DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communication filed 7-2-26.
Claims 1-27, 47-49, 52-56 are pending in the instant application.
Election/Restrictions
Claims 1-27, 47-49, 52 and 56 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 7-2-26.
Applicant’s election without traverse of Group IV, claims 53-55, in the reply filed on 7-2-26 is acknowledged.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 53-55 are rejected under 35 U.S.C. 112, first paragraph, because the specification, while being enabling for the in vitro testing of selected mutants of Cas12i2 of SEQ ID No. 922, comprising SEQ ID Nos. 924, 927, 1089 and 1090, in combination with various HAO1-Targeting RNA Guides, does not reasonably provide enablement of methods for treating primary hyperoxaluria (PH) in a subject comprising administering a gene editing system for editing a hydroxyacid oxidase 1 (HAO1) gene comprising (i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide comprising an amino acid sequence at least 95% identical to SEQID NO: 922 and optionally comprising one or more mutations relative to SEQ ID NO: 922;
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 and/or use the invention commensurate in scope with these claims.
The following factors have been considered in determining that the specification does not enable the skilled artisan to make and/or use the invention over the broad scope claimed.
The breadth of the claims:
The claims are drawn to methods for treating primary hyperoxaluria (PH) in a subject comprising administering a gene editing system for editing a hydroxyacid oxidase 1 (HAO1) gene comprising (i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide comprising an amino acid sequence at least 95% identical to SEQID NO: 922 and optionally comprising one or more mutations relative to SEQ ID NO: 922; (ii) an RNA guide or a second nucleic acid encoding the RNA guide comprising a spacer sequence specific to a target sequence within an HAO1 gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5'-TTN-3', which is located 5' to the target sequence.
Teachings in the specification:
The specification teaches the following in vitro studies of reduction of expression of the target gene HAO1:
Exemplary Casl2i polypeptide sequences and corresponding nucleotide sequences are listed in Table 6.
FIG. 1 shows HAO1 indels in HEK293T cells following RNP delivery… Following delivery, indels were detected within and/or adjacent to each of the HAO1 target sites with each of the RNA guides. Delivery of E1T2, E1T3, E1T6, E1T7, E1T13, T1T17, E2T4, E2T5, E2T9, E2T10, E3T6, E3T19, E3T22, and E3T28 resulted in indels in over 70% of the NGS reads. Therefore, HAO1-targeting RNA guides induced indels in exon 1, exon 2, and exon 3 in HEK293T cells.
This Example thus shows that HAO1 can be individually targeted by Cas 12i2 RNPs in mammalian cells such as HEK293T cells.
Example 2 - Cas12i2-Mediated Editing of HA01 Target Sites in HepG2 cells
This Example describes the genomic editing of the HAO1 gene using Cas 12i2 introduced into HepG2 cells by RNP. RNP complexation reactions were performed as described in Example 1 with various RNA guides of Table 7. HepG2 cells were harvested using TRYPLE™ (recombinant cell- dissociation
After 3 days, wells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf).
Samples were then frozen at -20°C. Samples were analyzed by NGS as described in Example 1.
FIG. 2 shows HAO1 indels in HepG2 cells following RNP delivery. Error bars represent the average of three technical replicates across one biological replicate. Following delivery, indels were detected within and/or adjacent to each of the HAO1 target sites with each of the RNA guides. Therefore, HAO1-targeting RNA guides induced indels in exon 1, exon 2, and exon 3 in HepG2 cells.
This Example thus shows that HA01 can be targeted by Cas12i2 RNPs in mammalian cells such as HepG2 cells.
Example 3 - Cas12i2-Mediated Editing of HA01 Target Sites in Primary Hepatocytes This Example describes the genomic editing of the HAO1 using Cas12i2 introduced into primary hepatocytes cells by RNP. RNP complexation reactions were performed as described in Example 1 with RNA guides of Table 7. Primary hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37°C …
After 4-5 days post RNP electroporation, media was aspirated and the cells were harvested by shaking (500 rpm) in a 37°C incubator with 2mg/ml collagenase IV
As shown in FIG. 3, each RNA guide tested induced indels within and/or adjacent to the HAO1 target sites. Indels were not induced with the non-targeting control. Therefore, HAO1- targeting RNA guides induced indels in primary hepatocytes. Indels were then correlated with mRNA levels for each target to determine whether indels lead to mRNA knockdown and subsequent protein knockdown. FIG. 4 shows %mRNA knockdown of HA01 in edited cells compared to unedited control cells. Although a higher percentage of NGS reads comprised indels using HAO1 E2T5 (SEQ ID NO: 989) compared to HAO1 E2T4 (SEQ ID NO: 988), HAO1 E2T4 resulted in a greater knockdown of HAO1 mRNA.
This Example thus shows that HA01 can be targeted by Cas12i2 RNPs in mammalian cells such as primary human hepatocytes.
Example 4 - Editing of HA01 Target Sites in HepG2 Cells with Cas12i2 Variants
This Example describes indel assessment on HAO1 targets using variants introduced into HepG2 cells by transient transfection.
The 12i2 variants of SEQ ID NO: 924 and SEQ ID NO: 927 were individually cloned into a pcda3. backbone (Invitrogen). Nucleic acids encoding RNA guides E1T2 (SEQ ID NO:967), E1T3 (SEQ ID NO: 968), E2T4 (SEQ ID NO: 988), E2T5 (SEQ ID NO: 989), E2T10(SEQ ID NO: 994) were cloned into a pUC19 backbone (New England Biolabs). The plasmids were then maxi-prepped and diluted. HepG2 cells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes … and counted. Cells were washed once with PBS and resuspended in SF buffer + supplement …
Approximately 16 hours prior to transfection, 25,000 HepG2 cells in EMEM/10%FBS were plated into each well of a 96-well plate. On the day of transfection, the cells were 70-90% confluent. For each well to be transfected, a mixture of Lipofectamine 3000 and … After incubation, the lipofectamine OptiMEM® mixture was added to a separate mixture containing nuclease plasmid and RNA guide plasmid and P3000 reagent (Solution 2). In the case of negative controls, the crRNA was not included in Solution 2. The Solution 1 and Solution 2 were mixed by pipetting up and down and then incubated at room temperature for 15 minutes.
Following incubation, the Solution 1 and Solution 2 mixture was added dropwise to each well of a 96 well plate containing the cells.
After 3 days, wells were harvested using TRYPLE and transferred to 96-well TWIN.TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 µL QUICKEXTRACT™ (DNA extraction buffer; Lucigen). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C and analyzed by NGS as described in Example 1.
As shown in FIG. 5A, comparable indel activity with the two Cas 12i2 variants was observed for E1T2, E1T3, E2T4, E2T5, E2T10. FIG. 5B shows the indel size frequency (left) and indel start position relative to the PAM for E1T3 and the variant Cas12i2 of SEQ ID NO: 924. As shown on the left, deletions ranged in size from 1 nucleotide to about 40 nucleotides. The majority of the deletions were about 6 nucleotides to about 27 nucleotides in length. As shown on the right, the target sequence is represented as starting at position 0 and ending at position 20. Indels started within about 10 nucleotides and about 35 nucleotides downstream of the PAM sequence. The majority of indels started near the end of the target sequence, e.g., about 18 nucleotides to about 25 nucleotides downstream of the PAM sequence.
Thus, this Example shows that HAO1 is capable of being targeted by multiple Cas 12i2 polypeptides.
Example 5 - Editing of HA01 in Primary Human Hepatocytes Using Cas12i2 mRNA Constructs This Example describes indel assessment on HAO1 target sites via delivery of Cas 12i2 mRNA and chemically modified HAO1-targeting RNA guides. mRNA sequences corresponding to the variant Cas 12i2 sequence of SEQ ID NO: 924 and the variant Cas 12i2 sequence of SEQ ID NO: 927 were synthesized … with 1- pseudo-U modified nucleotides …
The Cas 12i2 mRNA sequences, shown in Table 8, further comprised a C-terminal NLS. Table 8. Cas12i2 mRNA Sequences
FIG. 6 shows editing of an HAO1 target site by a variant Cas12i2 mRNA and 3' end modified E2T5 (SEQ ID NO: 1091) or 5' and 3' end modified E2T5 (SEQ ID NO: 1092). Indels in the HAO1 target site were introduced following electroporation of the Cas 12i2 mRNA of SEQ ID NO: 1089 or SEQ ID NO: 1090 and either the RNA guide of SEQ ID NO: 1091 or SEQ ID NO: 1092. Approximately 50% NGS reads comprised an indel following electroporation of the Cas 12i2 mRNA of SEQ ID NO: 1090 and the RNA guide of SEQ ID NO: 1091 or SEQ ID NO: 1092. Statistically significant higher % indels were observed using variant 12i2 mRNA of SEQ ID NO: 1090 compared to variant mRNA of SEQ ID NO: 1089. No statistical difference was observed using 5' and 3' versus 3' only modifications to RNA guide E2T5.
This Example thus shows that HAO1 can be targeted by Cas12i2 mRNA constructs and chemically modified RNA guides in mammalian cells.
Example 6 - Off-Target Analysis of Cas12i2 and HAO1-Targeting RNA Guides
This Example describes on-target versus off-target assessment of a Cas12i2 variant and an HAO1-targeting RNA guide. HEK293T cells were transfected with a plasmid encoding the variant Cas 12i2 of SEQ ID NO: 924 or the variant Cas 12i2 of SEQ ID NO: 927 and a plasmid encoding E2T5 (SEQ ID NO: 989), E1T2 (SEQ ID NO: 967), E1T3 (SEQ ID NO: 968), and E2T10 (SEQ ID NO: 994) according to the method described in Example 16 of PCT/US21/25257. The tagmentation-based tag integration site sequencing (TTISS) method described in Example 16…was then carried out.
FIG. 7A and FIG. 7B show plots depicting on-target and off-target TTISS reads. The black wedge and centered number represent the fraction of on-target TTISS reads. Each grey wedge represents a unique off-target site identified by TTISS. The size of each grey wedge represents the fraction of TTISS reads mapping to a given off-target site. FIG. 7A shows TTISS reads for variant Cas 12i2 of SEQ ID NO: 924, and FIG. 7B shows TTISS reads for variant Cas 12i2 of SEQ ID NO: 927.
As shown in FIG. 7A, variant 12i2 of SEQ ID NO: 924 paired with E2T5 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads mapped to the on- target. No TTISS reads mapped to potential off-target sites. E1T2 also showed a low likelihood of off-target editing. For E1T2, 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads. For E5T10, 95% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 5% of TTISS reads. E2T10 demonstrated a higher likelihood of off-target editing using the TTISS method. For E2T10, only 65% of TTISS reads mapped to the on-target and 4 potential off-target sites represented the remaining combined 35% of TTISS reads. One potential off-target represented the majority of potential off-target TTISS reads for E2T10. Therefore, this Example shows that compositions comprising Cas 12i2 and HAO1- targeting RNA guides comprise different off-target activity profiles.
Example 7 - HA01 Protein Knockdown with Cas12i2 and HAO1-Targeting RNA Guides This Example describes use of a Western Blot to identify knockdown of HAO1 protein using variant 12i2 of SEQ ID NO: 924 and HAO1-targeting RNA guides.
Primary hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37°C water bath. The cells were added to pre-warmed hepatocyte recovery media and resuspended in appropriate volume of hepatocyte plating Medium …,
For the RNP reactions, E2T5 (SEQ ID NO: 989) was used as the HAO1-targeting RNA guides. RNPs were added to each reaction at a final concentration of 20 µM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 µM. Unelectroporated cells and cells electroporated without cargo were used as negative controls.
Knockdown of HAO1 protein was observed in primary human hepatocytes at Day 7 post editing by Cas 12i2 RNPs targeting the HAO1 gene with E2T5 (lanes 1-3 of FIG. 8). HAO1 knockdown was not observed for the buffer only controls (lanes 4-7).
This Example thus shows that HAO1 protein levels were decreased following editing with Cas 12i2 and HAO1-targeting RNA guides. Cas 12i2 RNA guides were designed and ordered from Integrated DNA Technologies (IDT) as having 3' end modified phosphorothioated 2' O-methyl bases or 5' end and 3' end modified phosphorothioated 2' O-methyl bases guides, as specified in Table 9. Each variant Cas 12i2 mRNA was mixed with a crRNA at a 1:1 (Cas12i2:crRNA) volume ratio (1050:1 crRNA:Cas12i2 molar ratio). The mRNA and crRNA were mixed immediately before electroporation. The primary human hepatocyte cells were cultured and electroporated as described in Example 3.
Table 9. Chemically modified RNA guide sequences
FIG. 6 shows editing of an HAO1 target site by a variant Cas12i2 mRNA and 3' end modified E2T5 (SEQ ID NO: 1091) or 5' and 3' end modified E2T5 (SEQ ID NO: 1092). Indels in the HAO1 target site were introduced following electroporation of the Cas 12i2 mRNA of SEQ ID NO: 1089 or SEQ ID NO: 1090 and either the RNA guide of SEQ ID NO: 1091 or SEQ ID NO: 1092. Approximately 50% NGS reads comprised an indel following electroporation of the Cas 12i2 mRNA of SEQ ID NO: 1090 and the RNA guide of SEQ ID NO: 1091 or SEQ ID NO: Statistically significant higher % indels were observed using variant 12i2 mRNA of SEQ ID NO: 1090 compared to variant mRNA of SEQ ID NO: 1089. No statistical difference was observed using 5' and 3' versus 3' only modifications to RNA guide E2T5.
This Example thus shows that HAO1 can be targeted by Cas12i2 mRNA constructs and chemically modified RNA guides in mammalian cells.
Example 6 - Off-Target Analysis of Cas12i2 and HAO1-Targeting RNA Guides
This Example describes on-target versus off-target assessment of a Cas12i2 variant and an HAO1-targeting RNA guide. HEK293T cells were transfected with a plasmid encoding the variant Cas 12i2 of SEQ ID NO: 924 or the variant Cas 12i2 of SEQ ID NO: 927 and a plasmid encoding E2T5 (SEQ ID NO: 989), E1T2 (SEQ ID NO: 967), E1T3 (SEQ ID NO: 968), and E2T10 (SEQ ID NO: 994)according to the method described in Example 16 of PCT/US21/25257. The tagmentation-based tag integration site sequencing (TTISS) method described in Example 16 of PCT/US21/25257 was then carried out.
FIG. 7A and FIG. 7B show plots depicting on-target and off-target TTISS reads. The black wedge and centered number represent the fraction of on-target TTISS reads. Each grey wedge represents a unique off-target site identified by TTISS. The size of each grey wedge represents the fraction of TTISS reads mapping to a given off-target site. FIG. 7A shows TTISS reads for variant Cas 12i2 of SEQ ID NO: 924, and FIG. 7B shows TTISS reads for variant Cas 12i2 of SEQ ID NO: 927.
As shown in FIG. 7A, variant 12i2 of SEQ ID NO: 924 paired with E2T5 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads mapped to the on- target. No TTISS reads mapped to potential off-target sites. E1T2 also showed a low likelihood of off-target editing. For E1T2, 98% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 2% of TTISS reads. For E5T10, 95% of TTISS reads mapped to the on-target, and two potential off-target sites represented a combined 5% of TTISS reads. E2T10 demonstrated a higher likelihood of off-target editing using the TTISS method. For E2T10, only 65% of TTISS reads mapped to the on-target and 4 potential off-target sites represented the remaining combined 35% of TTISS reads. One potential off-target represented the majority of potential off-target TTISS reads for E2T10.
As shown in FIG. 7B, variant 12i2 of SEQ ID NO: 927 paired with E2T5 demonstrated a low likelihood of off-target editing, as 100% of TTISS reads mapped to the on- target. No TTISS reads mapped to potential off-target sites. Variant Cas 12i2 of SEQ ID NO: 927 paired with the E1T2 or E1T3 also demonstrated a low likelihood of off-target editing.
Therefore, this Example shows that compositions comprising Cas 12i2 and HAO1- targeting RNA guides comprise different off-target activity profiles.
Example 7 - HA01 Protein Knockdown with Cas12i2 and HAO1-Targeting RNA Guides This Example describes use of a Western Blot to identify knockdown of HAO1 protein using variant 12i2 of SEQ ID NO: 924 and HAO1-targeting RNA guides.
Primary hepatocyte cells from human donors were thawed from liquid nitrogen very quickly in a 37°C water bath. The cells were added to pre-warmed hepatocyte recovery media
For the RNP reactions, E2T5 (SEQ ID NO: 989) was used as the HAO1-targeting RNA guides. RNPs were added to each reaction at a final concentration of 20 µM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 µM.
Knockdown of HAO1 protein was observed in primary human hepatocytes at Day 7 post editing by Cas 12i2 RNPs targeting the HAO1 gene with E2T5 (lanes 1-3 of FIG. 8). HAO1 knockdown was not observed for the buffer only controls (lanes 4-7). This Example thus shows that HAO1 protein levels were decreased following editing with Cas 12i2 and HAO1-targeting RNA guides.
[Emphases added][Citations omitted].
The examples provided in the instant specification, of the in vitro testing of selected mutants of Cas12i2 of SEQ ID No. 922, comprising SEQ ID Nos. 924, 927, 1089 and 1090, in combination with various HAO1-Targeting RNA Guides, are not representative or correlative of the ability to treat any disease or condition in any subject comprising any mutation relative to SEQ ID No. 922.
In light of the teachings in the specification, one skilled in the art would not accept on its face the examples provided in the instant disclosure as being correlative or representative of the ability to adequately deliver and express the myriad of Cas12i2 mutants and corresponding HAO1 targeting guides to a subject, via any route of administration, or to provide treatment effects in any subject as instantly claimed.. Since the specification fails to provide the requisite guidance for the treatment of any disease in any subject using the large genus of mutants of Cas12i2 claimed, and since determination of the factors required for accomplishing this in any subject is highly unpredictable, it would require undue experimentation to practice the invention over the broad scope claimed.
For these reasons, the instant rejection for lacking enablement over the full scope claimed is proper.
Claims 53-55 are 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.
The breadth of the claims:
The claims are drawn to methods for treating primary hyperoxaluria (PH) in a subject comprising administering a gene editing system for editing a hydroxyacid oxidase 1 (HAO1) gene comprising (i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide comprising an amino acid sequence at least 95% identical to SEQID NO: 922 and optionally comprising one or more mutations relative to SEQ ID NO: 922; (ii) an RNA guide or a second nucleic acid encoding the RNA guide comprising a spacer sequence specific to a target sequence within an HAO1 gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5'-TTN-3', which is located 5' to the target sequence.
The specification fails to provide the requisite guidance for using the large genus of therapeutic agents and mutants instantly claimed, and further whereby treatment is provided in any subject. The specification teaches the in vitro testing of selected mutants of Cas12i2 of SEQ ID No. 922, comprising SEQ ID Nos. 924, 927, 1089 and 1090, in combination with various HAO1-Targeting RNA Guides. These examples do not provide a representative number of species for the multitude of Cas12i2 mutants encompassed by the claims.
Since the disclosure fails to describe the common attributes and characteristics concisely identifying members of the proposed genus of mutants, and because the claimed genus is highly variant, the description provided is insufficient. One of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the broad genus of mutants of Cas12i2 instantly claimed.
Thus, Applicant was not in possession of the broadly claimed genus.
Conclusion
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
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Jane Zara
7-23-26
/JANE J ZARA/Primary Examiner, Art Unit 1637
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