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 .
Applicant’s amendments to the claims dated 12/11/25 are acknowledged. Claims 76-77, 82, 88-90, and 93-113 are pending and subject to prosecution. Claims 76-77, 90, 95 and 104 are amended. Claims 91-92 are cancelled. Claims 112-113 are new.
PRIORITY
The instant application, filed 12/13/2021, is a 371 National Stage Application of PCT/US2020/040756, filed 07/02/2020; which claims priority to US Provisional Application No. 63/009,385, filed 04/13/2020; US Provisional Application No. 62/935,507, filed 11/14/2019; and US Provisional Application No. 62/869,907, filed 07/02/2019. Thus, the earliest possible priority for the instant application is 7/02/2019.
STATUS OF PREVIOUS OBJECTIONS/REJECTIONS OF RECORD
The objection to the specification is WITHDRAWN in light of Applicant’s amendment to the specification dated 12/11/25.
The 112b rejection over claims 91-92 is WITHDRAWN in light of the cancellation of the claims.
The 102(a)(1) rejection of claims 76-77, 82, 88-95, 98, 103-104 and 107 as anticipated by Li 2018, as evidenced by Sandig and Wang is WITHDRAWN in light of Applicant’s amendments to the claims or cancellation of a rejected claim.
The 102(a)(1) rejection of claims 76-77, 82, 88-95 and 103-104 as anticipated by Li 2018 (Li-2), as evidenced by Wang is WITHDRAWN in light of Applicant’s amendments to the claims or cancellation of a rejected claim.
The 103 rejection of claims 98-99, 101-102, 107-108 and 110-111 as obvious over Li-2 as evidenced by Wang, Palmer, Breda and Li-3 is WITHDRAWN in light of Applicant’s amendments to the claims.
The 103 rejection of claims 100 ad 109 as obvious over Li-2 as evidenced by Wang, Palmer, Breda, Li-3 and Conway is WITHDRAWN in light of Applicant’s amendments to the claims.
The 103 rejection of claims 96-97 and 105-106 as obvious over Li-2 as evidenced by Wang, Palmer, Andreani, Durost, and Yotnda is WITHDRAWN in light of Applicant’s amendments to the claims.
RESPONSE TO ARGUMENTS
Applicant’s arguments with regard to a WITHDRAWN objection or rejection of record are moot. Applicant argues that the claims have been amended to delete the requirement of a CRISPR endonuclease, and thus require use of a base editor. Applicant argues that none of the cited art teaches or suggests use of a base editing enzyme. The Examiner agrees, and the rejections of record are withdrawn.
Applicant argues the present invention, drawn to the use of delivering a base editor targeted by gRNA via an HD-Ad vector, alleging the HD-Ad vectors of the cited prior art were associated with “potential” toxicities not demonstrated by the HD-Ad vectors of the present application (page 9 of the Reply). Applicant alleges use of the HD-Ad vectors of the present application also reduced off-target deletions by the expressed editing enzymes, compared to the double-strand break enzymes of the prior art. Applicant alleges the reduction in both the toxicities and off-targeted deletions were unexpected results.
Applicant’s allegation of unexpected results is not persuasive. In addition, when considering evidence of non-obviousness, “the applicant should establish a nexus between the rebuttal evidence and the claimed invention, i.e., objective evidence of nonobviousness must be attributable to the claimed invention. Additionally, the evidence must be reasonably commensurate in scope with the claimed invention. MPEP 2145. Any differences between the claimed invention and the prior art may be expected to result in some differences in properties. The issue is whether the properties differ to such an extent that the difference is really unexpected. MPEP 716.02. Further, the burden is on Applicant to establish that the evidence provided are unexpected and significant. The evidence relied upon should establish "that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance." MPEP 716.02(b).
It is noted that the claims do not require any specific HD-Ad virus, and thus the properties associated with an unclaimed specific virus are not required or read into the claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
In addition, the viruses of the specification are significantly narrower in scope than the claimed viruses. As such, the alleged evidence is not commensurate in scope with the claimed invention.
CLAIMS
Claims 76 and 77 are directed to a helper dependent adenoviral “donor” vector and/or helper dependent adenoviral “donor” genome. The specification does not define the narrowest definition of a “donor” vector or “donor” genome. The broadest reasonable interpretation of a helper dependent adenoviral “donor” genome comprises two adenoviral ITRs flanking a heterologous gene and a functional adenoviral packaging sequence, that is capable of being packaged, and a helper dependent adenoviral “donor” vector comprises the packaged “donor” genome. The “donor” aspect of the helper dependent adenoviral vectors/genomes appears to be the encoded heterologous gene, which is later expressed in a transduced cell. This appears to be consistent with the teachings of the specification, for example at paragraphs [0279]-[0281] and [0310].
PNG
media_image1.png
200
400
media_image1.png
Greyscale
The previous claims of record recited wherein the HD-Av encoded “a CRISPR-associated RNA-guided endonuclease and/or a base editor.” Claims 76 and 77 have been amended to delete the alternative embodiment comprising the CRISPR endonuclease, and thus newly require only the presence of a base editor.
The specification teaches a base editor is a base-editing enzyme (i.e., a cytosine deaminase or adenosine deaminase) fused to a DNA-binding domain (DBD) protein (i.e., a cas9 nickase or an inactivated cas9), wherein a guide RNA directs the fusion protein to a genomic location, and the base-editing enzyme converts a specific cytosine-guanine pair to a thymine-adenosine pair, or an adenosine-thymine pair to a guanine-cytosine pair (paragraphs [0440]-[0457]).
PNG
media_image2.png
50
501
media_image2.png
Greyscale
A non-limiting representation of the claimed helper-dependent adenoviral genome of claims 76 and 77 can be visualized as:
Claim Rejections - 35 USC § 103
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 76-77, 82, 88-90, 93-95, 103-104 and 112-113 are rejected under 35 U.S.C. 103 as being unpatentable over by Li et al. HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells. Molecular Therapy: Methods & Clinical Development, June 2018. 9: 390-401, “Li-2” of record, cited on Applicant’s IDS dated 12/13/2021 and as evidenced by Wang et al. In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46. Journal of Virology, 2008. 82(21):10567-10579, of record, cited on Applicant’s IDS dated 12/13/2021, taken in view of WO2019/079347 to Liu.
The publication date of Li-2 is June 28, 2018, which is greater than one year from the earliest priority date of the instant application, July 2, 2019, and is thus not eligible for a 102(b)(1) exception. MPEP § 717.02. The publication date of Liu is April 25, 2019, and is thus a 102(a)(1) and 102(a)(2) dated reference.
With regard to claims 76 and 77, Li-2 discloses helper-dependent adenoviral (HD-Ad) donor vectors comprising a donor genome comprising a 5’ and 3’ Ad ITR, an Ad packaging signal, a gene encoding a CRISPR enzyme (i.e., a donor), and an sgRNA (i.e., a donor) that targets a bcl11A enhancer nucleic acid sequence (HDAd-globin-CRISPR-1) or an sgRNA that targets the HBG1 promoter nucleic acid sequence (HDAd-globin-CRISPR-2) (Abstract, page 391, 397; FIG 1A). Li-2 shows the HD-Ad donor vectors are Ad5/35++ vectors comprising an Ad5 genome, devoid of all viral genes, packaged into viral particles with an Ad5/35++ helper virus that comprises an Ad35 fiber (page 390, second column). Thus, the Ad ITR and Ad packaging sequence in FIG. 1A of Li-2 are necessarily Ad5 sequences, absent evidence to the contrary. Thus, a non-limiting embodiment of Li-2 can be visualized accordingly:
PNG
media_image3.png
84
616
media_image3.png
Greyscale
Li-2 discloses advantages of using HD-Ad5/35++ vectors to deliver Cas9 including the ability to transduce HSCs, low cytotoxicity associated with the adenoviral gene transfer process, and large insert capacity (page 395). Li-2 notes that cytotoxicity in HSCs can also be caused by extended periods of Cas9 expression in the cell, and by the double-stranded breaks (DSBs) caused by Cas9 cleavage, and suggests reducing the number of off-target DSBs may further reduce cytotoxicity (page 395, last paragraph – page 396, first paragraph). Li-2 discloses the expression of cas9 and the sgRNA directed to HBG1 and/or BCL11a have the potential to treat β-thalassemia and sick cell disease by re-activation of fetal γ-globin (Abstract). However, the adenoviral vector encodes a cas9 endonuclease, but does not disclose wherein the adenoviral vector encode a base editor, as required by claims 76 and 77.
Liu discloses targeted gene editing systems comprising a base-editing enzyme fused to a programmable DNA-binding protein, and a guide RNA which targets the HBG1 and/or the HBG2 gene to genetically modify the fetal γ-globin gene (paragraphs [0002]-[0004], [0006]-[0007], [0014], [0036]-[0037], [0060], [0066], [0137], [0152], [0215]-[0216]). The base-editing enzymes are adenosine deaminases, which generate a single point mutation, converting adenosine-thymine pairs to guanine-cytosine pairs (paragraphs [0006], [0032], [0036], [0069]). The programmable DNA-binding protein comprises a Cas9 nickase or an inactivated dCAs9 (paragraphs [0006], [0008], [0036], [0040]-[0055], [0109]-[0133]]). Liu discloses when expressed in a cell, the guide RNA directs the base-editing enzyme-cas9 fusion protein to a target genomic sequence, such as the HBG1 and/or HBG2 promoter, generates a single-strand break, and converts a single adenosine base to a guanine, which results in the reactivation of fetal -γ-globin, and has the potential to treat β-thalassemia and sick cell disease (paragraphs [0007], [0153]-[0157], [0185]-[0193], [0216], [0224], [0301]). Liu discloses the targeted gene editing system is provided as nucleic acid molecule constructs including vectors (paragraphs [0014], [0067], [0238]-[0239]).
Liu discloses genomic editing comprising base-editing enzymes fused to DNA binding proteins generates direct, irreversible conversion of one base pair to another at a target genomic locus without requiring double-stranded DNA breaks, homology direct repair processes, and no donor sequence (paragraph [0246]). Liu discloses targeted base-editing cytosine deaminases fused to programmable DNA binding domains and guide RNA were already known, and have been shown generate point mutations with higher efficiency and fewer undesired indels or translocations compared to use of Cas9-mediated genomic engineering (paragraphs [0247]-[0248]). Liu discloses the targeted gene editing systems comprising base-editing adenosine deaminases and guide RNA generate point mutations with higher efficiency and fewer undesired indels compared to use of Cas9-mediated homology driven repair comprising guide RNA (paragraph [0274]-[0275], Examples 6, 7).
PNG
media_image4.png
103
330
media_image4.png
Greyscale
Thus, Liu discloses nucleic acids encoding base editors and guide RNA targeted to HBG1 and/or HBG2 accordingly:
It would have been obvious to substitute the nucleic acids encoding a cas9 endonuclease and guide RNA targeted to HBG1 and/or HBG2 in the adenoviral vector constructs of Li-2, with the nucleic acids encoding a base-editing enzyme fused to a DNA-binding cas9 protein and guide RNA targeted to HBG1 and/or HBG2 of Liu. A person of ordinary skill in the art would have had a reasonable expectation of success in substituting the nucleic acids encoding the targeted base-editor system of Liu for the Cas9 system of Li-2 because both explicitly taught as being useful for generating targeted genetic modifications in HBG1 and/or HBG2 genes to re-activate γ-globin. Therefore, these compositions are functional equivalents in the art, and substituting one for the other would have been obvious at the time of the invention. “When a patent ‘simply arranges old elements with each performing the same function it had been known to perform’ and yields no more than one would expect from such an arrangement, the combination is obvious.” See KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (U.S. 2007) at 1395-1396, quoting Sakraida v. AG Pro, Inc., 425 U.S. 273 (1976) and In re Fout, 675 F.2d 297, 301 (CCPA 1982) (“Express suggestion to substitute one equivalent for another need not be present to render such substitution obvious”). In addition, a skilled artisan would have been motivated to use the base-editing enzyme fused to a DNA-binding cas9 protein and guide RNA targeted to HBG1 and/or HBG2 of Liu in the HD-Ad virus vectors of Li-2 because the base-editing systems, which generate single-strand breaks, have been shown to generate point mutations with higher efficiency and fewer undesired indels compared to use of Cas9-mediated homology driven repair comprising guide RNA as shown by Liu, and Li-2 discloses HD-Ad vectors advantageously have large capacity, and DSBs cause cytotoxicity, and reducing DSBs in modified cells may be one way to reduce cytotoxicity.
With regard to claim 82, Li-2 discloses the viral particles infect CD34+ cells (FIG 2A-B, FIG 3A-G).
With regard to claims 88 and 103, Li-2 discloses the sgRNA targeted to the Bcl11A enhancer and the HBG1 promoter result in the upregulation of γ-globin using a Cas9 endonuclease (FIG 3D, FIG 5; FIG 7). However, Liu discloses the sgRNA targeted to the HBG1 and/or HBG2 promoter result in the upregulation of γ-globin using the base-editing enzyme fused to a DNA-binding cas9 protein and guide RNA targeted to HBG1 and/or HBG2 (Examples 6, 7). Thus, this claim is obvious for the same reasons as stated above for claims 76, 77.
With regard to claim 89, Li-2 discloses the Ad5/35++ vectors there comprise an Ad35 Fiber shaft and Ad35 knob (page 398, first column).
With regard to claims 90, and 112-113, Li-2 discloses all the Ad5/35++ vectors comprise an affinity enhanced Ad35++ fiber knob (page 398, first column) with mutations that increase affinity to CD46, citing to Wang (page 390, second column, Ref. 12 therein). Wang is cited to show the Ad35++ knob comprises Asp207Gly and Thr245Ala substitutions (page 10571), which reads on a Ad35++mutant fiber knob.
With regard to claims 93-94, Li-2 discloses Ad35/35++ vectors, which read on the claimed “Ad5/35 capsid” and “Ad5/35++ capsid”.
With regard to claims 95 and 104, Li-2 discloses the HDAd-globin-CRISPR-1 and HDAd-globin-CRISPR-2 encoding the Cas9 and sgRNA are non-integrating (page 390, second column). The non-integrating properties of the HD-Ad viral vector of Li-2 remain following the substitution of the base-editing enzyme fused to a DNA-binding cas9 protein and guide RNA targeted to HBG1 and/or HBG2 of Liu into the HD-Ad.
Claims 98-99, 101-102, 107-108 and 110-111 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells. Molecular Therapy: Methods & Clinical Development, June 2018. 9: 390-401, “Li-2” of record, cited on Applicant’s IDS dated 12/13/2021 and as evidenced by Wang et al. In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46. Journal of Virology, 2008. 82(21):10567-10579, of record, cited on Applicant’s IDS dated 12/13/2021, in view of WO2019/079347 to Liu as applied to claims 76-77, 82, 88-90, 93-95, 103-104 and 112-113 above, and further in view of Palmer et al. Production of CRISPR/Cas9-Mediated Self-cleaving Helper-Dependent Adenoviruses. Molecular Therapy: Methods & Clinical Development, June 2019. 13:432-439, of record, Breda et al. Combining Gene Therapy and Fetal Hemoglobin Induction for Treatment of Beta-thalassemia. Expert Reviews in Hematology, 2013. 6(3):255-264, of record; and Li et al, Integrating HDAd5/35++ Vectors as a New Platform for HSC Gene Therapy of Hemoglobinopathies. Molecular Therapy: Methods & Clinical Development, June 2018. 9: 142-153, and “Li-3” of record, cited on Applicant’s IDS dated 12/13/2021.
Claims 99 and 108 are directed to an embodiment wherein the helper-dependent adenoviral (HD-Ad) vector and genome encoding a base-editor fused to a DBD (i.e., a donor) and an sgRNA (i.e., a donor), targeted to the Bcl11a enhancer, HBG1 and/or HBG2 promoter, of claims 76 and 77, further comprises a nucleic acid encoding a β-globin protein or γ-globin protein comprising an integration element (i.e., a donor that is to be integrated into a recipient genome). A non-limiting visualization of
PNG
media_image5.png
95
550
media_image5.png
Greyscale
the HD-Ad vector of claims 99 and 108 includes:
The disclosures of Li-2, as evidenced by Wang, and Liu are applied as in the 103 rejection above, the content of which is incorporated herein in its entirety.
With regard to claims 76-77, Li-2 as evidenced by Wang, and take in view of Liu render obvious a HD-Ad vector and genome encoding a base-editor fused to a DBD (a genome editing enzyme) and an sgRNA, targeted to the bcl11a enhancer, HBG1 and/or HGB2 promoter. Li-2 and Liu disclose expression of targeted enzyme (cas9 in Li-2; base-editor fused to DBD in Liu) and the sgRNA result in the induction of endogenous γ-globin gene expression in transduced cells, and is useful to treat β-thalassemia (Abstract in Li-2; Abstract, paragraphs [0007], [0185]-[0193], [0301]). Li-2 discloses the ultimate goal of such systems includes the integration of transgenes using donor templates for HSC gene therapy (page 396 last paragraph, bridging page 397). Li-2 discloses advantages of using HD-Ad5/35++ vectors to deliver Cas9 including the ability to transduce HSCs, low cytotoxicity associated with the adenoviral gene transfer process, and large insert capacity (page 395). Thus, Li-2 as evidenced by Wang, and taken in view of Liu render obvious an HD-Ad vector accordingly:
PNG
media_image6.png
87
363
media_image6.png
Greyscale
However, Li-2 as evidenced by Wang, nor Liu disclose wherein the HD-Ad vector, encoding a bade-editor fused to a DBD and an sgRNA, further comprises a nucleic acid encoding a β-globin protein or a γ-globin protein comprising an integration element (i.e. a donor that is to be integrated into a target genome), as required by instant claims 99 and 108.
Palmer discloses an “all-in-one” HD-Ad vector encoding a CRISPR enzyme (a targeted genome editing enzyme), an sgRNA and a donor template, wherein the sgRNA targets the chromosome to increase the efficiency of homology-dependent repair by the donor template (page 432, FIG 1). Palmer discloses including the donor on the HD-Ad vector encoding the CRISPR enzyme and sgRNA reduces the need for multiple transduction vectors and/or steps, thus reducing costs and increasing efficiency (page 437, second column). Thus, Palmer discloses a non-limiting HD-Ad vector accordingly:
PNG
media_image7.png
60
615
media_image7.png
Greyscale
Palmer discloses HD-Ad vectors are excellent for gene- and cell-therapy applications because they mediate high-efficiency transduction of many cell types, both in vivo and in vitro, regardless of cell cycle, have an enormous cloning capacity of 36 Kb, do not integrate into the genome, and provide long-term expression with reduced toxicity (page 432).
Breda discloses there are two major therapeutic strategies for treating β-thalassemia includes gene therapy and the induction of fetal hemoglobin (Abstract). Breda discloses gene therapy methods include β-globin or γ-globin transgene delivery from viral vectors (page 256, 259; Table 1). The methods of inducing fetal hemoglobin include the reactivation of the γ-globin gene, by directly activating the γ-globin locus, or by inhibiting factors that repress γ-globin synthesis, such as by inhibiting bcl11a, using small molecules or delivering gene constructs encoding the activators/inhibitors to cells (page 258-259; Table 1).
Breda discloses the effect of combining gene therapy to express a β-globin or γ-globin transgene with the induction of γ-globin gene expression appears to be additive. The combination not only increases both β-globin and γ-globin to concentrations sufficient to result in therapeutic hemoglobin, but may reduce excessive α-globin expression and/or concentration (page 260). Thus, Breda discloses the combination of methods of treating β-thalassemia include:
PNG
media_image8.png
90
431
media_image8.png
Greyscale
Li-3 discloses helper-dependent Ad5/35++ vectors encoding a heterologous γ-globin gene (i.e., a donor) for HSC gene therapy with potential to treat hemoglobinopathies (Abstract, pages 142, 143). Li-3 discloses the heterologous γ-globin gene is flanked by integration elements (i.e. a donor that is to be integrated into a target genome) (Abstract, page 145, FIG 3). Li-3 discloses that the integration of the γ-globin gene is flanked by transposon ITRs (page 143, Fig 1). Thus, Li-3 discloses a non-limiting HD-Ad vector accordingly:
PNG
media_image9.png
64
614
media_image9.png
Greyscale
It would have been obvious to combine the HD-Ad vector of Li-2 as evidenced by Wang, and taken in view of Liu, useful to treat β-thalassemia, encoding a base-editor fused to a DBD protein (a genome editing enzyme) and sgRNA targeted to increase expression from an endogenous γ-globin gene, further with the disclosures of Palmer, Breda and Li-3. A skilled artisan would have been motivated to include the integrating donor transgene encoding γ-globin of Li-3 on the HD-Ad vector of Li-2 as evidenced by Wang, and taken in view of Liu because: 1) Li-2 discloses the systems are ultimately designed for gene therapy insertion of donors; 2) Palmer discloses placing a donor on the same HD-Ad vector encoding cas9 (a genome editing enzyme) and sgRNA reduces the need for multiple transduction vectors and/or steps, thus reducing costs and increasing efficiency; and 3) Breda teaches combining gene therapy methods of β-globin or γ-globin transgenes with methods of inducing endogenous γ-globin in β-thalassemia patients has additive benefits not seen by the methods individually. A skilled artisan would have had a reasonable expectation of success in practicing the claimed invention, as HD-Ad vectors encoding genome editing enzymes and sgRNA to induce γ-globin expression were known and/or obvious, HD-Ad vectors encoding integrating globin donor transgenes were known, all-in-one HD-Ad vectors encoding genome editing enzymes, sgRNA and donors were all known, and the large cloning capacity of HD-Ad were known at the time of the invention.
With regard to claims 101-102, 110 and 111, Li-3 discloses the heterologous γ-globin gene is flanked by integration elements, wherein the transposon elements are transposon ITRs, and wherein the transposon ITRs are flanked by FRT recombinase repeats (FIG 1). Thus, these claims are obvious for the same reasons as stated above for claims 99 and 108.
With regard to claims 98 and 107, none of Li-2, Wang, Liu, Palmer or Breda disclose the use of a selection cassette on the HD-Ad. However, Li-3 discloses the integrating γ-globin cassette further comprises a selection cassette comprising an mgmtP140K gene, which allows for the drug-controlled increase of γ-globin expressing cells (Abstract, page 143, FIG 1). Li-3 discloses use of the selection cassette maximizes the production of γ-globin expressing erythroid cells (page 143, 144; FIG 2A-2G). It would have been obvious to include the selection cassette of Li-3 on the HD-Ad vector of Li-2, as evidenced by Wang taken in view of Liu, in order to increase the production of γ-globin expressing cells.
Claims 100 and 109 are rejected under 35 U.S.C. 103 as being unpatentable over Li-2 as evidenced by Wang, in view of Liu, Palmer, Breda and Li-3, as applied to claims 76-77, 82, 88-90, 93-95, 98-99, 101-104, 107-108 and 110-113 above, further in view of US Patent Application No. 2015/0056705 to Conway, of record.
Claims 100 and 109 are directed to an embodiment wherein the HD-Ad vector and genome encoding a genome editing enzyme – a base-editor fused to a DBD protein (i.e., a donor) and an sgRNA (i.e., a donor), targeted to the bcl11a enhancer, HBG1 and/or HBG2 promoter, further comprises a nucleic acid encoding a β-globin protein or γ-globin protein and an integration element, wherein the integration element is engineered for targeted integration into a genome, and wherein the integration element is flanked by homology arms that correspond to contiguously linked sequences of the target genome.
Li-2, as evidenced by Wang, taken in view of Liu, Palmer, Breda and Li-3 combine to render obvious HD-Ad vector and genome encoding a genome editing enzyme (a base-editor enzyme fused to a DBD protein from a cas9 protein), an sgRNA targeted to the bcl11a enhancer, HBG1 and/or HBG2 promoter, wherein the HD-Ad vector further comprises a nucleic acid encoding a β-globin protein or γ-globin protein comprising an integration element according to claims 99 and 108, as iterated in the 103 rejection above, the content of which is incorporated herein in its entirety. Li-3 discloses the integration element comprises transposon ITRs, wherein the integration of the globin transgene cassette occurs via random integration (FIG 1, Abstract, page 143). Li-3 discloses future modifications to the HD-Ad vector include targeted integration of the globin transgene into a safe harbor site (page 148, second column, citing to reference 39 therein). Thus, the cited art combines to render obvious a HD-Ad vector accordingly:
PNG
media_image5.png
95
550
media_image5.png
Greyscale
However, none of Li-2, as evidenced by Wang, Palmer, Breda or Li-3 disclose wherein the integration cassette encoding the β-globin protein or γ-globin protein and an integration element further comprises homology arms that correspond to contiguously linked sequences of the target genome, wherein the integration element is engineered for targeted integration into a genome, as required by instant claims 100 and 109.
Conway discloses compositions and methods of using CRISPR/cas and gRNA systems for the genetic alteration of cells treat hemoglobinopathies, including β-thalassemia and/or sickle cell anemia, wherein the CRISPR system modifies target genomes via non-homologous end joining or homology-directed repair utilizing an optional donor transgene (Abstract, paragraphs [0015]-[0016], [0022]-[0024], [0028], [0041]-[0050], [0099]-[0105], [0131], [0144]-[0148], [0175]-[0176], [0181]-[0185], [0229]-[0231], Table 1, Table 2). Conway discloses embodiments wherein the Cas9 protein is a cas9 nickase or an inactivated Cas9, and fused to a heterologous functional domain in order to generate targeted DNA-binding proteins (paragraphs [0027], [0158]-[0173]).
PNG
media_image10.png
94
393
media_image10.png
Greyscale
Conway discloses the methods include expressing sgRNA targeted to the promoter/enhancer domain of bcl11a to decrease bcl11a expression, thus increasing γ-globin (paragraphs [0048]-[0049], Table 1). Conway discloses the methods include expressing sgRNA targeted to regulatory domains of HGB1 to increase γ-globin (paragraph [0050], Table 1). Conway discloses the methods include the targeted integration of a γ-globin or a β-globin transgene, targeted to a safe harbor gene, wherein the transgene is flanked on the 5’ and 3’ ends with a homology arm that correspond to contiguously linked sequences in the target genome (paragraphs [0030], [0056], [0185]). Thus, Conway discloses the methods of genetically modifying cells to treat beta-thalassemia comprise providing a heterologous functional domain fused to a Cas9 DNA-binding domain, an sgRNA and an integration cassette accordingly:
56. Conway discloses the nucleotides encoding the CRISPR enzyme, sgRNA and the donor transgene can be encoded on the same vector, including HD-Ad vectors (paragraphs [0190], [0196],[0198], [0202]-[0203]).
It would have been obvious to substitute the integration cassette encoding a globin gene, flanked by transposon ITRS in the method of Li-2, as evidenced by Wang, in view of Liu, Palmer, Breda and Li-3 with the integration cassette encoding a globin gene flanked by homology arms of Conway, because both cassettes are explicitly taught as being useful for integrating a globin transgene into a genome. Therefore, these compositions are functional equivalents in the art, and substituting one for the other would have been obvious at the time of the invention. “When a patent ‘simply arranges old elements with each performing the same function it had been known to perform’ and yields no more than one would expect from such an arrangement, the combination is obvious.” See KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (U.S. 2007) at 1395-1396, quoting Sakraida v. AG Pro, Inc., 425 U.S. 273 (1976) and In re Fout, 675 F.2d 297, 301 (CCPA 1982) (“Express suggestion to substitute one equivalent for another need not be present to render such substitution obvious”). A skilled artisan would have been motivated to substitute the integration cassette for random integration of Li-2, as evidenced by Wang, Liu, Palmer, Breda and Li-3 for the integration cassette for targeted integration of Conway because Li-3 explicitly suggests further modifications include targeted integration of the globin transgene into a safe harbor site. A skilled artisan would have had a reasonable expectation of success in practicing the claimed invention as targeted integration of a transgene encoding a globin gene using homology arms was known at the time of the invention.
Claims 96-97 and 105-106 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells. Molecular Therapy: Methods & Clinical Development, June 2018. 9: 390-401, “Li-2” of record, cited on Applicant’s IDS dated 12/13/2021 and as evidenced by Wang et al. In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46. Journal of Virology, 2008. 82(21):10567-10579, of record, cited on Applicant’s IDS dated 12/13/2021, in view of WO2019/079347 to Liu as applied to claims 76-77, 82, 88-90, 93-95, 103-104 and 112-113 above, and further in view of Palmer et al. Production of CRISPR/Cas9-Mediated Self-cleaving Helper-Dependent Adenoviruses. Molecular Therapy: Methods & Clinical Development, June 2019. 13:432-439 of record, Andreani et al. Mixed Chimerism in Haemoglobinopathies: From Risk of Graft Rejection to Immune Tolerance. Tissue Antigens, 2014. 83(3):137-146 of record, Durost et al. Gene Therapy with an Adeno-Associated Viral Vector Expressing Human Interleukin-2 Alters Immune System Homeostasis in Humanized Mice. Human Gene Therapy, 2018. 29(3):352-365 of record; and Yotnda et al. Efficient Infection of Primitive Hematopoietic Stem Cells By Modified Adenovirus. Gene Therapy, 2001. 8:930-937 of record.
PNG
media_image11.png
93
550
media_image11.png
Greyscale
Claims 96-97 and 105-106 are directed to an embodiment wherein the HD-Ad vector and genome encoding a genome editing enzyme comprising a base-editor fused to a DBD (i.e., a donor) and an sgRNA (i.e., a donor), targeted to the bcl11a enhancer, HBG1 and/or HBG2 promoter, further comprises a nucleic acid encoding the cytokine IL-2. A non-limiting visualization of the HD-Ad vector includes:
The disclosures of Li-2, as evidenced by Wang, and Liu are applied as in the 103 rejection above, the content of which is incorporated herein in its entirety.
With regard to claims 76-77, Li-2 as evidenced by Wang, and taken in view of Liu render obvious a HD-Ad vector and genome encoding a base-editor fused to a DBD (a genome editing enzyme) and an sgRNA, targeted to the bcl11a enhancer, HBG1 and/or HGB2 promoter. Thus, Li-2 as evidenced by Wang, and taken in view of Liu render obvious an HD-Ad vector accordingly:
PNG
media_image6.png
87
363
media_image6.png
Greyscale
Li-2 transduces isolated human CD34+ HSC cells with the HD-Ad vector to increase the expression of γ-globin from the HSCs (FIG 2A-B, FIG 3A-G, FIG 5; FIG 7). NSG mice are subjected to an HSC transplant comprising the transduced human CD34+ HSC, and the engrafted human cells express γ-globin (FIG 4-5). Li-2 discloses the HD-Ad vectors and CD34+ human HSCs modified to increase expression of γ-globin have potential use for treating β-thalassemia following HSC transplantation (Abstract, page 391). Li-2 also discloses the ultimate goal of such systems includes the integration of transgenes using donor templates for HSC gene therapy (page 396 last paragraph, bridging page 397).
However, none of Li-2, Wang or Liu disclose wherein the virus further comprises a nucleic acid encoding IL-2, as required by instant claims 96-97 and 105-106.
Palmer discloses an “all-in-one” HD-Ad vector encoding a CRISPR enzyme (a targeted genome editing enzyme), an sgRNA and a donor template, wherein the sgRNA targets the chromosome to increase the efficiency of homology-dependent repair by the donor template (page 432, FIG 1). Palmer discloses including the donor on the HD-Ad vector encoding the CRISPR enzyme and sgRNA reduces the need for multiple transduction vectors and/or steps, thus reducing costs and increasing efficiency (page 437, second column). Thus, Palmer discloses a non-limiting HD-Ad vector accordingly:
PNG
media_image7.png
60
615
media_image7.png
Greyscale
Palmer discloses HD-Ad vectors are excellent for gene- and cell-therapy applications because they mediate high-efficiency transduction of many cell types, both in vivo and in vitro, regardless of cell cycle, have an enormous cloning capacity of 36 Kb, do not integrate into the genome, and provide long-term expression with reduced toxicity (page 432).
Andreani discloses treatment of β-thalassemia includes hematopoietic stem cell (HSC) transplant, wherein a recipient, pretreated to remove endogenous HSCs (with reduced or absent β-globin), is administered donor HSCs (genetically modified, or allogenic, that encode wild-type β-globin) which engraft, expand, differentiate and express β-globin at therapeutic levels (Abstract, pages 139, 142). Andreani discloses successful therapy can result even with the persistence of mixed chimerism (wherein donor cells and recipient cells co-exist) in the recipient, when donor cells survive longer than 2 years after HSC transplantation and wherein the recipient does not require RBC transfusions (Abstract, pages 140, 142).
However, Andreani discloses mixed chimerism, often the result following donor HSC transplant, can also cause graft-vs-host disease and donor graft rejection (Abstract, pages 137, 139). A simplified visualization of donor graft rejection in β-thalassemia patients exhibiting mixed chimerism following donor HSC transplant is provided below:
PNG
media_image12.png
348
953
media_image12.png
Greyscale
Andreani discloses donor graft survival, in the presence of mixed chimerism, can be increased by inducing donor-specific immune tolerance via infusing expanded regulatory T cells into the recipient, preventing GVHD (Pages 142-143). A simplified visualization of donor graft survival in β-thalassemia patients exhibiting mixed chimerism following donor HSC transplant and induced tolerance, wherein the induced tolerance results from increasing Treg concentrations in the patient, is provided below:
PNG
media_image13.png
380
959
media_image13.png
Greyscale
Durost discloses humanized mice, comprising immunodeficient mice with engrafted human CD34+ HSC, generate functional human immune systems, including the generation of human effector and regulatory T cells, and human innate NK immune cells, making these humanized mice candidates for studying the effect of gene therapy treatment of human diseases in vivo or the effect of a gene therapy treatment on specific human immune cell populations (abstract, page 352, 361-0362). Durost discloses low-dose IL-2 therapy has been shown to increase functional FOXP3+CD4+ Tregs in both human patients and mouse models, a population of Tregs essential to T cell tolerance (page 353; page 362, second column). Such increases in functional FOXP3+CD4+ Tregs induces T cell tolerance between host and donor cell transplants, and has been used to reduce autoimmune disease symptoms and progression, reduce transplant rejection, and increase transplant engraftment, in both human patients and mouse models (page 353, first column; page 362, second column, page 363). The presence of host and donor cells co-existing reads on mixed chimerism, as discussed by Andreani above.
A simplified visualization of donor HSC graft survival in HSC recipients exhibiting mixed chimerism following donor HSC transplant and induced tolerance, wherein the induced tolerance results from increasing Treg concentrations following administration of a viral vector encoding IL-2, in the recipient is provided below:
PNG
media_image14.png
416
954
media_image14.png
Greyscale
Durost notes delivery of IL-2 to the recipient includes administering of IL-2 peptides, plasmids encoding IL-2 and AAV viruses encoding IL-2 to patients or animals (page 353, first column, page 362). Thus, Durost establishes an rAAV vector encoding IL-2 accordingly:
PNG
media_image15.png
83
588
media_image15.png
Greyscale
Yotnda discloses Ad5/35 adenoviral vectors encoding an interleukin-2 transgene that is capable of transducing CD34+ hematopoietic stem cells (Abstract, page 933-934; FIG 5,6). Yotnda shows transduced CD334+ HSC secrete therapeutic levels of IL-2 (page 933, FIG 5).
It would have been obvious to combine the HD-Ad vector encoding a base-editing enzyme and sgRNa, used to transduce human CD34+ HSCs, wherein the transduced human CD34+ HSC have upregulated γ-globin expression of Li-2 as evidenced by Wang and Liu further with the disclosures of Palmer, Andreani, Durost and Yotnda. A skilled artisan would have been motivated to include an IL-2 donor transgene of Yotnda on the HD-Ad vector of Li-2 as evidenced by Wang and Liu because 1) Li-2 discloses the transduced human CD34+ HSC cells have potential use for treating β-thalassemia following HSC transplantation, and the vector CRISPR systems will ultimately include transgenes using donor templates for HSC gene therapy; 2) Palmer discloses placing a donor on the same HD-Ad encoding a genome editing enzyme and sgRNA reduces the need for multiple transduction vectors and/or steps, thus reducing costs and increasing efficiency; 3) Andreani discloses successful β-thalassemia treatments include transplant and engraftment of allogenic donor CD34+ HSC; and discloses rejection of donor grafts can be prevented by increasing Tregs in the recipient; and 4) Durost discloses methods of increasing Tregs in CD34+ HSC transplant recipients includes administering a viral vector encoding IL-2 providing low-dose IL-2 to the recipient. A skilled artisan would have had a reasonable expectation of success in practicing the claimed invention because HD-Ad vectors encoding genome editing enzymes and sgRNA, viral vectors encoding IL-2, combining CRISPR systems with donor transgenes, and the role of CRISPR/cas9, sgRNA, and IL-2 in HSC transplant survival of β-thalassemia patients, or HSC transplant patients, and the large cloning capacity of HD-Ad vectors were all known at the time of the invention.
Conclusion
No claims are allowed.
FINAL REJECTION
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KIMBERLY A ARON whose telephone number is (571)272-2789. The examiner can normally be reached Monday-Friday 9AM-5PM.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Christopher Babic can be reached at 571-272-8507. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/CHRISTOPHER M BABIC/Supervisory Patent Examiner, Art Unit 1633