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 .
Election/Restrictions
Applicant’s election without traverse of Group I (Claims 1-6, 8, 10-20, and 38-40; drawn to a hematopoietic stem or progenitor cell (HSPC) genetically modified to express a receptor conferring a selective proliferation advantage on the genetically modified HSPC on introduction into a subject relative to endogenous HSPCs) in the reply filed on August 2, 2024, is acknowledged.
Claims 21-37 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention (Group II), there being no allowable generic or linking claim.
DETAILED ACTION
The amended claims filed on December 3, 2025, have been acknowledged. Claims 1-15 and 38-41 were cancelled. Claims 16, 43, and 47 were amended. Claim 48 is new. Claims 21-37 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention (Group II), there being no allowable generic or linking claim. Claims 16-20 and 42-48 are pending and examined on the merits.
Information Disclosure Statement
The information disclosure statement (IDS) filed on December 3, 2025, has been considered.
Claim Rejections - 35 USC § 112
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 16-20 and 42-48 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. This is a new rejection made in response to Applicant’s amendments that is substantially similar to a previous rejection. Any aspect of Applicant’s traversal that is relevant to the rejection as newly written is addressed below.
The term “at least 95% sequence identity for the human wildtype form” of CD47/c-Kit in claims 16, 42-43, and 48 is a relative term which renders the claim indefinite. The term “at least 95% sequence identity for the wildtype form” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Applicant does not identify a specific sequence that corresponds to the wildtype form. Although Applicant identifies specific Swiss Prot accession numbers, this is not a controlling definition for the wild type sequence. Furthermore, regarding CD47, UniProt (CD47 Human) evidences that wildtype human CD47 has four isoforms (page 11) and Kaur et al. (Atlas Genet Cytogenet Oncol Haematol. 25: 83–102. 2021) evidences that there six isoforms (page 2). Additionally, UniProt and Kaur cite different sequences for the “canonical” wild type sequence with UniProt (page 11) citing Q08722-1 as the canonical sequence while Kaur identifies Homo sapiens CD47 molecule (CD47), transcript variant 2 (page 2 of Kaur and whole document of NCBI (Homo sapiens CD47 molecule (CD47), transcript variant 2, mRNA) cited by Kaur) (corresponds to Q08722-3 of UniProt). Therefore, it is not clear which isoform and resulting sequence is considered the wild type sequence for CD47. Regarding c-Kit, UniProt identifies at least 3 and up to 10 isoforms (pages 13-14) and NCBI (KIT proto-oncogene, receptor tyrosine kinase) evidences that there are eight isoforms (pages 7-9). Additionally, UniProt identifies a different “canonical” sequence (P10721-1) (page 13) (corresponds to isoform 1 identified by NCBI (KIT proto-oncogene, receptor tyrosine kinase)) than NCBI (KIT proto-oncogene, receptor tyrosine kinase) which compares each of the isoforms to isoform 3 (pages 7-9). Therefore, it is not clear which isoform and resulting sequence is considered the wild type sequence for c-Kit. As such, there are multiple sequences that could be considered the “canonical” wildtype sequence for determining the 95% sequence identity and no defined wildtype reference sequence.
Response to Arguments
Applicant's arguments filed December 3, 2025, are acknowledged.
Applicant argues that although specific sequences for CD47 and c-Kit are not provided in the specification, the specification indeed provides Swiss Prot access numbers for CD47 and c-Kit: "Examples of suitable receptors are CD47 (e.g., Swiss Prot Q08722), c-Kit (e.g., Swiss Prot P10721)..." (see paragraph [0043] of the published application). In addition, paragraph [0043] of the published application further discloses that "Reference to such receptors should be understood as referring to the human forms, such as those of the accession numbers provided."
Thus, Applicant contends that the specification indeed particularly points out the relevant CD47 and c-Kit sequences that are to serve as references and the relevant species from which they are derived.
Further, although, there are many isoforms of CD47 and c-Kit, UniProt readily identifies one canonical sequence for each of CD47 and c-Kit. Specifically, per the UniProt database, the canonical sequence of CD47 is Q08722-1 and the canonical sequence of c-Kit is P10721-1.
Thus, in view of the above disclosure and arguments, one of ordinary skill in the art would recognize that the term "at least 95% sequence identity for the human wildtype form" as recited in the claims refers to the canonical sequence of the human wildtype form of CD47 or c-Kit (page 9, paragraph 1-page 10, paragraph 5).
Applicant's arguments and cited references have been fully considered but they are not persuasive.
As stated in the rejection above, although Applicant identifies specific Swiss Prot accession numbers, this is not a controlling definition for the wild type sequence as these are identified as examples only and not as the definitive wild type sequences. Furthermore, as identified above, these accession numbers cite multiple isoforms for each gene and there are conflicting opinions regarding what is considered the “canonical” wildtype sequence.
Withdrawn Claim Rejections - 35 USC § 103
The prior rejection of claims 16-20, 41-42, and 44-45 under 35 U.S.C. 103 as being unpatentable over Jahn et al. (Oncogene 21: 4508-4520. 2002) and Pino-Barrio et al. (bioRxiv. 1-41. July 2018), as evidenced by Bougherara et al. (Mol Cancer Res 7:1525-1533. 2009), Lennartson et al. (Physiol Rev 92. 1619-1649. 2012), and Qiu et al. (The EMBO Journal 7: 1003-1011. 1988) is withdrawn in light of Applicant’s amendments to claim 16 to recite that the mutant form shows at least 95% sequence identity for the human wildtype form of c-Kit.
New Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 16-17, 42, and 44-45 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Zhao et al. (Blood 119: 1511-1521. 2012), as evidenced by Bougherara et al. (Mol Cancer Res 7:1525-1533. 2009), Lennartson et al. (Physiol Rev 92. 1619-1649. 2012), Bougherara et al. (Clinical Lymphoma, Myeloma & Leukemia 13:62-69. 2013), and Qiu et al. (The EMBO Journal 7: 1003-1011. 1988). This is a new rejection made in response to Applicant’s amendments to claim 16. Applicant’s traversal has been fully considered but is moot in response to the new rejection.
Regarding claims 16(ii), 42, and 44-45, Zhao teaches that they transduced bone marrow cells (including hematopoietic stem and progenitor cells) with vectors carrying c-Kit with a D816V or D816Y mutation which led to increased proliferation (abstract, page 1512, column 1, paragraphs 3-4, page 1513, column 1, paragraph 3- column 2, paragraph 2, and Figure 1).
Bougherara 2009 evidences that that the D816V and D816Y mutations are in the catalytic (intracellular) domain (Figure 1). Lennartsson evidences that stem cell factor binds to the first three Ig-like domains of the extracellular domain (Figure 2). Regarding the interpretation of “retains binding to stem cell factor”, this is broadly interpreted to include any mutant receptor that has the capability to bind to stem cell factor and not to just mutant receptors localized to the cell membrane that bind to stem cell factor. As the D816V and D816Y mutations are in the intracellular catalytic domain and not in any of the three Ig-like domains that bind stem cell factor, D816V and D816Y mutant c-Kit receptors would still retain binding to stem cell factor. As such, HSPCs expressing mutant c-Kit receptors with the D816V and D816Y mutations have a proliferative advantage over the wild type receptor and retain binding to stem cell factor. Furthermore, Bougherara 2009 evidences that 5.4% of cells expressing the mutant c-Kit express the receptor on the cell surface (Figure 3) where it would bind to stem cell factor as well. Bougherara 2013 evidences that this intracellular localization is consistent across multiple cell lines and primary cells, including the hematopoietic murine B cell progenitor BaF/3 cells (page 63, column 1, paragraph 2-column 2, paragraph 3, page 64, column 2, paragraph 2-page 68, column 2, paragraph 1, and Figures 1-4). Therefore, the c-Kit mutants of Zhao would retain to capability to bind to stem cell factor, as evidenced by Lennartsson.
Regarding the limitation “wherein the c-Kit in mutant form has reduced binding to an antibody relative to a wildtype form of the c-Kit receptor, and wherein the c-Kit in mutant form shows at least 95% sequence identity for the wildtype form”, Bougherara 2009 evidences that D816V mutants exhibited increased intracellular localization (Figure 4). Bougherara 2013 evidences that this intracellular localization is consistent across multiple cell lines and primary cells, including the hematopoietic murine B cell progenitor BaF/3 cells (page 63, column 1, paragraph 2-column 2, paragraph 3, page 64, column 2, paragraph 2-page 68, column 2, paragraph 1, and Figures 1-4). Thus, the D816V leads to an increase in internalization of the c-Kit receptor compared to the wild type receptor which would make these cells more resistant to depletion by an immunotherapeutic agent relative to the HSPC before modification.
Lennartsson evidences that the c-Kit protein consists of 976 amino acids (page 1623, column 1, paragraph 3). As such the single amino acid mutation of D816V and D816Y mutant c-Kit receptors represents a ~0.1% change in the amino acid sequence (i.e. ~99.9% sequence identity).
Zhao teaches that they used retroviruses to stably transduce the cells and express the mutant receptors (abstract, page 1512, column 1, paragraphs 3-4, page 1513, column 1, paragraph 3- column 2, paragraph 2, and Figure 1).
Regarding claim 17, Zhao teaches that they used MSCV retroviral vectors to transduce the bone marrow cells. MSCV vectors contain a promoter (a regulatory sequence) to drive expression of the transgene. Regarding the signal peptide, Qiu evidences that c-Kit comprises an N-terminal signal peptide (abstract). Signal peptides are known to direct secretion of receptors for incorporation into the cell membrane. As stated supra, Bougherara 2009 evidences that 5.4% of cells expressing the mutant c-Kit express the receptor on the cell surface (Figure 3). Bougherara 2013 evidences that there are some D816V receptors that are expressed on the cell surface across multiple cell lines and primary cells, including the hematopoietic murine B cell progenitor BaF/3 cells (Figures 1-4).
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 16-20 are rejected under 35 U.S.C. 103 as being unpatentable over Zhao et al. (Blood 119: 1511-1521. 2012) as applied to claim 16 above and further in view of Pino-Barrio et al. (bioRxiv. 1-41. July 2018), as evidenced by Bougherara et al. (Mol Cancer Res 7:1525-1533. 2009), Lennartson et al. (Physiol Rev 92. 1619-1649. 2012), Bougherara et al. (Clinical Lymphoma, Myeloma & Leukemia 13:62-69. 2013), and Qiu et al. (The EMBO Journal 7: 1003-1011. 1988). This is a new rejection made in response to Applicant’s amendments to claim 16. Applicant’s traversal has been fully considered but is moot in response to the new rejection.
Regarding claim 18, the teachings of Zhao are as discussed above. As stated supra, Zhao teaches that they used a retrovirus for transducing and expressing the mutant receptor in the cells.
Zhao does not teach wherein they used homologous recombination at the endogenous gene to incorporate the mutant receptor into the genome.
However, Pino-Barrio teaches a method of genetically modifying murine HPCs with TALENs and a donor expression vector to the Mbs85 locus (ortholog of the hAAVS1 safe harbor locus) for efficient gene targeting. Their results demonstrate the feasibility of implementing a targeted integration strategy in a murine safe harbor locus, such as the Mbs85 gene, of murine HPCs (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the retroviral based insertion of the D816V c-Kit with the targeted integration strategy using homologous recombination at a murine safe harbor locus of Pino-Barrio to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute the insertion methods with a reasonable expectation of success because Pino-Barrio successfully reduces to practice that a method of genetically modifying murine HPCs with TALENs and a donor expression vector to the Mbs85 locus (ortholog of the hAAVS1 safe harbor locus) is capable of efficient gene targeting. Their results demonstrate the feasibility of implementing a targeted integration strategy in a murine safe harbor locus, such as the Mbs85 gene, of murine HPCs. Furthermore, retroviral vectors are known to undergo random insertion which can cause negative mutations that disrupt expression of proteins whereas targeted insertion at a safe harbor site avoids this issue. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 19, as stated above, Pino-Barrio teaches that TALENs (a nuclease) is nucleofected with the donor sequence for cleaving the DNA to stimulate homologous recombination. The therapeutic donor DNA includes homology arms on either side of the construct for homologous recombination to occur (Figure 1A and 1B).
Regarding claim 20, Pino-Barrio teaches that the TALEN monomers are nucleofected in expression plasmids (page 4, paragraph 1).
Regarding claim 17, Pino-Barrio teaches that the donor expression vector comprises a PGK promoter (a regulatory sequence) in front of the transgene (Figure 4, page 40), which would have been an obvious promoter to include because of its ability to drive constitutive and robust expression in HPCs. Regarding the signal peptide, Qiu evidences that c-Kit comprises an N-terminal signal peptide (abstract). Signal peptides are known to direct secretion of receptors for incorporation into the cell membrane. As stated supra, Bougherara evidences that 5.4% of cells expressing the mutant c-Kit express the receptor on the cell surface (Figure 3). Bougherara 2013 evidences that there are some D816V receptors that are expressed on the cell surface across multiple cell lines and primary cells, including the hematopoietic murine B cell progenitor BaF/3 cells (Figures 1-4).
Claim 48 is rejected under 35 U.S.C. 103 as being unpatentable over Zhao et al. (Blood 119: 1511-1521. 2012) and Sugase et al. (Gastric Cancer 21: 968-976. April 2018), as evidenced by Bougherara et al. (Mol Cancer Res 7:1525-1533. 2009), Lennartson et al. (Physiol Rev 92. 1619-1649. 2012), and Bougherara et al. (Clinical Lymphoma, Myeloma & Leukemia 13:62-69. 2013). This is a new rejection for new claim 48.
Zhao teaches that they transduced bone marrow cells (including hematopoietic stem and progenitor cells) with vectors carrying c-Kit with a D816V or D816Y mutation which led to increased proliferation (abstract, page 1512, column 1, paragraphs 3-4, page 1513, column 1, paragraph 3- column 2, paragraph 2, and Figure 1). Zhao teaches that these mutant cells led to leukemia in most of the experimental mice (abstract).
Bougherara 2009 evidences that that the D816V and D816Y mutations are in the catalytic (intracellular) domain (Figure 1). Lennartsson evidences that stem cell factor binds to the first three Ig-like domains of the extracellular domain (Figure 2). Regarding the interpretation of “retains binding to stem cell factor”, this is broadly interpreted to include any mutant receptor that has the capability to bind to stem cell factor and not to just mutant receptors localized to the cell membrane that bind to stem cell factor. As the D816V and D816Y mutations are in the intracellular catalytic domain and not in any of the three Ig-like domains that bind stem cell factor, D816V and D816Y mutant c-Kit receptors would still retain binding to stem cell factor. As such, HSPCs expressing mutant c-Kit receptors with the D816V and D816Y mutations have a proliferative advantage over the wild type receptor and retain binding to stem cell factor.
Furthermore, Bougherara 2009 evidences that 5.4% of cells expressing the mutant c-Kit express the receptor on the cell surface (Figure 3) where it would bind to stem cell factor as well. Bougherara 2013 evidences that this intracellular localization is consistent across multiple cell lines and primary cells, including the hematopoietic murine B cell progenitor BaF/3 cells (page 63, column 1, paragraph 2-column 2, paragraph 3, page 64, column 2, paragraph 2-page 68, column 2, paragraph 1, and Figures 1-4). Therefore, the c-Kit mutants of Zhao would retain to capability to bind to stem cell factor, as evidenced by Lennartsson.
Regarding the limitation “wherein the c-Kit in mutant form has reduced binding to an antibody relative to a wildtype form of the c-Kit receptor, and wherein the c-Kit in mutant form shows at least 95% sequence identity for the wildtype form”, Bougherara 2009 evidences that D816V mutants exhibited increased intracellular localization (Figure 4). Bougherara 2013 evidences that this intracellular localization is consistent across multiple cell lines and primary cells, including the hematopoietic murine B cell progenitor BaF/3 cells (page 63, column 1, paragraph 2-column 2, paragraph 3, page 64, column 2, paragraph 2-page 68, column 2, paragraph 1, and Figures 1-4). Thus, the D816V leads to an increase in internalization of the c-Kit receptor compared to the wild type receptor which would make these cells more resistant to depletion by an immunotherapeutic agent relative to the HSPC before modification.
Lennartsson evidences that the c-Kit protein consists of 976 amino acids (page 1623, column 1, paragraph 3). As such the single amino acid mutation of D816V and D816Y mutant c-Kit receptors represents a ~0.1% change in the amino acid sequence (i.e. ~99.9% sequence identity).
Zhao teaches that they used retroviruses to stably transduce the cells and express the mutant receptors (abstract, page 1512, column 1, paragraphs 3-4, page 1513, column 1, paragraph 3- column 2, paragraph 2, and Figure 1).
Zhao does not teach further modifying the HSPCs to express of functional human protein as a result of which the HSPCs can alleviate a genetic disorder.
However, Sugase teaches that most of the gastrointestinal stromal tumors (GIST) have mutations in the KIT gene. Imatinib, a receptor tyrosine kinase inhibitor, is the first-line therapy for unresectable and metastatic GISTs. Despite the revolutionary effects of imatinib, some patients are primarily resistant to imatinib and many become resistant because of acquisition of secondary mutations in KIT. This study investigated the antitumor effects of SOCS1 gene therapy, which targets several signaling pathways. AdSOCS1 significantly decreased the proliferation and induced apoptosis (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the D816V mutant expressing hematopoietic cells by expressing the SOCS1 human gene in these cells, as identified by Sugase, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Zhao and Sugase are focused on treating cancers caused by c-Kit mutations (i.e. a genetic disorder caused by gene mutations) and Sugase has successfully reduced to practice that genetic modifications of cancer cells expressing KIT mutations to overexpress SOCS1 using AdSOCS1 significantly decreased the proliferation and induced apoptosis in these cells. Therefore, it would be obvious to further modify the cells of Zhao to determine whether overexpressing SOCS1 improves treatment efficacy in their leukemia model. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 16-20, 43-44, and 46-47 are rejected under 35 U.S.C. 103 as being unpatentable over Tena et al. (American Journal of Transplantation 14: 2713-2722. 2014), in view of United States Patent Application No. 20180155717 (Valamehr) as evidenced by Zhang et al. (Front. Immunol. 15: 1-11. 2024). This is a new rejection that is substantially similar to a previous rejection. Applicant’s traversal has been fully considered but is moot in response to the new rejection.
Regarding claims 16(i), 43-44, and 46, Tena teaches the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages (i.e. HSPC cells) (abstract). Transgenic porcine cells were generated through genetic Knock-in of human CD47 under an EF1-alpha promoter to produce hCD47 Knock-in pigs (page 2716, column 2, paragraph 2-page 2717, column 1, paragraph 1). The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable chimerism levels were observed in the peripheral blood of these recipients. In contrast, transplantation of WT progenitor cells resulted in little or no bone marrow engraftment and no detectable peripheral chimerism. These results demonstrate a substantial protective effect of hCD47 expression on engraftment and persistence of porcine cells in this model, presumably by modulation of macrophage phagocytosis (abstract). As such, the hCD47 HSPCs show increased proliferation compared to the endogenous HSPCs (see also Table 1 demonstrating increased recovery of HSPCs).
Regarding the limitation “wherein the CD47 in mutant form has reduced binding to an antibody relative to a wildtype form of the CD47 receptor, and wherein the CD47 in mutant form shows at least 95% sequence identity for the wildtype form, as stated in the 112b rejection above, there is no defined wild type sequence. Therefore, any wild type sequence can be used to determine the 95% sequence identity. Tena teaches that they used the human CD47 coding sequence of splice form 1 of the hCD47 gene. Zhang evidences that isoform 1 has 12, 19, 30, and 8 fewer amino acids than isoforms 2-5 (Table 1 and Figure 1). Therefore, the splice (also known as isoform) 1 human CD47 used by Tena would represent a mutant form compared to isoforms 2-5 of the wildtype human CD47 and would have at least 95% sequence identity with isoforms 2 (12 amino acid difference) and 5 (8 amino acid difference). Furthermore, as there is a difference in the sequence between isoform 1 and isoforms 2 and 5 of 12 and 8 amino acids, respectively, an antibody that binds to these missing regions would have reduced binding to the mutant isoform 1 used by Tena, leading to a selective proliferative advantage by resisting depletion from an immunotherapeutic agent targeting those missing regions.
Tena teaches that they transplanted their genetically modified cells into immunodeficient and irradiated mice and were able to generate cell engraftment (abstract and page 2716, column 2, paragraphs 2-3).
Tena does not teach wherein the HSPC is genetically modified by introducing into the HSPC a construct that is incorporated into the genome of the HSPC.
However, Valamehr discloses a method of modifying the genome of a an HSPC and wherein the genome-engineered cell comprises introduced expression of at least one protein such as CD47 (paragraph 0034). Valamehr discloses that the introduced expression is obtained via integrated methods for introducing exogenous protein encoding polypeptides (paragraph 0034). Furthermore, Valamehr discloses a construct comprising: (1) one or more polynucleotides of interest operatively linked to one or more promoters comprised in the construct; and (2) a pair of homology arms specific to the selected site and flanking the exogenous polynucleotides of interest for targeted integration via homologous recombination (paragraphs 0008, 0150, and 0164). Finally, Valamehr discloses that the targeted editing of the genome can be achieved through a nuclease dependent approach (paragraphs 0150, 0151 and 0165).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the method of producing transgenic HSPCs by making a transgenic pig that expresses hCD47 and purifying transgenic HSPCs of Tena with the method of genetically modifying HSPCs through nuclease mediated homologous recombination directly in the HSPCs identified by Valamehr to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because Tena teaches that their cloning method produced a single healthy piglet and one that dies after 1 week and involved a multitude of cloning steps (page 2716, column 1, paragraph 3-page 2717, column 1, paragraph 1). On the other hand, directly modifying HSPCs through the method of Valamehr requires significantly less work, in comparison, as this can be done with any HSPCs. As such, it would have been obvious that one would want to genetically modify HSPCs through homologous recombination to express hCD47. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 17, as stated supra, Transgenic porcine cells were generated through genetic Knock-in of human CD47 under an EF1-alpha promoter to produce hCD47 Knock-in pigs (page 2716, column 2, paragraph 2-page 2717, column 1, paragraph 1). Furthermore, Zhang evidences that CD47 has a signal peptide sequence (page 2, column 2, paragraph 3)
Regarding claim 18, Valamehr discloses a construct comprising: a pair of homology arms specific to the selected site (an endogenous locus) and flanking the exogenous polynucleotides of interest for targeted integration via homologous recombination (paragraph 0008)
Regarding claim 19, as stated supra, Valamehr discloses that the targeted editing of the genome can be achieved through a nuclease dependent approach (paragraphs 0150-0151).
Regarding claim 20, Figure 34A shows an example of a guided nuclease (ZFN) in a vector (i.e. a construct) and a donor vector for homologous directed recombination, which would have been obvious for high-efficiency targeted integration.
Regarding claims 43 and 46, Tena teaches that to test the efficacy of hCD47 expression on engraftment and survival of porcine hematopoietic cells, they transplanted progenitor enriched cells from either transgenic founder 18286 into NOD background mice. Mice on the NOD background express an SIRPa receptor allele which can productively bind hCD47, enabling engraftment of human CD34 cells in mice also bearing the scid mutation (page 2717, column 2, paragraph 1). Therefore, the hCD47 of Tena would retain binding to SIRPα.
Regarding claim 47, the teachings of Tena are as discussed above. Tena teaches that they performed their engraftment experiments in CHEF-NSG mice which have severe immunodeficiency. These mice are deficient not only in T and B cells but also NK cells as a result of their genetic background (i.e. a genetic disorder of blood cells) (page 2717, column 2, paragraph 1-page 2718, column 1, paragraph 1).
Tena does not teach further modifying the HSPC to express a functional human protein as a result of which the HSPC can alleviate a genetic disorder.
However, Valamehr teaches that CD47 can be expressed in combination with proteins promoting engraftment (paragraph 0221).
Therefore, it would have been obvious to also express an additional functional human protein in the HSPCs of Tena that further promotes engraftment of the hCD47 expressing HSPCs in the immunodeficient CHEF-NSG mice of Tena to improve engraftment of the chimeric HSPCs and alleviate the immunodeficiency of the CHEF-NSG mice. As Tena is focused on improving the engraftment of human hematopoietic cells in a murine model with a genetic immunodeficiency (page 2714, column 1, paragraph 2), it would have been obvious additionally modify the chimeric HSPCs to express at least one additional human protein that improves engraftment to further increase the engraftment of the chimeric HSPCs of Tena.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KEENAN A BATES whose telephone number is (571)270-0727. The examiner can normally be reached M-F 7:30-5:00.
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/KEENAN A BATES/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631