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 .
This action is in response to the amendment filed 07/17/2026, in which claims 1-3, 7-9, 11, 12 and 18 were amended and claims 4-6, 10, 16, 17 and 19 were previously presented. Claims 1-12 and 16-19 are currently pending.
Applicant’s arguments have been thoroughly reviewed, but are not persuasive for the
reasons that follow. Any rejection and objections not reiterated in this action have been
withdrawn. This action is FINAL.
Specification
The substitute specification filed 07/17/2026 has not been entered because it does not conform to 37 CFR 1.125(b) and (c) because: it does not include a statement of no new matter.
Therefore, the previous objection to the specification mailed in the previous Office Action on 04/20/2026 is maintained.
Response to Amendments - Claim Objections
The previous objection of claims 3, 11, 12 and 18 for minor informalities has been withdrawn in view of Applicant’s amendments to the claims filed on 07/17/2026.
Response to Amendments - Claim Rejections - 35 USC § 112
The previous rejection of claims 18 and 19 under 35 U.S.C. 112(a) has been withdrawn in view of Applicant’s amendments to the claims filed on 07/17/2026.
The previous rejection of claims 2, 3, 7, 8 and 9 under 35 U.S.C. 112(b) has been withdrawn in view of Applicant’s amendments to the claims filed on 07/17/2026.
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 complexes (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 resulted in 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 resulting 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 resulting 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.
Regarding claims 10-12, the claim is interpreted that “a sequence” means two or more consecutive nucleotides and not the entirety of the sequence.
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.
Response to Arguments - Claim Rejections - 35 USC § 103
The previous rejection of claims 1-5, 8, 9 and 16-19 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) has been maintained in view of Applicant’s arguments filed on 07/17/2026. Applicant’s arguments have been fully considered but have not been found to be persuasive.
Applicant argues Li does not teach using two gRNAs but instead using a single guide RNA targeting the BCL11A binding motif in the HBG promoter region. Applicant continues to argue that the primary mechanism of action is the disruption of BCL11A binding via small indels, thus, because the targeted sequence is present in both the HBG1 and HBG2 promoters, this single sgRNA cuts at both locations simultaneously, and a 4.9kb deletion can arise as an incidental byproduct. Applicant argues Li detects this deletion at only “approximately 2%” frequently in clinically relevant CD34+ cells and explicitly flags it as a concern for clinical translation, stating “the fact that this large deletion occurs should be considered in the potential clinical translation of the approach because it is not known whether the intergenic HBG2-HBG1 region bears critical functional elements”. Applicant argues Li’s primary teaching is therefore BCL11A binding disruption via small indels, not creation of the LCR-HBG proximity via a deliberate deletion and Li therefore provides no teaching or motivation to deliberately create a deletion of at least 6kn for the purpose of operably linking the LCR to HBG1/HBG2.
However, Li teaches increasing expression of gamma globin 1 (HBG1) by using two CRISPR-Cas9 complexes (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). Although Li describes the 4.9 kb deletion as an incidental byproduct, the current claim language does not specify that the deletion cannot be an incidental byproduct. The claim requires a broad method of deletion using two site-specific endonucleases that provide cuts directed by sgRNAs resulting in three fragments. Applicant points to the deletion at only “approximately 2%” frequency in clinically relevant CD34+ cells and that this explicitly flags it as a concern for clinical translation, however, that is only one cell type experimented on within Li. Li also teaches detected -20% deletions in HUDEP-2 cells transduced with HDAd-HBG-CRISPR (Page 2924, Column 2). Li is not specifically relied on for it teaching the LCR-HBG proximity but rather that the deletion would result in the closer proximity of the LCR to the HBG1 gene. It should also be noted that Li teaches HPLC analyses showed that the ratio of-y-globin to adult a- and 13-globin
protein was increased compared with untransduced cells, indicating a partial switch from adult globin to fetal -y-globin, confirming that the HDAd-HBG-CRISPR vector is functional (Page 2920, Column 1).
Applicant argues that Antoniani targets “a 13.6-kb genomic region encompassing the δ- and β-globin genes are located downstream of the HBG1 on chromosome 11. The genomic order is, whereby Antoniani’s deletion is underlined: 5’-LCR-HBG2(Gγ)-HBG1(Aγ)-HBD(ϭ)-HBB(β)-3’ which could be compared to Fig, 2B of the as-filed application. Applicant continues to argue that Antoniani’s deletion therefore does not reduce the distance between the LCR and HBG1. This confirmed by Antoniani’s description of the mechanism in the Discussion: “A factor contributing to HbF reactivation […] may be the juxtaposition of the potent 3’ β-globin enhancer to the γ-globin genes”, i.e. not the LCR. Applicant argues the additional contributing mechanisms are removal of a putative HbF silencer and inactivation of the adult β-globin gene. Applicant continues to argue Antoniani therefore does not teach or suggest deleting a fragment between the LCR and the HBG2/1 and instead Antoniani points towards a fully different downstream deletion strategy. Applicant argues that hence the combination of Li and Antoniani fails to teach or render obvious each and every element of independent claim 1.
However, Antoniani is only relied on for the teachings that a deletion resulting in a juxtaposition of the LCR to the HBG1 gene resulted in 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. Therefore, the combination of the teachings of Li that recite a deletion between the LCR and HBG1 that would result in the two loci being brought closer in proximity and Antoniani teaching that juxtaposition of the LCR to the HBG1 gene resulted in increased expression of the gamma globin 1 gene due to proximity of the gene to enhancer elements would be obvious to one of ordinary skill in the art.
The previous rejection of claims 6 and 7 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) has been maintained in view of Applicant’s arguments filed on 07/17/2026.
The previous rejection of claims 10-12 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) has been maintained in view of Applicant’s arguments filed on 07/17/2026.
Applicant argues that neither Koenig nor Porteus remedy the deficiencies of Li and Antoniani as set forth above and therefore the rejections should be withdrawn.
However, and as stated above, the rejection of the claims over Li and Antoniani has been maintained and therefore, the rejections of the claims further in view of Koenig or Porteus is therefore maintained as teaching the current limitations of the instant claims.
Conclusion
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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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/CELINE X QIAN/Primary Examiner, Art Unit 1637
