DETAILED ACTION
Notice of Pre-AIA or AIA Status
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
The Examiner for this Application has changed. Please direct all future
correspondence to Juliana Candelaria, AU 1634. Additional contact information can be
found at the end of this paper.
This action is in response to the papers filed on 07/30/2025. Claims 71-75, 78-80, 86, 90, 93, 96-98, 101, 102, 109-118 are currently pending as per claims filed on 07/30/2025. Claims 74, 86, 90, 93, 96-98 are withdrawn and Claim 71 has been amended by Applicants’ amendment filed on 07/30/2025. Claim 71 is an independent claim.
Applicants’ election without traverse of Group I was acknowledged in the Office action filed on 7/10/2019. Additionally, applicants’ election of SEQ IS NOS: 4 and 15 as the pair of gRNAs in the reply filed on Feb 20, 2019 was previously acknowledged.
Claims 40-52, 74, 86-98 and 106 (claims 40-52, 87-89, 91-92 and 106, now canceled) were previously withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on February 20, 2019.
Therefore, claims 71-73, 79-80, 101-102, and 109-118 are pending and under examination are pending and under examination to which the following grounds of rejection are applicable.
Priority
The instant application is a 371 of PCT/IB2016/000282 filed 02/23/2016 and claims benefit to PRO 62/119,754 filed on 02/23/2015.
Thus, the earliest possible priority for the instant application is 02/23/2015.
Response to Arguments
Withdrawn objections/rejection in response to Applicants’ arguments or amendment
Drawing Objection
In view of Applicants’ amendment of the drawings, the drawing objection has
been withdrawn.
Maintained and/or modified rejections in response to Applicants’ arguments or amendment
Claim Rejections - 35 USC § 103
Claims 71-73, 75, 78-80, 101-102, 109-113, and 116-118 remain rejected under 35 U.S.C. 103 as being unpatentable over Takeuchi (US20150166969A1, earliest filing date to provisional application, 61/603231 filed on February 24, 2012) in view of Conway (WO2014186585A2, PG Pub date 11/20/2014), Camaschella (Blood. 1990, Vol. 75(4):1000-1005), Solovieff (Blood. Volume 115, Issue 9, 4 March 2010, Pages 1815-1822), Forget (Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management, Chapter 3: The Normal Structure and Regulation of Human Globin Gene Clusters, Aug 17, 2009, page 46-55), and Wu (Quantitative Biology. Volume 2. No. 2, July 10, 2014, pages 59-70). This rejection has been modified in consideration of the applicant's amendment filed on 7-30-2025.
Regarding claim 71, Takeuchi teaches an invention concerned with compositions and methods for the treatment of hemoglobinopathies such as thalassemia and sickle cell disease (SCD) wherein the compositions and methods comprise one or more endonuclease(s) or endonuclease fusion protein(s), and/or CRISPR endonuclease(s) ad/or CRISPR endonuclease fusion protein(s) to disrupt a Bcl11a coding region, regulatory region, to modify an adult human β-globin locus; to disrupt a HbP silencing DNA regulatory element or pathway, to mutate one or more γ-globin gene promoter(s) to achieve increased expression of a γ-globin gene; and to mutate one or more δ-globin gene promoter(s) to achieve increased expression of a δ-globin gene; and/or (g) to correct one or more β-globin gene mutation(s) (Abstract). Takeuchi teaches that five genes of the β-globin locus reside in a cluster on chromosome 11, wherein genes are expressed in an erythroid, and developmentally stage specific manor and the ε, A65 and δ and β genes being expressed primarily during the embryonic, fetal and post-natal periods respectively ([0007]). Takeuchi teaches methods of gene editing steps by introducing one or more CRISPR endonucleases, e.g. Cas9 endonucleases (SEQ ID NO: 37 in Fig. 26) in combination with one or more RNA guide strands (gRNAs) to mutate (cause a deletion or inversion) to the region that encodes the increased the expression of ʏ-globin (pg. 2, [0022]). Takeuchi teaches that homing endonucleases (HEs) that are selected for achieving high-efficiency, multiplex gene disruption and gene editing functions ([0087]) recognize multiple targets at a critical region within the β-globin locus that suppresses HbF function, specifically targeting the γ- and δ -promoters ([0090]). Takeuchi teaches that HEs generate 3′ overhangs at the site of targeted double-strand breaks, which results in an enhanced rate of end processing following HE cleavage ([0098]). Takeuchi teaches compositions and methods that comprise one or more endonuclease(s), including one or more HEs and/or one or more Cas9 endonuclease(s) to achieve the disruption of a sequence that encodes Bcl11a or its key regulatory sequences ([0101]). Takeuchi teaches that a 3.6 kb HbF silencing region associated with Bcl11a genomic location is disrupted by the upstream breakpoint of HPFH ([0110]). Takeuchi further teaches that HEs target sequences are evenly distributed throughout the 350 bp region that the region of Bcl11a occupancy within the HbF silencing region in adult erythroid cells that is disrupted in the HPFH deletion throughout the specified region of chromosome 11 and these target sequences comprise DNA sequence modules for which pools of highly active endonuclease variants ([0167], Example 5, Table 4).
Takeuchi does not teach a method wherein the Cas9 and the pair of RNA guides are electroporated into the cell; wherein the pair of RNA guides consists of a first RNA guide and a second RNA guide and the pair of DSBs consists of a 5' DSB locus and a 3' DSB locus; wherein the first RNA guide comprises a spacer sequence complementary to a segment of the 5' DSB locus and the second RNA guide comprises a spacer sequence complementary to a segment of the 3' DSB locus and wherein the chromosomal DNA between the 5' DSB locus and the 3' DSB locus comprises the 5' boundary and 3' boundary of a region deleted in a Hereditary Persistence of Fetal Hemoglobin (HPFH) deletion, wherein the HPFH deletion is a deletion within the region from Chr1 1:5224779 to Chr1 1:5237723, and wherein the first RNA guide comprises a spacer sequence selected from the nucleic acid sequence of any one of SEQ ID NOs: 4-6, and the second RNA guide comprises a spacer sequence selected from the nucleic acid sequence of any one of SEQ ID NOs: 15-20.
However, one of ordinary skill in the art would have considered the teachings of Conway, Camaschella, Solovieff, Forget and Wu as these references are analogous prior art pertaining to genetic condition or gene editing approach associated with beta hemoglobinopathy and guide RNA design.
Conway discloses an invention related to a method modifying the expression of an endogenous gene in a cell (Abstract). Conway teaches that correction of the human HBB gene that encodes beta globin is accomplished with the CRISPR/Cas system wherein regions of chromosome 11 near a protospacer adjacent motif or PAM site are targeted preferably by using S. pyogenes Cas9 system ([0046]). Regarding Cas9 mediated gene-editing approach, Conway teaches that guide RNA complex comprising crRNA:tracrRNA directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to PAM, wherein tracrRNA base pairs with the crRNA at its 3' end, and this association triggers Cas9 mediated cleavage of the target DNA ([0143]). Conway teaches methods of treating sickle cell disease (SCD) hemoglobinopathies with the CRISPR/Cas system wherein the single guide RNA comprises sequences to target HbF genes and knocking out of the target genes is achieved through double strand cleavage ([0048]). Conway teaches that Cas9 nuclease protein is introduced into a cell with the desired sgRNA by a non-viral nucleic acid delivery mechanism such as electroporation ([0028]) and while naked DNA can be administered wherein the administration is done by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation ([0199]).
Camaschella teaches an invention concerned with a molecular comparison of the new deletion with others of similar size and location but associated with a delta beta-thalassemia phenotype suggests that the residual enhancer element, relocated near gamma genes, increases the fetal hemoglobin (HbF) expression above the level observed in delta beta-thalassemia (Abstract). Camaschella teaches the restriction map of the normal β-globin gene cluster with breakpoint indicated as potential sites from 5’ to 3’ region for genome modifications of Hereditary Persistence of Fetal Hemoglobin (HPFH);) (pg. 1002, Fig. 2) see β and δ at the breakpoint.In further support of Camaschella’s teaching of a restriction map for the normal β-globin gene cluster showing the breakpoint, Forget teaches a detailed map of the human globin gene complex, which includes genomic features and representative deletions on chromosome 11 that cause an array of thalassemia and HPFH disorders.
Forget teaches the chromosome nucleotide ranges that encompass the β-globin cluster and show distinct chromosome regions corresponding to the β and δ genes which span the region of approximately Chr11:5230000 and Chr11:5200000 as well as regions of regulatory potential, enhancers, promoters, etc (page 49; Figure 3.3A; shown below with a red box indicating region of interest).
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Solovieff is drawn to a genome-wide association (GWAS) study of 848 blacks with sickle cell anemia that identifies single nucleotide polymorphisms (SNPs) associated with fetal hemoglobin concentration (Abstract). Solovieff teaches that GWAS analysis identifies SNPs in several regions including Bcl11a (a developmentally regulated silencer of hemoglobin subunit gamma which forms HbF) and regions on chromosome 11: 5235931-5284996, both of which are associated with elevated HbF
The combined teachings of Takeuchi , Conway, Camaschella, Forget, and Solovieff fail to disclose that “the first RNA guide comprises a spacer sequence selected from the nucleic acid sequence of any one of SEO ID NOs: 4-6, and the second RNA guide comprises a spacer sequence selected from the nucleic acid sequence of any one of SEO ID NOs: 15-20”.
However, Wu is prior art that teaches that several tools (eleven tools are cited in Wu; page 66, left col, para 2) have been developed for designing guide RNAs such that off-target effects are mitigated. Wu teaches that the tools take into consideration the input sequence, a genomic region, or a gene and predicts guide RNAs with minimal off-target effects. Wu teaches that these tools can also consider the presence of SNPs and secondary structures, the genomic context of the guide, and GC content, all of which can impact the effectiveness of the gRNA (page 66, left col, para 2-3).
Therefore, it would be obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of Takeuchi for Conway using S. pyogenes Cas9 and gRNAs that are electroporated into cells as an efficient non-viral nucleic acid delivery mechanism for treating hemoglobinopathies. One of ordinary skill in the art would have been motivated to make this modification because Conway teaches a technique for electroporating Cas9 protein and one or more gRNAs into cells while knowing Camaschella’s restriction map of normal β-globin gene with breakpoints and the region to target for deletion would include Chr11: 5224779 to Chr11: 5237723 as taught by Solovieff and Forget. Furthermore, Forget’s disclosed structure, regulation, and genomic features of the human globin gene clusters and Solovieff’s teachings of particular locations of SNPs associated with elevated HbF would also lead one of ordinary skill in the art to target the Chr11: 5224779 to 5237723 region. Furthermore, combining with teachings by Wu of designing gRNAs for targeting genomic regions of interest, one of ordinary skill in the art would recognize that SEQ ID NO 4-6 and 15-20 to be just one of many possible guide RNAs that would be produced in the course of routine optimization of guide RNA design when the target domain a Hereditary Persistence of Fetal Hemoglobin (HPFH) for a deletion within the region from Chr11 :5224779 to Chr11 :5237723 is known in the art, absent evidence of any factual data, and one would have reasonable expectations of success as guide RNA design is known to be a part of gene editing methods.
It would also have been obvious to one of ordinary skill in the art at the time of invention to have replaced the sequence on chromosome 11 of Takeuchi, for the sequence on chromosome 11 of Camaschella, and Forget. A person of ordinary skill in the art would have had a reasonable expectation of success because Takeuchi does teach a CRISPR endonucleases target a 3.6Kb region with the β-globin gene locus Chr11: 5212342-5213944 in HG18 (site for regulatory protein BCL-11a) (as described above and pg. 6, [0087]) which is directed to the HPFH deletion group (pg. 3, [0025]) and the substitution of chromosome 11 deletion regions would have been obvious to modify because it is well known in the art as taught in Camaschella, Solovieff, and Forget teachings and that there are over 200 mutations responsible for β-thalassemia, for example Sickle cell disease is caused by a single nucleotide substitution within the β-globin gene (Takeuchi, pg. 1, [0008 -0009]). The disruption of these regions leads to a decrease in repressive complexes, which results in an elevated level of y-globin gene expression, and a corresponding increase in HbP (fetal) protein production to levels that are sufficient to achieve therapeutic efficacy in methods for the treatment of hemoglobinopathies, including β-thalassemia and sickle cell disease (Takeuchi, pg. 8, [0109]). Therefore, modifying the sequence on chromosome 11 of Takeuchi with the method of Conway, for the sequence on chromosome 11 of Camaschella, Solovieff, and Forget and the design of guide RNAs from Wu would have resulted in the claimed invention with a predictable outcome of success.
Regarding claims 72 -73 and 75, Takeuchi teaches an embodiment of treatments for patients with hemoglobinopathies that includes β-hemoglobinopathy which is also known as Sickle Cell disease (pg. 8, [0104].
Regarding claims 72 -73 and 75, Takeuchi teaches an embodiment of treatments for patients with hemoglobinopathies that includes β-hemoglobinopathy which is also known as Sickle Cell disease (pg. 8, [0104].
Regarding claims 78-79, Takeuchi teaches methods wherein one or more naturally occurring deletions of the β-globin gene and/or regions wherein the pair of RNA guides causes the deletion of the chromosomal DNA between the 5' DSB locus and the 3' DSB locus (methods for ameliorating thalassemia and sickle cell disease that are disclosed herein achieve therapeutic efficacy by introducing one or more mutation that result in increased HbF and/or HbA.sub.2 and/or HbA protein production. Exemplified herein are compositions and methods for recapitulating one or more naturally-occurring deletion(s) of the β-globin gene and/or regions, which activate ʏ-globin gene expression thereby increasing levels of fetal hemoglobin (pg. 9, [0122]).
Regarding claim 80, Takeuchi teaches a method wherein CRISPR endonucleases target a 3.6 Kb region with the β-globin gene locus Chr 11: 5212342-5213944 in HG18 of which is directed to the HPFH deletion group (pg. 3, [0025]).
Regarding claims 101-102, Takeuchi teaches a method wherein Cas9 endonucleases in combination with one or more RNA guide strands and/or CRISPR endonuclease fusion protein(s) (i.e. Cas9 endonuclease fusion protein(s) in combination with one or more RNA guide strands) (pg. 2, [0022] & Fig. 27).
Regarding claims 109-112, Takeuchi teaches a method wherein cells (iPSCs) are infused (systematic administration) after in vivo growth evidence to determine functionality (pg. 11, [0142]- [0143]).
Regarding claim 113, Takeuchi teaches a method of genome editing wherein HSCs and iPSCs, may be transplanted into a patient to treat one or more hemoglobinopathies, such as a thalassemia and/or sickle cell disease (pg. 3, [0029]).
Regarding claims 116-118, Takeuchi teaches methods wherein hematopoietic cells include CD34+ cells (pg. 10, [0129]) that may be infused (systematic administration) after in vivo growth evidence to determine functionality (pg. 11, [0142]- [0143]).
Response to Applicant’s arguments as they apply to rejection of claims 71-73, 75, 78-80, 101-102, 109-113-118 under 35 USC § 103
Applicant's amendments to the claims and arguments filed 07/30/2025 have
been fully considered but have not been found persuasive in overcoming the rejection
for reasons of record as discussed in detail below.
On page 8-10 of Applicant’s Remarks filed 07/30/2025, applicant argues that 1) amended claim 71 states that “the first guide comprises a spacer sequence selected from the nucleic acid sequence of any one of SEQ ID NOs: 4-6, and the second RNA guide comprises a spacer sequence selected from the nucleic acid sequence of any one of SEQ ID NOs: 15-20", that Takeuchi does not teach or suggest editing within the claimed region of Chr1 1:5224779 to Chr1l :5237723, Takeuchi also does not teach or suggest the use of guide RNA comprising spacer sequences selected from any one of SEQ ID NOs: 4-6 or 15-20 and Conway, Camaschella, and Solovieff fail to correct this deficiency of Takeuchi. Therefore, a person of ordinary skill in the art would not be able to combine the teachings of Takeuchi, Conway, Camaschella, and Solovieff to arrive at the claimed invention. Applicant argues 2) the teachings of Yannaki does not remedy the deficiencies of the combination of Takeuchi, Conway, Camaschella, and Solovieff.
Regarding argument 1), upon consideration of the amendment made to claim 71 which now recites specific SEQ ID NOs corresponding to the first and second guide RNA, new prior art of Forget and Wu have been applied to the modified 103 rejection in view of the previously cited prior art Takeuchi, Conway, Solovieff and Camaschella. Examiner states that Camaschella and Forget indeed teach the human globin gene cluster genomic regions of chromosome 11 and, specifically, the locations for γ, β, δ globin genes which are within said cluster and are associated with Hereditary Persistence of Fetal Hemoglobin. As recited in the 103 rejection above, Camaschella teaches that deletion of the δ and β genes is associated with the HPFH phenotype (i.e. increase level of fetal hemoglobin) (page 1000, left col, para 2; Fig 2 shows restriction map) and Solovieff teaches SNPs in regions associated with elevated HbF that are in the claimed region of Chr11 :5224779 to 5237723. As recited in the 103 rejection above, Forget further teaches the specific nucleotide regions in chromosome 11 where various deletions have led to the HPFH phenotype of elevated fetal hemoglobin due to increased γ gene expression, which further supports Camaschella’s teaching of the restriction map demonstrating a known deletion that causes HPFH. While the combined teachings do not teach the specific guide RNA sequences required to elicit the deletion for gene editing, the genomic target region to delete the δ and β genes was taught by Camaschella, Solovieff, and Forget, and one could design guide RNAs to target said known region by the teachings of various tools and the parameters to consider for the guide RNA design from Wu. Therefore, It would have been prima facie obvious to one of ordinary skill, in the art at the time of the effective filing date to arrive at one of many guide RNA sequences, including those in in instant claim 71, by the teachings and motivations of deleting the δ and β gene region to increase fetal hemoglobin from Camaschella, Solovieff, and Forget and optimizing gRNAs to achieve the gene edit from Wu.
With regard to Applicant’s argument 2 that Yannaki does not teach the entire claimed subject matter, the Examiner agrees. However, Yannaki is not applied alone, but in combination with Takeuchi, Conway, Camaschella, Solovieff , Forget, and Wu, and the claimed invention becomes obvious when the references are considered together as a whole rather than each alone.
Claims 114-115 are rejected under 35 U.S.C. 103 as being unpatentable over Takeuchi, Conway, Camaschella, Solovieff, Forget, and Wu (previously cited), as applied to claim 113 above, and further in view of Yannaki (Human Gene Therapy. 2013. Vol. 24: 852-860).
Regarding claims 114-115, the teachings of Takeuchi, Conway, Camaschella, Forget, Solovieff, and Wu are full detailed above and are fully incorporated here.
However, they do not teach a method wherein the hematopoietic progenitor cell is obtained from the patient having a hemoglobinopathy following treatment of the patient with granulocyte colony stimulating factor (GCSF) performed in combination with Plerixafor wherein the hematopoietic progenitor cell is a CD34+ cell by injection, infusion, or combinations thereof, by systemic administration.
Yannaki teaches a stem cell gene therapy method wherein CD34+ cell from patients with hemoglobinopathies that had been engineered and administered in combination with Plerixafor subcutaneously to patients for treating GCSF (pg. 853, col 2, para 2).
It would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of genome editing with Cas9 and gRNAs, plasmid-free electroporation targeting HPFH deletions taught by Takeuchi, Conway, Camaschella, Solovieff, Forget, and Wu to include gene edited hematopoietic progenitor cell treatment as a follow for patients with GCSF by administering Plerixafor treatments subcutaneously. One of ordinary skill in the art would have been motivated to make this modification because Yannaki teaches that it a novel mobilizing agent that increases yields by several folds, therefore one would have reasonable expectations of success.
Nonstatutory Double Patenting Rejection
Claims 71 and 101-102 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 5-6 of U.S. Patent No. 10738305B2 of Porteus and in view of Takeuchi, Conway, Camaschella, Forget, Solovieff, and Wu as applied to claims 71-73, 75, 78-80, 101-102, 109-113, and 116-118 above. Although the claims at issue is not identical, they are not patentably distinct from the claims of Patent No. 10738305 for the following reasons. This rejection has been modified as necessitated by applicant’s amendment filed on 7/30/2025
Regarding claim 71, Porteus claim 1 teaches a method of increasing the level of fetal hemoglobin (HbF) in a human cell by genome editing using DNA endonuclease to effect the double stranded break (DSB) at one or more loci within the δβ-globin region of human chromosome 11, causing a deletions or insertions of chromosomal DNA at the one or more loci that results in increased expression y-globin, thereby increasing the level of HbF in the cell, wherein one of the loci comprises a sequence that is complementary to the nucleic acid sequence of SEQ ID NO: 139 (claim 32). Furthermore Porteus teaches the same cell and chromosomal locations and describes the gRNA SEQ ID NOs recited in the instant claims.
Porteus does not teach a method wherein S. pyogenes Cas9 DNA endonucleases and a pair of guide RNAs (gRNA) are electroporated into the cell, nor does it teach wherein the pair of RNA guides consists of a first and a second RNA guide (SEQ ID NO 4-6 for first guide RNA and SEQ ID NO: 15-20 for second guide RNA) and a pair of DSBs consists of a 5’ DSB locus and a 3’DSB locus wherein the RNA guides have complementary sequences to comprise a spacer sequence for 5’ or 3’ locus, respectively. Porteus also does not teach wherein the chromosomal DNA between the 5’ DSB and 3’ DSB locus comprises the boundary 5’ and 3’ regions of a Heredity Persistence of Fetal Hemoglobin (HPFH) deletion. However, the previously listed limitations are met by the combine teachings of Takeuchi, Conway, Camaschella, Solovieff, Forget, and Wu (as described above).
It would have been obvious to modify as to combine the cited references to try the genome editing technique of Takeuchi to use the electroporation method of Conway to insert the Cas9 protein with gRNAs in the cells, the method of using one or more RNA guides with 5’ and 3’ complementary sequencing to the spacers that are the 5’ and 3’ boundaries for the mapped HPFH gene deletion taught by Conway, Solovieff, Forget, and Camaschella and the guide RNA sequences can be optimized to instead target the same known chromosome region using gRNAs such as SEQ ID NO 4-6 and 15-20 by the teachings of Wu. One of ordinary skills would have reasonable expectations for success because Conway taught the desire to electroporate Cas9 proteins with gRNA in cells with the knowledge of the targeted deletion region for HPFH for genome editing.
Regarding claim 101-102, Porteus teaches a method wherein the DNA endonucleases comprise one or more guide RNAs; a single molecule or combination (claims 36-37).
Response to Applicant’s arguments as they apply to rejection of claims 71 and 101-102 under Non-statutory double patenting (NSDP)
Applicant’s amendments to the claims and arguments filed 07/30/2025 have
been fully considered but have not been found persuasive in overcoming the rejection
for reasons of record as discussed in detail below.
Examiner notes that the provisional NSDP is updated to reflect that the co-pending applications have been allowed.
On page 10 of Applicant’s Remarks filed 07/30/2025, applicant argues that 1)
SEQ ID NO: 139 recited in claims 1 and 5-6 of the ‘305 patent does not target the currently claimed chromosomal region. SEQ ID NO: 139 cuts at Chr11:5249972. See e.g., Figure 14C of US 10,738,305. There is no suggestion in the claims of the ‘305 patent to target the claimed region using the spacer sequences of SEQ ID Nos: 4-6 or 15-20.
Regarding argument 1), while examiner acknowledges that SEQ ID NO: 139 does not target the currently claimed chromosomal region, the region where SEQ ID NO: 139 does cut (Chr11:5249972) is still within the known region of interest as disruption of said region causes HPFH and, therefore, causes increased fetal hemoglobin, as taught by Camaschella and Forget recited above. Moreover, as recited above, the teachings of Wu for designing guide RNAs for a known target region would permit one of ordinary skill in the art to design an array of gRNAs such as SEQ ID NO: 139 which, similar to SEQ ID NO 4-6 and 15-20, can target and disrupt the region of interest for HPFH. Therefore, the SEQ ID NO: 139 of claim 1 of ‘305 patent is in fact obvious over the prior art of Camaschella, Forget, and Wu, as the genomic region to be disrupted to cause elevated fetal hemoglobin was known and the design of guide RNAs to achieve said disruption could be achieved by the teachings of Wu with routine optimization.
Claims 71, 75, 78-80, 86, 101-102 and 109-118 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 9, 19, 22, 32, 40, 49, 57 and 68 of U.S. Patent No. 11268077 of Chakraborty and in view of Takeuchi, Conway, Camaschella, Solovieff, Forget, and Wu (all previously cited). Although the claims at issue are not identical, they are not patentably distinct from each other for the following reasons. This rejection has been modified in consideration of now issued patents and applicant’s amendment.
Regarding claim 71, Chakraborty teaches a method for editing the β-globin locus of chromosome 11 wherein one or more DNA endonucleases and one or more guide RNAs are introduced into the human cell to increase the fetal hemoglobin (HbF) levels (claims 1-3, 19, 22, 32 and 68).
Chakraborty does not teach a method wherein S. pyogenes Cas9 DNA endonucleases and a pair of guide RNAs (gRNA) are electroporated into the cell, nor does it teach wherein the pair of RNA guides consists of a first and a second RNA guide (SEQ ID NO 4-6 for first guide RNA and SEQ ID NO: 15-20 for second guide RNA) and a pair of DSBs consists of a 5’ DSB locus and a 3’DSB locus wherein the RNA guides have complementary sequences to comprise a spacer sequence for 5’ or 3’ locus, respectively. Chakraborty also does not teach wherein the chromosomal DNA between the 5’ DSB and 3’ DSB locus comprises the boundary 5’ and 3’ regions of a Heredity Persistence of Fetal Hemoglobin (HPFH) deletion. However, the previously listed limitations are met by the combine teachings of Takeuchi, Conway, Camaschella, Solovieff, Forget, and Wu (all previously cited and detailed above).
It would have been obvious to modify as to combine the cited references to try the genome editing technique of Takeuchi to use the electroporation method of Conway to insert the Cas9 protein with gRNAs in the cells, the method of using one or more RNA guides with 5’ and 3’ complementary sequencing to the spacers that are the 5’ and 3’ boundaries for the mapped HPFH gene deletion taught Conway, Solovieff, Forget, and Camaschella. One of ordinary skills would have reasonable expectations for success because Conway taught the desire to electroporate Cas9 proteins with gRNA in cells with the knowledge of the targeted deletion region for HPFH for genome editing and the guide RNA sequences can be optimized to instead target the same known chromosome region using gRNAs such as SEQ ID NO 4-6 and 15-20 by the teachings of Wu.
Regarding claim 75, Chakraborty teaches an in vivo method wherein the hemoglobinopathy is selected from a group consisting of Sickle-cell Anemia and thalassemia (α, β, δ, ʏ and any combination thereof) (claim 57).
Regarding claim 101-102, Chakraborty teaches a method wherein the DNA endonucleases comprise one or more guide RNAs; a single molecule or combination (claims 2-3 and 9).
Regarding claims 109-113, Chakraborty teaches an ex vivo method for treating patients with hemoglobinopathies wherein induced pluripotent stem cells (iPSC) are differentiated to hematopoietic progenitor cells and implanted into patients using systemic administration (claim 40).
Regarding claims 114-118, Chakraborty teaches an ex vivo method for treating patients with hemoglobinopathies wherein GCSF patients are treated with Plerixafor wherein the hematopoietic progenitor cell is CD34+ implanted via systemic administration (claim 49).
Claims 71-72, 75, 78, 101 and 113 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 33, 78-79, 81-83, 86-88 and 92-93 of Patent No. US 12129471 B2 (patent ‘471- corresponding to US Application 15/550/954) and in view of Takeuchi, Conway, Solovieff, Camaschella, Forget, and Wu (all previously cited). Although the claims at issue are not identical, they are not patentably distinct from each other for the following reasons. This rejection has been modified in consideration of now issued patents and applicant’s amendmentRegarding claim 71, patent ‘471 teaches a method for gene editing wherein at least one Cas9 endonuclease and two guide RNAs are introduced into a human cell to target the double strand breaks (DSBs) at the first 5’ locus and second 3’ locus within the δβ-globin region of human chromosome 11, wherein the first gRNA comprises a a spacer sequence that hybridizes to the same target sequence of the δβ-globin region of human chromosome 11 as a nucleic acid having the nucleic acid sequence of any one of SEQ ID NO: 1-117 and 129-137, the second gRNA comprises a spacer sequence that hybridizes to the same target sequence of the δβ-globin region of human chromosome 11 as a nucleic acid having the nucleic acid sequence of any one of SEQ ID NO: 1-117 and 129-137.
Patent ‘471 does not teach a method wherein S. pyogenes Cas9 DNA endonucleases and a pair of gRNAs are electroporated into the cell, the intended effect to increase the expression of ʏ-globin with the deletions or inversions within the δβ-globin region of chromosome 11, nor does it teach the 5’ and 3’ boundary for the HPFH deletion and the pair of RNA guides consists of a first and a second RNA guide (SEQ ID NO 4-6 for first guide RNA and SEQ ID NO: 15-20 for second guide RNA). However, the teaching Takeuchi, Conway, Camaschella, Solovieff, Forget, and Wu teach those limitations as previously detailed above.
It would have been obvious to modify the cited references to combine the genome editing of S. pyogenes Cas9 DNA endonucleases taught by Takeuchi to include the electroporation method of the Cas9 protein and gRNAs taught by Conway to focus on the 5’ and 3’ boundary for the HPFH deletion to increase the expression of ʏ-globin with the deletions or inversions within the δβ-globin region of chromosome 11 and the guide RNA sequences can be optimized to instead target the same known chromosome region using gRNAs such as SEQ ID NO 4-6 and 15-20 by the teachings of Wu. One of ordinary skill would have been motivative to try this combination of methods with a reasonable expectation of success because of the evidence for support provided by the cited references.
Regarding claims 72 and 75, patent ‘471 teaches a method wherein the human cell is from a patient with β-hemoglobinopathy which is Sickle-cell disease or a β-thalassemia (claims 10).
Regarding claim 78, patent ‘471 teaches a method wherein there is a deletion of the chromosomal DNA between the pair of DSBs within the δβ-globin region of human chromosome 11 (claim 9).
Regarding claim 101, patent ‘471 teaches a method wherein the first gRNA and/or the second gRNA are single-molecule gRNAs (sgRNAs). (claim 5).
Regarding claim 113, patent ‘471 teaches a wherein the isolated progenitor cell is a hematopoietic progenitor cell. (claim 7).
Claims 71-72, 75 and 80 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 57, 62, 67, 72, 75, 77 of US 12043843 B2 (patent ‘843 corresponding to US Application 15/762700) in view of Takeuchi, Conway, Camaschella, Solovieff, Forget, and Wu (all previously cited). Although the claims at issue are not identical, they are not patentably distinct from each other for the following reasons. This rejection has been modified in consideration of now issued patents and applicant’s amendment
Regarding claim 71, patent ‘843 teaches a method comprising introducing the Cas9 endonuclease and the one or more gRNAs to the human cell to effect one or more double-strand breaks (DSBs) at one or more loci within the δβ-globin region of human chromosome 11, causing deletions or insertions of chromosomal DNA at the one or more loci, wherein one of the one or more gRNAs comprise a spacer sequence (claim 1).
Patent ‘843 does not teach a method wherein Cas9 protein and gRNAs are electroporated into the cell, the complementary 5’ and 3’ spacers for the first and second gRNAs nor does it teach the DNA boundary between the 5’ and 3’ locus of the HPFH deletion and the pair of RNA guides consists of a first and a second RNA guide (SEQ ID NO 4-6 for first guide RNA and SEQ ID NO: 15-20 for second guide RNA). However, the teachings of Takeuchi, Conway, Camaschella, Solovieff, Forget, and Wu are detailed above and are fully incorporated here.
It would have been obvious to modify the cited references to combine the genome editing of S. pyogenes Cas9 DNA endonucleases taught by Takeuchi to include the electroporation method of the Cas9 protein and gRNAs taught by Conway to focus on the 5’ and 3’ boundary for the HPFH deletion to increase the expression of ʏ-globin with the deletions or inversions within the δβ-globin region of chromosome 11 and the guide RNA sequences can be optimized to instead target the same known chromosome region using gRNAs such as SEQ ID NO 4-6 and 15-20 by the teachings of Wu. One of ordinary skill would have been motivative to try this combination of methods with a reasonable expectation of success because of the evidence for support provided by the cited references.
Regarding claim 72, patent ‘843 teaches a method wherein the human cell is derived from a patient having β-hemoglobinopathy (claim 10).
Conclusion
Claims 71-75, 78-80, 86, 90, 93, 96-98, 101, 102, 109-118 are rejected.
THIS ACTION IS MADE FINAL. 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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/JULIANA IRENE CANDELARIA/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634
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