPR technology (pg 2, ¶4). Claim 1 requires a 1st gRNAs that targets a coding region of exon 1 of the B2M gene, and a 2nd gRNAs that targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene.
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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Fig. 5D:
However, the target sequences of these gRNA pairs used for Fig. 4C, 5D required for achieving “about 9%” inactivation of a B2M gene cannot be found in the specification. There is nothing in Fig. 4C that says a 1st gRNA targets a coding region of exon 1 of the B2M gene or that a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1. Support has not been provided and none can be found.
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 have the pattern 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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Applicants arguments filed 5-4-26 say gRNA L1 targets a coding sequence in exon 1; gRNA L2 targets a 5’ UTR region of exon 1, gRNA 8 targets intron 1, and gRNA 10 targets exon 2 (pg 6-7 of the response). However, Fig. 5D and the rest of the specification do not teach the specific structures of “L1”, “L2”, “8”, “10 within the B2M gene or the specific structure of any target sequences for gRNA pairs that target the sequences 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 gRNA pairs that target the sequences claimed and would inactivate the B2M gene are not disclosed in Fig. 5D or elsewhere in the specification. There is nothing in Fig. 5D that says a 1st gRNA targets a coding region of exon 1 of the B2M gene was used in combination with a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1.
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 There is nothing in Fig. 4C that says the “guide combination” is a 1st gRNA targets a coding region of exon 1 of the B2M gene and a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1. 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 gRNA pairs having the structures claimed 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 a 1st gRNA targets a coding region of exon 1 of the B2M gene and a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1. 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. 8. The specific sequence of any 1st gRNA targets a coding region of exon 1 of the B2M gene and a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene that would inactivate the B2M gene are not disclosed in Fig. 8 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 target the sequences 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 are those claimed, 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 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 inactivating 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 target sequences for the B2M gene in Fig. 4C were the target sequences claimed or elsewhere in the specification required to obtain the results in Fig. 6B, 8A, or 8C.
Response to arguments
Applicants argue Fig. 5D shows that gRNA L1 targets a coding sequence in exon 1; gRNA L2 targets a 5’ UTR region of exon 1, gRNA 8 targets intron 1, and gRNA 10 targets exon 2 (pg 6-7 of the response). Applicants’ argument is not persuasive. Fig. 5D and the rest of the specification do not teach the specific structures of “L1”, “L2”, “8”, “10 within the B2M gene or the specific structure of any target sequences for gRNA pairs that target the sequences 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 gRNA pairs that target the sequences claimed and would inactivate the B2M gene are not disclosed in Fig. 5D or elsewhere in the specification. There is nothing in Fig. 5D that says a 1st gRNA targets a coding region of exon 1 of the B2M gene was used in combination with a 2nd gRNA targets a 5’ UTR of exon 1, exon 2, or intron 1 of the B2M gene as required in claim 1.
B) The specification lacks written description for any 1st gRNA that targets a coding sequence in exon 1 and a 2nd gRNA that targets a 5’ UTR region of exon 1, intron 1, or exon 2 and which is approximately 2.2 kb away from the target of the 1st gRNA as required in claim 16. Pg 47, para 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 that they target sequences for gRNA pairs 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 gRNA pairs that bind the locations claimed, are at least 2.2 kb apart, and 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 their structure, and 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 gRNAs having the target sequences claimed that are at least 2.2 kb apart and capable of inactivating the B2M gene in at least 9% of the cells as required in claim 1.
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 have the targets 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 gRNA pairs that target the sequences claimed and 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.
Applicants argue the assembly of chromosome 37 (GRCh37) mRNA reveals the coding region of exon 1 has 4 NGG target motifs, the 5’UTR of exon 1 has 2 NGG target motifs, exon 2 has 9 NGG target motifs, and intron 1 has a handful of NGG target motifs (pg 8 of response). Applicants’ argument is not persuasive because the “B2M mRNA track of the GRCh37 genome assembly (released in February 2009)” cannot be found. There is nothing in the specification that shows the sequence of the B2M gene or the delineation of the exon 1 UTR, the exon 1 coding region, intron 1, or exon 2. The IDSs and prior art listings in the application have been reviewed for a clue about the “genome assembly” to which applicants refer, but none can be found. More importantly, the specification does not teach the specific structure of any gRNA that immediately precedes either of the 5’UTR of exon 1 NGG target motifs, any of the 4 exon 1 NGG target motifs, the 9 exon 2 NGG target motifs, or the handful of intron 1 NGG target motifs.
New rejection
C) The specification lacks written description for any gRNAs that are complementary to sequences “that each immediately precede an NGG motif” as required in claim 1. The NGG motif is part of the gRNA target sequence (pg 2, ((N)20NGG), so saying the gRNA targets immediately before the NGG motif does not make sense. The target sequences is 20 nucleotides + the NGG motif which is not what applicants have claimed. Furthermore, while the specification says “immediately preceding”, the specification does not define when a sequence is “immediately preceding” the NGG motif. The term “immediate” is relative and does not necessarily mean adjacent. It is unclear if the 20 nucleotide target could be one or two nucleotides away from the NGG motif or if the claim is limited to a 20 nucleotide target that is adjacent to the NGG motif. Finally, the specification is limited to a 20 nucleotide target + NGG motif being the complete target sequence for gRNA; however, the claim encompasses any size gRNA. It is unclear if the gRNA in claim 1 must target a 20 nucleotide sequence + the NGG motif or if any size sequence “immediately preceding” the NGG motif + the NGG motif is encompassed by claim. Accordingly, the concept lacks written description.
Indefiniteness
Claims 1, 2, 6-13, 16, 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.
Withdrawn rejection
The rejection regarding the metes and bounds of when target sequences are “offset” in claim 1 has been withdrawn because the concept has been replaced with a 1st gRNA that targets a sequence in exon 1 coding region while a 2nd gRNA targets a 5’ UTR of exon, exon 2, or intron 1.
The rejection regarding 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 has been withdrawn because the claim has been canceled.
The rejection regarding the phrase “the offset sequences to which the two gRNAs bind are offset by approximately 2.2 kb” in claim 16 has been withdrawn in view of the amendment.
The rejection regarding 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 has been withdrawn because the claim has been canceled.
Pending rejections
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 Beauchesne Declaration filed 5-4-26 says the metes and bounds of “primary” cells were immediately recognized because they are “directly isolated or harvested from living tissue or organs”. Applicants’ argument and the Declaration are not persuasive. The argument and Declaration are illogical because cell lines and “fresh” cells are both “directly isolated and harvested from living tissue or organs”. It is unclear when cells are “directly” isolated or “fresh”. Even if applicants were just trying to exclude “cell lines”, the specification and the art do not teach when cells are in culture long enough to be considered “cell lines”.
Applicants point to Hayflick who discussed a “Hayflick limit” and replicative senescence. Applicants’ argument is not persuasive. Hayflick did not define when cells are “primary” as claimed or tie the “Hayflick limit” to “primary” cells, specifically CD4+ Tcells as required in claim 1. While “many kinds of cultured cells” exhibit limited replication of less than a year, some of them have more. Kayflick and applicants do not teach when a cell culture is or no longer “primary”. Hayflick, Roth, the specification, and the art at the time of filing fail to teach the amount of “replicative senescence” that defines “primary” cells.
Applicants point to Roth who described senescence and genetic instability in CD4+ T cells that express TERT. Applicants’ argument and Roth are not persuasive. Roth did not define when cells are “primary” as claimed or define the amount of senescence in CD4+ T cells that makes them “primary” as required in claim 1. While CD4+ T-cells may exhibit limited replication, not all of them do. Roth, Hayflick, the specification, and the art at the time of filing do not teach when CD4+ T-cells are or no longer “primary” using “replicative senescence” or any other measure.
New rejection
C) The metes and bounds of gRNAs that are complementary to sequences “that each immediately precede an NGG motif” as required in claim 1. The NGG motif is part of the gRNA target sequence (pg 2, ((N)20NGG), so saying gRNA targets immediately before the NGG motif does not make sense. NGG is part of the target, not before as claimed. This alone makes the claim indefinite because the target sequences is 20 nucleotides plus the NGG motif; that’s not what applicants have claimed. Furthermore, while the specification says “immediately preceding”, the specification does not define when a sequence is “immediately” preceding the NGG motif. The term “immediate” is relative and does not necessarily mean adjacent. It is unclear if the 20 nucleotide target could be one or two nucleotides away from the NGG motif or if the claim is limited to a 20 nucleotide target that is adjacent to the NGG motif. Accordingly, the concept makes the claim indefinite.
Claim Rejections - 35 USC § 103
Withdrawn rejections
The rejection of claims 1, 2, 6, 8-13, 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), 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) has been withdrawn because it addresses genetically modifying hematopoietic stem cells which have been deleted from the claim set.
The rejection of claim 7 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, 16 and further in view of Hoerr (WO 2008/052770) has been withdrawn because it addresses genetically modifying hematopoietic stem cells which have been deleted from the claim set.
The rejection of claim 17 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) as applied to claims 1, 2, 6, 8-13, 16, 17 and further in view of Cannon (Curr. Opin HIV AIDS, 2011, Vol. 6, No. 1, pg 74-79) has been withdrawn in favor of the following rejection.
New rejections
A) 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, 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 Cannon (Curr. Opin HIV AIDS, 2011, 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 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 an exon 1 coding sequence and an exon 1 5’ UTR, exon 2, or intron 1 of 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”). The exon 1 coding sequence, exon 1 5’ UTR, exon 2, and intron 1 were all disclosed by Arnould. 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). It would also have been obvious to those of ordinary skill in the art at the time of filing to make target an exon 1 coding sequence and exon 2 of the B2M gene to inactivate the B2M by insuring no protein would be expressed.
Those of ordinary skill would have had a reasonable expectation of success because 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 inactivate 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, e.g. targeting exons 1 and 2. Those of ordinary skill in the art at the time of filing would have been motivated to use a pair of gRNAs that target exons 1 and 2 to ensure deletion occurs.
Cho did not teach applying the technology to human CD4+ T-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). And knocking out genes in human CD4+ T-cells was well known as described by Cannon (pg 2, 2nd full paragraph; pg 4, last 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 exons 1 and 2 of an endogenous B2M gene to make a deletion in the B2M gene of isolated human cells as described by Cho, Arnould and Mali 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 human CD4+ T-cells for in vitro research of the role of B2M in T-cell activation.
Cho used a Cas9 from Streptococcus 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).
The combined teachings of Cho, Arnould, Mali did not teach the gRNA targets are separated by 2.2 kb as required in claim 16. 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.
Cannon taught the T-cells were from peripheral blood as required in claim 17 (pg 4, last paragraph). Claim 17 is also included because it embeds a product-by-process into the claim without a clear active step of isolating CD4+ T-cells from peripheral blood. Claim 17 is further included because it would have been obvious to do so as it was well-known to be the least invasive way of isolating CD4+ T-cells. Those of ordinary skill in the art at the time of filing would have been motivated to isolate CD4+ T-cells from peripheral blood to avoid surgical procedures.
Thus, Applicants' claimed invention as a whole is prima facie obvious in the absence of evidence to the contrary.
Response to arguments
Applicants argue Arnauld teaches away from performing the invention in hematopoietic cells because “hematopoietic stem cells from β2m -/- mice were quickly rejected” (pg 14). Applicants’ argument is not persuasive. The claims are not drawn to β2m -/- mouse hematopoietic stem cells. 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 compare and contrast the differences between Carroll or Ando and the claims (pg 15-16). Applicants’ arguments are not persuasive. Mali, Carroll, and Ando need not teach every limitation as claimed.
Applicants conclude the amendment overcomes the rejection (pg 16). Applicants’ argument is not persuasive for reasons set forth above.
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 Cannon (Curr. Opin HIV AIDS, 2011, Vol. 6, No. 1, pg 74-79) as applied to claims 1, 2, 6, 8-13, 16, 17 and further in view of Hoerr (WO 2008/052770).
The combined teachings of Cho, Arnould, Mali, Carroll, and Cannon taught contacting a population of isolated primary human CD4+ T cells with a nucleic acid sequence encoding Cas9 and a pair of gRNAs that target exons 1 and 2 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 Cannon 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 exons 1 and 2 of an endogenous B2M gene to make a deletion in the B2M gene of isolated human CD4+ T cells as described by Cho, Arnould, Mali, Carroll, and Cannon 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.
Thus, Applicants' claimed invention as a whole is prima facie obvious in the absence of evidence to the contrary.
C) Claims 1, 2, 6, 8-13, 16, 17 remain rejected under 35 U.S.C. 103 as being unpatentable over Kucherlapati (CA 2037907) 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 Cannon (Curr. Opin HIV AIDS, 2011, Vol. 6, No. 1, pg 74-79).
Arnould taught inactivating a B2M gene in human cells using a nuclease (“Meganuclease variants cleaving a DNA target sequence from the beta-2-microglobulin gene and uses thereof”).
Arnould did not teach applying the technology to human CD4+ T-cells or using Cas9 and gRNA as encompassed by claim 1. However, Carroll described “A CRISPR approach to gene targeting” (Title) and using Cas9 and gRNA in human hematopoietic lineages: “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), and Cannon taught knocking out genes in isolated human CD4+ T-cells (pg 2, 2nd full paragraph; pg 4, last paragraph). Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to inactivate an endogenous B2M gene in an isolated human cell as described by Arnould in a human CD4+ T-cells using Cas9 and gRNA as described by Carroll and Cannon. Those of ordinary skill in the art at the time of filing would have been motivated to replace the cells of Arnould with human CD4+ T-cells for in vitro research of the role of B2M in T-cell activation. Those of ordinary skill in the art at the time of filing would have been motivated to replace the meganuclease with the CRISPR system of Carroll for increased efficiency.
Arnould did not teach targeting exon 1 and exon 2 of the B2M gene with two different gRNAs as encompassed by 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 inactivate an endogenous B2M gene in isolated human cells as described by Arnould using a pair of gRNAs that target exons 1 and 2 as encompassed by claim 1. Those of ordinary skill in the art at the time of filing would have been motivated to use a pair of gRNAs that target exons 1 and 2 to ensure deletion occurs.
Mali used a Cas9 from Streptococcus pyogenes as required in claim 2 (references 6, 31, 32, 34).
Mali used a sequence encoding Cas9 that is codon-optimized for expression in human cells (pg 823, col. 1, 2nd para) which is a “modified nucleic acid” as required in claim 6.
Mali used 20 base targets (“X20”) as required in claim 8 (pg 823, col. 1, 2nd para).
Mali used 20 base targets (“X20”) (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.
Targeting exons 1 and 2 was obvious as required in claim 12 for reasons cited above.
Mali used a plasmid as required in claim 13 (pg 6, supplemental materials).
The combined teachings of Arnould, Carroll, Cannon, and Mali did not teach the gRNA targets are separated by 2.2 kb as required in claim 16. 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.
Cannon taught the T-cells were from peripheral blood as required in claim 17 (pg 4, last paragraph). Claim 17 is also included because it embeds a product-by-process into the claim without a clear active step of isolating CD4+ T-cells from peripheral blood. Claim 17 is further included because it would have been obvious to isolate CD4+ T-cells from peripheral blood because it was well-known to be the least invasive way of isolating CD4+ T-cells. Those of ordinary skill in the art at the time of filing would have been motivated to isolate CD4+ T-cells from peripheral blood to avoid surgical procedures.
Thus, Applicants' claimed invention as a whole is prima facie obvious in the absence of evidence to the contrary.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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Michael C. Wilson
/MICHAEL C WILSON/
Primary Examiner, Art Unit 1638
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