DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Acknowledgment is made of applicant’s claim for priority based on a foreign application filed as EP21156461.2 on 02/11/2021.
All claims are given the priority date of 02/11/2021.
Application Status
Receipt is acknowledged of amendment, filed 0/08/2023. Claims 1-19 are currently pending.
Information Disclosure Statement
Receipt of acknowledgment of the information disclosure statement filed on 08/08/2023 have been received and all references have been considered.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claims 3, 11, 12 and 18 are objected to because of the following informalities:
Claim 3 comprises the abbreviation of “CRISPR”. All first instances of abbreviations must comprise the spelled-out term with the abbreviation in parenthesis next to it. For example: “Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease complex”.
Claim 11 recites “a sequence having SEQ ID NO: 7” as well as claim 12 recites “a sequence having SEQ ID NO: 7” and “a sequence having SEQ ID NO: 3”. This is improper grammar of the claim and should be amended to recite “a guide RNA having the sequence of SEQ ID NO: 7” and “a guide RNA having the sequence of SEQ ID NO: 3”, respectively.
Claim 18 recites “A method for the treatment of prevention of a haemoglobinopathy”. It seems the claim should recite “A method for the treatment or prevention of a haemoglobinopathy”.
Appropriate correction is required.
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 18 and 19 are 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 a method for the treatment of a haemoglobinopathy, the method comprising administering a first and a second site-specific endonuclease, or one or more vectors encoding the same in a subject in need thereof wherein the first and second site-specific endonuclease can generate a first and a second double stranded break in a chromosome, thereby generating a first, an intervening second and a third chromosomal fragment, wherein i) the first fragment comprises a beta globin locus control region (LCR), or a functional fragment thereof; and ii) the third fragment comprises a sequence encoding HBG1 and/or HBG2; wherein the length of the second intervening fragment is at least 6 kb, and wherein joining the first fragment to the third fragment results in operably linking the beta globin LCR to the sequence encoding HBG1 and/or HBG2, thereby increasing HBG1 and/or HBG2 protein expression, does not reasonably provide enablement for method for the treatment of and/or prevention of a haemoglobinopathy, the method comprising administering a first and a second site-specific endonuclease, or one or more vectors encoding the same in a subject in need thereof wherein the first and second site-specific endonuclease can generate a first and a second double stranded break in a chromosome, thereby generating a first, an intervening second and a third chromosomal fragment, wherein i) the first fragment comprises a beta globin locus control region (LCR), or a functional fragment thereof; and ii) the third fragment comprises a sequence encoding HBG1 and/or HBG2; wherein the length of the second intervening fragment is at least 6 kb, and wherein joining the first fragment to the third fragment results in operably linking the beta globin LCR to the sequence encoding HBG1 and/or HBG2, thereby increasing HBG1 and/or HBG2 protein expression. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(A)). These include: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and the quantity of experimentation needed to make or use the invention. All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature of the invention: The claims are drawn to a method for the treatment and/or prevention of a haemoglobinopathy comprising administering a first and second site-specific endonuclease, or one or more vectors encoding the same in a subject in need thereof wherein the first and second site-specific endonuclease can generate a first and second double stranded break in a chromosome, thereby generating a first, an intervening second and a third chromosomal fragment. The nature of the invention is complex in that the function of prevention of a haemoglobinopathy is not specified or described.
Breadth of the claims: The claims broadly encompass a method for treatment and/or prevention of a haemoglobinopathy comprising administering a first and second site-specific endonuclease, or one or more vectors encoding the same in a subject in need thereof wherein the first and second site-specific endonuclease can generate a first and second double stranded break in a chromosome, thereby generating a first, an intervening second and a third chromosomal fragment wherein the first fragment comprises a beta globin locus control region (LCR), or a fragment thereof and the third fragment comprises a sequence encoding HBG1 and/or HBG2; wherein the length of the second intervening fragment is at least 6kb, and wherein joining the first and third fragment results in operably linking the beta globin LCR to the sequence encoding HBG1 and/or HBG2, thereby increasing HBG1 and/or HBG2 protein expression. Dependent claim 19 specifies that the haemoglobinopathy is either sickle cell disease or β-thalassemia. The complex nature of the subject matter of this invention is greatly exacerbated by the breadth of the claims.
Guidance of the specification and existence of working examples: The specification teaches the definition of “prevent”, “preventing” and “prevention” as the prevention or reduction of the recurrence, onset, development or progression of a disease, preferably a haemoglobinopathy, preferably a haemoglobinopathy as defined herein, or the prevention or reduction of the severity and/or duration of the haemoglobinopathy or one or more symptoms thereof (Page 17, Lines 13-17).
The working examples are only predictive of the efficacy of in vivo and ex vivo treatment of sickle cell disease and β-thalassemia, however the working examples do not teach prevention of any haemoglobinopathy by the present invention.
Working example one teaches how genomic distance affects the functionality of the beta globin LCR to enhance gene expression by using a GFP reporter gene driven by the HBG1/2 promoter, and a micro-LCR to determine that transcriptional output increases with decreased enhancer distance before and after hemin-induced maturation of K562 cells (human erythroleukemia cells) (Page 20, Lines 3-12). Working example one continues to teach that altered the distance between a fetal globin gene and the LCR, and we observed that (1) expression levels increase with decreased linear distance between the LCR and close linear juxtaposition of the endogenous LCR to the fetal globin genes, accomplished through genetic deletion of intervening sequences, reactivates the fetal globin genes, e.g. to levels that may cure SCD and thal (Page 20, Lines 26-31).
Working example two teaches several guide RNA combinations were evaluated for their ability to enable Cas9-mediated DNA simultaneous cutting near or inside an HBG promoter and near or inside HS2 of the LCR in K562 cells that would result in the deletion of the intervening chromosomal segment and thereby the immediate juxtaposition of the HS2-HS5 of the LCR to an HBG gene promoter (Page 20, Lines 34-40). Working example two continues to teach that guide RNA no. 3 enables Cas9-mediated cutting near a HBG promoter and guide RNA no. 7 enables Cas9-mediated cutting inside HS2 of the LCR, simultaneously (Page 21, Lines 9-15).
Predictability and state of the art: For some relevant background, Yao et al (Mol Ther. 2024 Dec 4;32(12):4167-4169) teaches the therapeutic “editing” of disease-causing mutations in HSCs in vivo, without the need for extracting and reintroducing HSCs back into the patient, has the potential to overcome many of the limitations of ex vivo therapy approaches, however current in vivo prevention in humans using gene editing for humans is not proven successful as well as challenging (Page 4167, Column 1).
Kabrah (J Blood Med. 2025 Nov 6;16: 493-507) teaches the emerging gene therapies have offered transformative potential by directly targeting the genetic basis of SCD (Page 494, Paragraph 2). Kabrah teaches that further research in this direction, including the maturation of in vivo editing technologies and comparative cost-effectiveness studies, is needed, and intensive follow-up of the results in patients on a post-marketing basis is necessary (Page 505, Paragraph 5). Kabrah teaches if translated safely into human applications, in vivo strategies could represent a paradigm shift, enabling minimally invasive, one-time curative interventions for SCD (Page 505, Paragraph 3).
Amount of experimentation necessary: In order to practice the claimed invention, an immense amount of experimentation would be required. As disclosed above, the specification itself provides the method of treatment of a haemoglobinopathy. No description is provided of how the method would be used and/or capable of prevention of a haemoglobinopathy within a human individual. Therefore, experiment could be conducted, but in view of the specification there does not appear to be any amount of experimentation that would be sufficient to reliably produce the exact product of the invention. Such experimentation would not be possible due to not having the steps or requirements of the method to be used for prevention of a haemoglobinopathy (such as sickle cell disease or β-thalassemia) within a human individual. Therefore, it would require immense amount of unpredictable experimentation to practice the claimed invention with such variants in the possible result.
In view of the breadth of the claims and the lack of guidance provided by the specification as well as the unpredictability of the art, the skilled artisan would have required an undue amount of experimentation to make and/or use the claimed invention. Therefore, claims 18 and 19 are not considered to be fully enabled by the instant disclosure.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2, 3, 7, 8 and 9 are 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.
Claim 3 contains the trademark/trade name “TALEN”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a transcription activator-like effector nuclease and, accordingly, the identification/description is indefinite.
Claims 2, 7 and 8 are vague and indefinite in that the metes and bounds of the phrase “preferably” are unclear. The phrase is unclear in that it makes it unknown and unclear in what limitations are actually required for the claim and thus, the claimed invention. It would be remedial to replace the phrase “preferably” with“optionally” or removing the phrase “preferably”.
Claim 9 is vague and indefinite in that the metes and bounds of the phrase “at least about” are unclear. The phrase is unclear in that it makes it unknown and unclear in what limitations are actually required for the claim and thus, the claimed invention. It would be remedial to replace the phrase “at least about” with “at least” or removing the phrase “at least about” entirely.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-5, 8, 9 and 16-19 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al (Blood (2018) 131 (26): 2915–2928; Visual Abstract Attached 1/1-1/1) in view of Antoniani et al (Blood. 2018 Apr 26;131(17):1960-1973; Supp. Material pages 1/41-41/41).
Regarding claims 1 and 9, Li teaches increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells (Page 2924, Column 1 bridging Column 2). Li teaches the first fragment comprises an beta globin locus control region (LCR) and the third fragment comprises a sequence encoding HBG1; wherein the first double stranded break is downstream of the LCR, wherein the second double-stranded break is located upstream of the HBG1 transcription start site, wherein the length of the deletion fragment is 4.9 kb, and wherein joining the first fragment to the third fragment results in decreasing the genomic distance and thereby increasing gamma globin 1 expression (Page 2925, Figure 7; and Visual Abstract, attached below article).
Li does not teach a deletion larger than 6 kb which would lead to operably linking the LCR to the HBG1 gene.
Antoniani teaches a deletion resulting in a juxtaposition of the LCR to the HBG1 gene results in the increased expression of the gamma globin 1 gene due to proximity of the gene to enhancer elements (Page 1960, Column 2). Therefore, removing the 34 kb portion between the 5’ HS1 LCR and the HBG1 would result in the juxtaposition of the LCR to the HBG1 gene to increase expression due to proximity to the enhancer elements.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Li to include the larger than 6kb deletion as taught by Antoniani because Li teaches it is within the ordinary skill in the art to use increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells and Antoniani teaches using a deletion to bring the LCR into a juxtaposition with the HBG1 gene results in increased expression due to the proximity of the gene to enhancer elements.
One would have been motivated to make such a modification in order to receive the expected benefit of increased expression of HBG1 due to close proximity to the LCR as taught by Antoniani.
Regarding claims 2-4, Li teaches increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells (Page 2924, Column 1 bridging Column 2).
Regarding claim 5, Li teaches the wild-type distance is 9.9kb therefore the deletion of 4.9kb sequence results in the remainder of 5kb between the LCR and HBG1 regions; therefore, the decrease in genomic distance is roughly 50% between the LCR and HBG1 (Page 2925, Figure 7D and Figure 7 description).
Regarding claim 8, Li teaches the second double strand base is directly prior to the HBG1 gene therefor it would fall within the range of -1 to 1,000 bp prior to the transcriptional start site (Page 2925, Figure 7A).
Regarding claims 16 and 17, Li teaches ex vivo methods of harvesting J3-YAC/CD46 Lin-cells and the transduction of the J3-YAC/CD46 Lin - cells with HDAd-HBGCRISPR (Page 2926, Column 1 and Page 2916, Figure 1). Li teaches increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells (Page 2924, Column 1 bridging Column 2). Li teaches the first fragment comprises an beta globin locus control region (LCR) and the third fragment comprises a sequence encoding HBG1; wherein the first double stranded break is downstream of the LCR, wherein the second double-stranded break is located upstream of the HBG1 transcription start site, wherein the length of the deletion fragment is 4.9 kb, and wherein joining the first fragment to the third fragment results in decreasing the genomic distance and thereby increasing gamma globin 1 expression (Page 2925, Figure 7; and Visual Abstract, attached below article).
Li does not teach a deletion larger than 6 kb which would lead to operably linking the LCR to the HBG1 gene.
Antoniani teaches a deletion resulting in a juxtaposition of the LCR to the HBG1 gene results in the increased expression of the gamma globin 1 gene due to proximity of the gene to enhancer elements (Page 1960, Column 2). Therefore, removing the 34 kb portion between the 5’ HS1 LCR and the HBG1 would result in the juxtaposition of the LCR to the HBG1 gene to increase expression due to proximity to the enhancer elements.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Li to include the larger than 6kb deletion as taught by Antoniani because Li teaches it is within the ordinary skill in the art to use increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells and Antoniani teaches using a deletion to bring the LCR into a juxtaposition with the HBG1 gene results in increased expression due to the proximity of the gene to enhancer elements.
One would have been motivated to make such a modification in order to receive the expected benefit of increased expression of HBG1 due to close proximity to the LCR as taught by Antoniani.
Regarding claims 18 and 19, Li teaches the treatment of sickle cell disease [SCD] and β-thalassemia (Page 2915, Column 1). Li teaches nonintegrating HDAd5/35++ vector for HSPC
genome editing in vitro and in vivo with the goal to reactivate human -y-globin expression in red blood cells (RBCs) in f3-YAC mice (Page 2918, Column 1). Li teaches increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells (Page 2924, Column 1 bridging Column 2). Li teaches the first fragment comprises an beta globin locus control region (LCR) and the third fragment comprises a sequence encoding HBG1; wherein the first double stranded break is downstream of the LCR, wherein the second double-stranded break is located upstream of the HBG1 transcription start site, wherein the length of the deletion fragment is 4.9 kb, and wherein joining the first fragment to the third fragment results in decreasing the genomic distance and thereby increasing gamma globin 1 expression (Page 2925, Figure 7; and Visual Abstract, attached below article).
Li does not teach a deletion larger than 6 kb which would lead to operably linking the LCR to the HBG1 gene.
Antoniani teaches a deletion resulting in a juxtaposition of the LCR to the HBG1 gene results in the increased expression of the gamma globin 1 gene due to proximity of the gene to enhancer elements (Page 1960, Column 2). Therefore, removing the 34 kb portion between the 5’ HS1 LCR and the HBG1 would result in the juxtaposition of the LCR to the HBG1 gene to increase expression due to proximity to the enhancer elements.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Li to include the larger than 6kb deletion as taught by Antoniani because Li teaches it is within the ordinary skill in the art to use increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells and Antoniani teaches using a deletion to bring the LCR into a juxtaposition with the HBG1 gene results in increased expression due to the proximity of the gene to enhancer elements.
One would have been motivated to make such a modification in order to receive the expected benefit of increased expression of HBG1 due to close proximity to the LCR as taught by Antoniani.
Claims 6 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al (Blood (2018) 131 (26): 2915–2928; Visual Abstract Attached 1/1-1/1) in view of Antoniani et al (Blood. 2018 Apr 26;131(17):1960-1973; Supp. Material pages 1/41-41/41), as applied to claims 1-5, 8, 9 and 16-19 above, and further in view of Koenig et al (Am J Hematol. 2009 Sep;84(9):603-6).
The teachings of Li and Antoniani are described above and applied as before.
Regarding claims 6 and 7, Li and Antoniani do not teach wherein the first fragment comprises LCR DNAse I hypersensitivity sites HS5, HS4, HS3, HS2 and HS1 and wherein the first double-stranded break is located less than 300 bp from HS1 and wherein the first double stranded break is located in the LCR, preferably wherein the first double stranded break is located within HS1, HS2 or HS3, or in between: i) LCR DNAse I hypersensitivity sites HS1 and HS2; ii) LCR DNAse I hypersensitivity sites HS2 and HS3; or iii) LCR DNAse I hypersensitivity sites HS3 and HS4; preferably wherein the first double stranded break is located in between HS1 and HS2.
Koenig teaches the single deletion of the 5’ HS1 resulted in minimal to no effects on the beta-like globin genes, however, the study confirmed that the 5’ HS2 and 5’ HS3 portions of the LCR were primarily responsible for conferring erythroid-specific high-level expression of cis-linked β-like globin genes (Page 603, Abstract and Page 604, Column 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Li and Antoniani to include the deletion within the LCR as taught by Koenig because Li teaches it is within the ordinary skill in the art to use increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells, Antoniani teaches using a deletion to bring the LCR into a juxtaposition with the HBG1 gene results in increased expression due to the proximity of the gene to enhancer elements and Koenig teaches the removal of the 5’HS1 portion of the LCR as well as the 5’ HS2 and 5’ HS3 portions of the LCR were primarily responsible for conferring erythroid-specific high-level expression of cis-linked β-like globin genes.
One would have been motivated to make such a modification in order to receive the expected benefit of bringing the most essential and dominant enhancer (5’ HS2/3) into juxtaposition with the HBG1 gene as taught by Antoniani and Koenig.
Claims 10-12 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al (Blood (2018) 131 (26): 2915–2928; Visual Abstract Attached 1/1-1/1) in view of Antoniani et al (Blood. 2018 Apr 26;131(17):1960-1973; Supp. Material pages 1/41-41/41), as applied to claims 1-5, 8, 9 and 16-19 above, and further in view of Porteus et al (WO 2017 /077394 A2).
The teachings of Li and Antoniani are described above and applied as before.
Li and Antoniani do not teach specifically, wherein the first CRISPR- nuclease complex comprises a guide RNA comprising a sequence having SEQ ID NO: 7 and the second CRISPR-nuclease complex comprises a guide RNA comprising a sequence having SEQ ID NO: 3.
Porteus teaches materials and methods for targeting portions of the HBB locus on chromosome 11 for treating sickle cell and β-thalassemia [0023]. Porteus teaches gRNAs for targeting within the HBB locus wherein SEQ ID NO: 11,988 is 100% identical to instant SEQ ID NO: 3 and SEQ ID NO: 42,352 100% identical to instant SEQ ID NO: 7 and these gRNAs are used for the purpose of recruiting the Cas9 proteins for targeted double strand cuts within chromosome 11 in the HBB locus ([0061]; See Appendix I and II).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the gRNAs taught in Li and Antoniani for the gRNAs as taught by Porteus because Li teaches it is within the ordinary skill in the art to use increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 systems (including two gRNAs) to generate two cuts on chromosome 11 for a 4.9 kb deletion within HUDEP-2 cells and CD34+ cells, Antoniani teaches using a deletion to bring the LCR into a juxtaposition with the HBG1 gene results in increased expression due to the proximity of the gene to enhancer elements and Porteus teaches gRNAs for targeting within the HBB locus wherein SEQ ID NO: 11,988 is 100% identical to instant SEQ ID NO: 3 and SEQ ID NO: 42,352 100% identical to instant SEQ ID NO: 7 and these gRNAs are used for the purpose of recruiting the Cas9 proteins for targeted double strand cuts within chromosome 11 in the HBB locus.
One would have been motivated to make such a modification in order to receive the expected benefit of the specific gRNAs for targeting sequences within the HBB locus for Cas9 recruitment as taught by Porteus.
Conclusion
No claims are allowed.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/CELINE X QIAN/Primary Examiner, Art Unit 1637
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