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 .
DETAILED ACTION
The amended claims filed on February 27, 2026, have been acknowledged. Claims 1-158, 160, 164, 166-168, 170-180, and 183-184 were cancelled. Claims 159 and 182 were amended. Claims 188-202 are new. Claims 159, 161-163, 165 169, 181-182, and 185-202 are pending and examined on the merits.
Priority
The applicant claims foreign priority from PCTCN2019087310 filed on May 16, 2019, and PCTCN2019087314, filed on May 16, 2019. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55, received November 16, 2021. Claims 159, 161-163, 165 169, 181-182, and 185-202 find support in foreign applications PCTCN2019087310 and PCTCN2019087314, filed on May 16, 2019.
Information Disclosure Statement
The information disclosure statements (IDS) filed on July 21, 2025, and February 27, 2026, have been considered.
Withdrawn Claim Rejections - 35 USC § 103
The prior rejection of claims 159, 161, 165, 169, 181-182, and 185-186 under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180249688 (Ayares) in further view of Hwang et al. (Lipids 53: 933-945. 2018) is withdrawn in light of Applicant’s amendments to claim 159 to recite that the first and second promoters are constitutive promoters and divergent promoters.
The prior rejection of claims 159, 162-163, and 187 under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180249688 (Ayares) and Hwang et al. (Lipids 53: 933-945. 2018), as applied to claim 159 above and further in view of Kwon et al. (Transgenic Res 26:153–163. 2017) is withdrawn in light of Applicant’s amendments to claim 159 to recite that the first and second promoters are constitutive promoters and divergent promoters.
New 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 159, 161-163, 165, 169, 181-182, 185-190, 192-195, 198-199, and 201 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180249688 (Ayares) in further view of Amendola et al. (Nature Biotechnology 23: 108-116. 2005), Hwang et al. (Lipids 53: 933-945. 2018), and Kwon et al. (Transgenic Res 26:153–163. 2017). This is a new rejection that is similar to a previous rejection. Any aspect of Applicant’s traversal that is considered relevant to the new rejection as currently written is addressed below.
Regarding claim 159, Ayares teaches a nucleic acid comprising at least six, at least seven, at least eight, at least nine, or at least ten transgenes or more. In certain embodiments, expression of the at least six, at least seven, at least eight, at least nine, or at least ten transgenes or more is controlled by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten promoters or more. In certain embodiments, one promoter controls expressions of one transgene. Alternatively, one promoter controls expressions of more than one transgene, e. g., one promoter controls expression of two transgenes (paragraph 0093). Ayares teaches that the at least four transgenes can be under the control of at least two promoters on the same nucleic acid molecule (paragraph 0302).
Ayares teaches a bicistronic nucleic acid encoding CD46 and CD55 (DAF) (Figure 16) and multicistronic nucleic acids encoding a combination of four transgenes encoding thrombomodulin (TBM), CD39, EPCR, CD55 (DAF), A20, TFPI, CD47, and HO-1 with either an ICAM-2 promoter or CAG promoter (different promoters) and a gene encoding a 2A peptide between genes within the same polycistronic cassette (i.e. under control of the same promoter) (paragraph 0518 and Figure 4).
Ayares teaches nucleic acids encoding CD46 and CD55 and encoding at least five additional transgenes. The additional transgenes are selected from an anticoagulant (TFPI, EPCR, CD39), an immunosuppressant (B2M, PD-L1, FasL, CD47, HLA-E), a cytoprotective gene (A20 and HO-1) or combination thereof (paragraphs 0276, 0279, 0289, 0298, 0317, and 0335-0337).
Ayares teaches that the nucleic acid can comprise at least four transgenes, wherein the at least four transgenes are incorporated and expressed at a single locus under the control of at least two promoters, wherein at least two of the promoters are constitutive (paragraph 0025).
Although Ayares contemplates a nucleic acid encoding six or more transgenes in a multicistronic expression cassette and exemplifies nucleic acids with four transgenes comprising two polycistronic cassettes comprising two transgenes, they do not reduce to practice a nucleic acid with six or more transgenes and wherein there are three polycistronic cassettes comprising at least two transgenes. Furthermore, Ayares does not teach wherein the first and second promoter are divergent promoters and wherein the three promoters are constitutive promoters.
However, Amendola teaches a bidirectional promoter system (i.e. divergent promoters) as part of a lentiviral vector comprising a miniCMV promoter driving expression of a first gene and a PGK or UBI-C promoter driving expression of a second gene in the opposite direction (see Figure 1). As can be seen in Figure 1, the combination of the miniCMV and PGK divergent promoters amplifies the expression of the gene driven by the miniCMV promoter while maintaining similar expression of the gene driven by the PGK or UBI-C promoter (Figure 1 and page 108, column 2, paragraph 3-page 110, column 2, paragraph 2).
Hwang teaches that nucleic acids can comprise three cistronic cassettes encoding separate transgenes for stable transduction of porcine cells (Title and Figure 1). As can be seen in Figure 1, the first cistron encodes rt-TA under the control of a CMV promoter, the second cistron encodes pSCD1 and GFP under the control of a Tre3G promoter, and the third cistron encodes puromycin under the control of a PGK promoter. Furthermore, as can be seen in Figure 1, the first and second promoters are divergent promoters encoding genes in opposite directions with a ubiquitous chromosome opening element (UCOE) between the divergent promoters. As can be seen in Figure 3, the bidirectional plasmid of Figure 1 resulted in expression of the SCD1 gene of interest.
Kwon teaches a nucleic acid comprising five genes (CD55 (hDAF), CD39, TFPI, C1 inhibitor, and A20 (TNFAIP3)) under control of a single promoter (a single polycistronic expression cassette) with each gene separated by a different 2A peptide P2A, T2A, E2A, or F2A (page 155, column 1, paragraph 3-column 2, paragraph 1 and Figure 1). Kwon teaches that this nucleic acid was knocked in to the GGTA1 locus of a pig and successfully expressed all five genes in the transgenic pig (Figure 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the nucleic acid of Ayares by modifying the first and second promoter to be divergent constitutive promoters, as identified by Amendola, and to incorporate a third polycistronic expression cassette (under control of a constitutive promoter) and at least two other genes under the control of the third promoter 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 Ayares already teaches multiple multicistronic nucleic acids comprising four transgenes and directly contemplates nucleic acids comprising six or more transgenes in a multicistronic nucleic acid with multiple polycistronic cassettes (i.e. transgenes under control of separate promoters) under the control of constitutive promoters. Amendola successfully reduces top practice that two divergent, constitutive promoters can be incorporated within a nucleic acid construct to drive expression of an upstream gene (under the control of the first constitutive promoter) and a downstream gene (under the control of the second constitutive promoter) and that the combination of the miniCMV promoter and PGK promoter leads to improved expression of the upstream gene. Furthermore, Hwang successfully reduces to practice that divergent promoters can be used to drive the expression of two transgenes under the control of one of the divergent promoters. Therefore, it would have been obvious to modify the construct of Ayares to incorporate the divergent miniCMV promoter and PGK promoter as this was known to generate increased expression compared to both promoters alone and Hwang shows that divergent promoters can drive expression of at least two transgenes under the control of the same promoter.
Furthermore, Hwang successfully reduces to practice that nucleic acids can include three cistronic cassettes. As showsn in Figures 1-2, the three cistronic cassettes include divergent promoters for the first and second cistron and a constitutively active promoter encoding a separate transgene for the third cistron and one polycistronic cassette encoding two transgenes. Additionally, Kwon successfully reduced to practice that five human transgenes (CD55 (hDAF), CD39, TFPI, C1 inhibitor, and A20 (TNFAIP3)) could be incorporated into the GGTA1 locus of a pig. Therefore, it would have been obvious that the four transgene multicistronic nucleic acids of Ayares could be expanded to include another polycitrsonic cassette with a constitutive promoter encoding two transgenes as Ayares and Hwang successfully reduce to practice that two transgenes can be expressed under the control of the same promoter within a polycistronic cassette as part of a larger multicistronic cassette comprising more than one promoter and Kwon teaches that five human transgenes can be expressed under the control of the same promoter and knocked in at the GGTA1 locus for expression.
Ayares directly identifies CD39, CD46, CD55, CD47, A20, HO-1, THBD, CD59, TFPI, HLA-E, B2M, PD-L1, FasL, and EPCR as potential transgenes within their nucleic acid (paragraphs 0134, 0207-211, 0289, and 0508). As such, it would have been obvious that one could make a nucleic acid comprising three polycistronic cassettes (with their own constitutive promoters) encoding two or more of these transgenes each and wherein the first and second promoters are divergent as Ayares directly contemplates encoding six or more transgenes in a single nucleic acid and directly contemplates these specific transgenes as the possible transgenes that could be encoded, Amendola reduces to practice bicistronic cassettes with divergent, constitutively active promoters, and Hwang reduces to practice the three polycistronic cassettes wherein the third cistron has a constitutive promoter encoding transgenes and the divergent first and second promoters.
Furthermore, in regard to choosing to use three promoters, Ayares provides multiple embodiments of “at least two promoters”, and “at least four promoters” ([0095, 0172], see also claims 16 & 17 of Ayares); thus, three promoters would have been immediately obvious choice considering the preferred embodiments of Ayares and would embrace the teaching that the nucleic acid can comprise at least four transgenes, wherein the at least four transgenes are incorporated and expressed at a single locus under the control of at least two promoters, wherein at least two of the promoters are constitutive. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 161, as the combined nucleic acid of Ayares, Amendola, Hwang, and Kwon teaches six transgenes, this would be less than twelve transgenes.
Regarding claims 162-163, and 187, Kwon, as stated supra, teaches a nucleic acid comprising five genes (CD55 (hDAF), CD39, TFPI, C1 inhibitor, and A20 (TNFAIP3)) under control of a single promoter (a single polycistronic expression cassette) with each gene separated by a different 2A peptide P2A, T2A, E2A, or F2A (page 155, column 1, paragraph 3-column 2, paragraph 1 and Figure 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the nucleic acid of Ayares to incorporate at least three transgenes within a single polycistronic expression cassette (under control of one promoter, as seen in Kwon) 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 Ayares already teaches multiple multicistronic nucleic acids comprising four transgenes and directly contemplates nucleic acids comprising six or more transgenes in a multicistronic nucleic acid with multiple polycistronic cassettes (i.e. transgenes under control of separate promoters). As Kwon teaches that the same genes (CD39, TFPI, CD55, and A20) can all be expressed under the same promoter in a single multicistronic expression cassette using different 2A peptides, it would have been obvious that the six transgene multicistronic nucleic acids (three polysistronic cassettes comprising two transgenes each) of Ayares, Amendola, Hwang, and Kwon could be expanded to express more transgenes as long as different 2A peptides are used in front of each new transgene (up to four different 2A peptides can be used to express five different transgenes, as identified by Kwon (page 155, column 1, paragraph 3-page 158, column 2, paragraph 1 and Figure 1)). Ayares directly identifies CD46, CD55, CD47, A20, HO-1, THBD, CD59 (paragraph 0207), TFPI, HLA-E, B2M, PD-L1, FasL, and EPCR as potential transgenes within their nucleic acid. Therefore, it would have been obvious that the seventh transgene could be any one of these. As such, it would have been obvious that one could make a nucleic acid comprising seven or more of these transgenes as Ayares directly contemplates encoding seven or more transgenes in a single nucleic acid and directly contemplates these specific transgenes as the possible transgenes that could be encoded. Furthermore, Kwon successfully reduces to practice that four of the above recited transgenes (CD55 (hDAF), CD39, TFPI, and A20) and an additional transgene (C1 inhibitor) can be expressed under a single promoter and Ayares, Amendola, and Hwang successfully reduce to practice that multicistronic nucleic acids with multiple promoters and transgenes under control of those promoters can be produced. As such, under this configuration, at least seven transgenes could be expressed and wherein the seventh transgene is expressed within the same cistronic cassette as two other transgenes, as Kwon teaches that up to five transgenes can be under the control of the same promoter. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 165, as can be seen in Figures 1 and 4 and as identified in paragraphs 0403-0405, Ayares uses 2A sequences between the two transgenes within each polycistronic cassette to allow co-expression of the linked transgenes.
Regarding claims 169 and 186, as stated supra, Hwang uses a ubiquitous chromatin opening element (UCOE) between the divergent promoters (Figure 1). As this was shown to be successful in expressing the transgenes under the control of the divergent promoters (Figure 3), it would have been obvious to include this as part of the combined nucleic acid molecule of Ayares, Amendola, Hwang, and Kwon.
Regarding claim 181, Ayares, as stated supra, teaches different combinations of multicistronic nucleic acids encoding CD55 (DAF), CD39, CD47, as examples (Figure 4). Furthermore, Ayares teaches nucleic acids encoding additional transgenes, such as an immunosuppressant (B2M and HLA-E) and a cytoprotective gene (A20 and HO-1) or combination thereof (paragraphs 0276, 0279, 0289, 0298, 0317, and 0335-0337).
Regarding claim 182, Ayares teaches that the multicistronic nucleic acids can comprise constitutive promoters for both polycistronic cassettes (paragraph 0417). As stated supra, Ayares directly identifies CAG as one possible constitutive promoter (Figure 4 and paragraph 0095).
Ayares does not identify Ef1α as a possible constitutive promoter, only identifying that other constitutive promoters outside of CAG could be used (paragraph 0095). However, Ef1α is a commonly used constitutive promoter that would have been readily envisioned as another potential constitutive promoter that could be used. Therefore, it would have been obvious to one of ordinary skill in the art that one polycistronic cassette could be under the control of a CAG promoter (as identified by Ayares) and another cassette could be under control of the constitutively active EF1α promoter (another constitutive promoter, which is contemplated by Ayares).
Regarding claim 185, the placement of CD55 within the first polycistronic cassette would have been readily contemplated as a known option as there are a limited number of possible combination of transgenes in each polycistronic cassette.
Regarding claim 188, Ayares teaches a method of making a transgenic pig expressing at least four transgenic genes but lacking expression of alpha
1, 3 galactosyltransferase, comprising ( i ) incorporating at least four transgenes under the control of at least two promoters at a single locus within the pig genome of a pig zygote to provide a polygene pig genome; ( ii ) permitting a cell comprising the
polygene pig genome to mature into a transgenic pig (paragraphs 0101-0102).
Regarding claim 189, Ayares teaches making a transgenic pig expressing at least four transgenic genes but lacking expression of alpha 1, 3 galactosyltransferase, comprising ( i ) incorporating at least four transgenes under the control of at least two promoters at a single locus within the pig genome of a pig zygote (a cell) to provide a polygene pig genome; ( ii ) permitting a cell comprising the polygene pig genome to mature into a transgenic pig (paragraphs 0101-0102)
Regarding claim 190, Ayares teaches that the genetically modified pig can include incorporation of the transgenes in the GGTA1 locus, inactivating the GGTA1 gene, and additional genetic knockouts of porcine genes β4GalNT2 and CMAH (paragraphs 0020-0035 and 0085-0096).
Regarding claims 192-195, 198-199, and 201, the present invention provides a transgenic animal (e.g., a transgenic porcine animal) that serves as a source for organs (including the kidney heart, or liver), organ fragments, tissues or cells for use in xenotransplantation. The present invention extends to the organs, tissues and cells derived from the transgenic animal (paragraphs 0167-0195 and 0461-0468).
Response to Arguments
Applicant's arguments filed February 27, 2026, are acknowledged.
First, Applicant argues that there was no reasonable expectation of success in combining Ayares and Hwang because capacity constraints and transcription interference is a known technical obstacle for generating transgenic pigs for safe xenotransplantation that others were not able to solve. Applicant argues that Ayares expresses doubt that greater than four transgenes can be expressed at a single locus and teaches away from doing so by expressing six transgenes at two different loci. Accordingly, a person of ordinary skill in the art at the time the application was filed, starting from Ayares, would doubt that more than four human transgenes could successfully be expressed from a single locus and would not be motivated to modify the nucleic acid constructs of Ayares to provide six or more human transgenes (page 10, paragraph 3-page 12, paragraph 1).
Applicant's arguments have been fully considered but they are not persuasive.
The claims at issue regarding the previous rejection require the generation of a nucleic acid, not integration at a specific locus to generate a multitransgenic pig. Furthermore, the success of making a nucleic acid with three polycistronic cassettes encoding at least two transgenes was high as Ayares successfully reduced to practice that they could make a bicistronic nucleic acid encoding four genes under the control of two promoters, two genes per promoter, Amendola successfully reduces to practice that divergent, constitutive promoters can be used to drive expression of genes of interest, and Hwang successfully reduce to practice that three cistronic cassettes can be incorporated into a nucleic acid to drive expression of transgenes and that divergent promoters could be used as part of that tricistronic structure. As such, all that would be required is to include additional genes in the nucleic acid construct by introducing a third polycistronic cassette with a constitutive promoter, which Hwang already reduces to practice, and modifying the first and second promoters to be divergent constitutively active promoters, which Amendola already reduces to practice. Therefore, there would be a reasonable expectation of success for one of ordinary skill in the art to expand the nucleic acid construct of Ayares to include an additional polycistronic cassette encoding at least two genes of interest and to modify he first and second promoters to be divergent constitutively active promoters. Furthermore, Kwon has already successfully reduced to practice that five genes under the same promoter and separated by 2A peptides can be integrated into the genome of a pig to generate a transgenic pig with five human transgenes at the same locus and Ayares successfully reduced to practice that bicistronic cassettes encoding two transgenes in each cistron can be incorporated at a single locus. Therefore, it would have also been expected that the number of transgenes at a single locus could be expanded to include more than just the four transgenes exemplified by Ayares.
Furthermore, although Ayares exemplifies using four transgenes at a single locus and two transgenes at another locus, they do not teach away from using more than four transgenes. Even in the section cited by the Applicant (page 10, paragraph 4 of Remarks), Ayares directly states that at least four transgenes under control of at least two promoters can integrate at one loci. Furthermore, Ayares directly contemplates at least six transgenes can be expressed at a single locus (paragraph 0093). As such, the combined teachings of Ayares, Amendola, Hwang, and Kwon do not teach away from expressing six transgenes on a single nucleic acid. MPEP 2141.02, (VI) states that "the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed…." In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004).
Second, Applicant argues that the nucleic acid of claim 159 achieves unexpected results because they produced pigs comprising the nucleic acid of claim 159 which have high transgene expression from a dingle locus, no detectable transcription interference, positive functional data for engineered organs, and successful xenotransplantation and xenoperfusion results (page 12, paragraphs 2-3).
Applicant's arguments have been fully considered but they are not persuasive.
Although Applicant argues unexpected results, the claims are not commensurate in scope with the experimental results cited by the Applicant. MPEP 716.02(d) discloses that whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980) (Claims were directed to a process for removing corrosion at "elevated temperatures" using a certain ion exchange resin (with the exception of claim 8 which recited a temperature in excess of 100°C). Appellant demonstrated unexpected results via comparative tests with the prior art ion exchange resin at 110°C and 130°C. The court affirmed the rejection of claims 1-7 and 9-10 because the term "elevated temperatures" encompassed temperatures as low as 60°C where the prior art ion exchange resin was known to perform well. The rejection of claim 8, directed to a temperature in excess of 100°C, was reversed.). See also In re Peterson, 315 F.3d 1325, 1329-31, 65 USPQ2d 1379, 1382-85 (Fed. Cir. 2003) (data showing improved alloy strength with the addition of 2% rhenium did not evidence unexpected results for the entire claimed range of about 1-3% rhenium); In re Grasselli, 713 F.2d 731, 741, 218 USPQ 769, 777 (Fed. Cir. 1983) (Claims were directed to certain catalysts containing an alkali metal. Evidence presented to rebut an obviousness rejection compared catalysts containing sodium with the prior art. The court held this evidence insufficient to rebut the prima facie case because experiments limited to sodium were not commensurate in scope with the claims.).
Regarding the claims at issue in the instant application, claim 159 encompasses a nucleic acid rather than a transgenic pig. As such, the claims as written are not commensurate in scope with alleged unexpected results. Furthermore, as shown by the combined teachings of Ayares, Amendola, Hwang, and Kwon, it would not have been unexpected to produce a nucleic acid with three polycistronic cassettes encoding at least two transgenes and wherein the first and second promoter are constitutive promoters and divergent and the third promoter is constitutive.
As stated supra, the success of making a nucleic acid with a polycistronic expression system was high as Ayares successfully reduced to practice that they could make a bicistronic nucleic acid encoding four genes under the control of two promoters, two genes per promoter, Amendola successfully reduces to practice that divergent, constitutive promoters can be used to drive expression of genes of interest, and Hwang successfully reduce to practice that three cistronic cassettes can be incorporated into a nucleic acid to drive expression of transgenes and that divergent promoters could be used as part of that tricistronic structure. As such, all that would be required is to include additional genes in the nucleic acid construct by introducing a third polycistronic cassette with a constitutive promoter, which Hwang already reduces to practice, and modifying the first and second promoters to be divergent constitutively active promoters, which Amendola already reduces to practice. Therefore, there would be a reasonable expectation of success for one of ordinary skill in the art to expand the nucleic acid construct of Ayares to include an additional polycistronic cassette encoding at least two genes of interest and to modify he first and second promoters to be divergent constitutively active promoters. Furthermore, Kwon has already successfully reduced to practice that five genes under the same promoter and separated by 2A peptides can be integrated into the genome of a pig to generate a transgenic pig with five human transgenes at the same locus and Ayares successfully reduced to practice that bicistronic cassettes encoding two transgenes in each cistron can be incorporated at a single locus. Therefore, it would have also been expected that the number of transgenes at a single locus could be expanded to include more than just the four transgenes exemplified by Ayares.
Claims 189, 191, 196-197, and 202 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180249688 (Ayares), Amendola et al. (Nature Biotechnology 23: 108-116. 2005), Hwang et al. (Lipids 53: 933-945. 2018), and Kwon et al. (Transgenic Res 26:153–163. 2017), as applied to claim 189 above and further in view of Dieckhoff et al. (Xenotransplantation 15: 36-45. 2008). This is a new rejection made in response to the new claims.
The teachings of Ayares, Amendola, Hwang, and Kwon are as discussed above. Ayares teaches that their transgenic porcine animals (pigs) are a useful source of organs, organ fragments, tissues or cells for xenotransplantation (paragraphs 0085-0096).
The combined teachings of Ayares, Amendola, Hwang, and Kwon do not teach using porcine cells modified to be free of porcine endogenous retroviruses.
However, Dieckhoff teaches that specified pathogen-free breeding of pigs can prevent transmission of most porcine microbes. However, porcine endogenous retroviruses (PERVs) are integrated in the porcine genome, and can be released as infectious particles from normal pig cells and can infect human cells in vitro. Retroviruses are tumorigenic and may cause immunodeficiencies in infected hosts. The risk of PERV infection has to be carefully assessed specifically in view of the fact that the human immunodeficiency viruses HIV-1 and HIV-2, causing AIDS in humans, are the result of multiple trans-species transmissions of retroviruses from non-human primates. Results of their study demonstrate a strong and long lasting inhibition of PERV mRNA expression in primary porcine fibroblasts using a lentiviral vector system expressing a PERV-specific shRNA and use in somatic cell nuclear transfer. This is the first report demonstrating shRNA mediated reduction of PERV expression in shRNA-transgenic pigs in vivo. The strategy applied here could lead to microbiological safer porcine xenotransplants and is therefore of considerable medical importance (page 3, paragraphs 1-2 and page 7, paragraph 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the pig cell of the combined teachings of Ayares, Amendola, Hwang, and Kwon by modifying the porcine cell to be free of porcine endogenous retroviruses 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 Ayares teaches using their porcine cells, organs, and pigs for xenotransplantation and Dieckhoff teaches that porcine endogenous retroviruses (PERVs) are integrated in the porcine genome, and can be released as infectious particles from normal pig cells and can infect human cells in vitro. Retroviruses are tumorigenic and may cause immunodeficiencies in infected hosts. Dieckhoff teaches that results of their study demonstrate a strong and long lasting inhibition of PERV mRNA expression in primary porcine fibroblasts using a lentiviral vector system expressing a PERV-specific shRNA and use in somatic cell nuclear transfer. The strategy applied here could lead to microbiological safer porcine xenotransplants and is therefore of considerable medical importance. Therefore, it would have been obvious to use porcine cells free of porcine endogenous retroviruses. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 189, 192, 198, and 200 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180249688 (Ayares), Amendola et al. (Nature Biotechnology 23: 108-116. 2005), Hwang et al. (Lipids 53: 933-945. 2018), and Kwon et al. (Transgenic Res 26:153–163. 2017), as applied to claim 189 above and further in view of Kulick et al. (The Journal of Thoracic and Cardiovascular Surgery 119: 690-699. 2000). This is a new rejection made in response to the new claims.
The teachings of Ayares, Amendola, Hwang, and Kwon are as discussed above. Ayares teaches that their transgenic porcine animals (pigs) are a useful source of organs, organ fragments, tissues or cells for xenotransplantation (paragraphs 0085-0096).
The combined teachings of Ayares, Amendola, Hwang, and Kwon do not teach wherein the genetically modified pig is a Yorkshire pig.
However, Kulick teaches that they used Yorkshire swine for generating transgenic pigs expressing human transgenes and identify the potential for xenotransplantation (whole document).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the genetically modified pig could be a Yorkshire pig as Kulick already teaches that this type of pig has been previously used for genetic modification with human transgenes and has been considered for xenotransplantation. Therefore, it would have been obvious it could have also been used to generate the pig of the combined teachings of Ayares, Amendola, Hwang, and Kwon. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 159, 161-163, 165 169, 181-182, 185-190, 192, 194, 198, and 201 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 54-58 and 72-89 of copending Application No. 17/790,797 in view of United States Patent Application No. 20180249688 (Ayares), Amendola et al. (Nature Biotechnology 23: 108-116. 2005), Kwon et al. (Transgenic Res 26:153–163. 2017), and Hwang et al. (Lipids 53: 933-945. 2018). This is a new rejection made in response to Applicant’s amendments to claim 159 and new claims 188-202. Applicant’s traversal has been fully considered but is considered moot in response to the new rejection.
Regarding claims 159, 161-163, 165, 181, and 189, ‘797 claims a method of expressing twelve polypeptides derived from a non-porcine mammalian species from a single construct in different cistrons in a genetically modified, transgenic porcine islet cell, wherein said twelve polypeptides comprise CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, A20, PD-L1, HO-1, and CD47, wherein transgenes within each of the different cistrons in said single construct are separated by a ribosomal skipping 2A peptide (claim 54). Although this is a method, the method comprises the product as claimed in the instant application.
‘797 is silent as to the overall design of the single construct and different cistrons.
However, Ayares teaches a nucleic acid comprising at least seven transgenes or more. In certain embodiments, expression of the at least seven transgenes is controlled by at least three promoters In certain embodiments, one promoter controls expressions of more than one transgene, e. g., one promoter controls expression of two transgenes (paragraph 0093). Ayares teaches that the at least four transgenes can be under the control of at least two promoters on the same nucleic acid molecule (paragraph 0302).
Ayares teaches a bicistronic nucleic acid encoding CD46 and CD55 (DAF) (Figure 16) and multicistronic nucleic acids encoding a combination of four transgenes encoding thrombomodulin (TBM), CD39, EPCR, CD55 (DAF), A20, TFPI, CD47, and HO-1 with either an ICAM-2 promoter or CAG promoter (different promoters) and a gene encoding a 2A peptide between genes within the same polycistronic cassette (i.e. under control of the same promoter) (paragraph 0518 and Figure 4).
Ayares teaches nucleic acids encoding CD46 and CD55 and encoding at least five additional transgenes. The additional transgenes are selected from an anticoagulant (TFPI, EPCR, CD39), an immunosuppressant (B2M, PD-L1, FasL, CD47, HLA-E), a cytoprotective gene (A20 and HO-1) or combination thereof (paragraphs 0276, 0279, 0289, 0298, 0317, and 0335-0337).
Ayares teaches that the nucleic acid can comprise at least four transgenes, wherein the at least four transgenes are incorporated and expressed at a single locus under the control of at least two promoters, wherein at least two of the promoters are constitutive (paragraph 0025).
Amendola teaches a bidirectional promoter system (i.e. divergent promoters) as part of a lentiviral vector comprising a miniCMV promoter driving expression of a first gene and a PGK or UBI-C promoter driving expression of a second gene in the opposite direction (see Figure 1). As can be seen in Figure 1, the combination of the miniCMV and PGK divergent promoters amplifies the expression of the gene driven by the miniCMV promoter while maintaining similar expression of the gene driven by the PGK or UBI-C promoter (Figure 1 and page 108, column 2, paragraph 3-page 110, column 2, paragraph 2).
Kwon teaches a nucleic acid comprising five genes (CD55 (hDAF), CD39, TFPI, C1 inhibitor, and A20 (TNFAIP3)) under control of a single promoter (a single polycistronic expression cassette) with each gene separated by a different 2A peptide P2A, T2A, E2A, or F2A (page 155, column 1, paragraph 3-column 2, paragraph 1 and Figure 1).
Hwang teaches that nucleic acids can comprise three cistronic cassettes encoding separate transgenes for stable transduction of porcine cells (Title and Figure 1). As can be seen in Figure 1, the first cistron encodes rt-TA under the control of a CMV promoter, the second cistron encodes pSCD1 and GFP under the control of a Tre3G promoter, and the third cistron encodes puromycin under the control of a PGK promoter. Furthermore, as can be seen in Figure 1, the first and second promoters are divergent promoters encoding genes in opposite directions with a ubiquitous chromosome opening element (UCOE) between the divergent promoters. As can be seen in Figure 3, the bidirectional plasmid of Figure 1 resulted in expression of the SCD1 gene of interest.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the nucleic acid of ‘797 to arrange the nucleic acid of ‘797 to have three polycistronic cassettes comprising the eight or more transgenes of ‘797 with two to five transgenes under control of each promoter and to incorporate divergent constitutive promoters for the first and second promoter and a third constitutive promoter 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 Ayares already teaches multiple multicistronic nucleic acids comprising four transgenes and directly contemplates nucleic acids comprising six or more transgenes in a multicistronic nucleic acid with multiple polycistronic cassettes (i.e. transgenes under control of separate promoters) under the control of constitutive promoters. Amendola successfully reduces top practice that two divergent, constitutive promoters can be incorporated within a nucleic acid construct to drive expression of an upstream gene (under the control of the first constitutive promoter) and a downstream gene (under the control of the second constitutive promoter) and that the combination of the miniCMV promoter and PGK promoter leads to improved expression of the upstream gene.
Hwang successfully reduces to practice that nucleic acids can include three cistronic cassettes encoding separate transgenes, including one polycistronic cassette encoding two transgenes and that the first and second promoters can be divergent promoters encoding genes in opposite directions with a ubiquitous chromosome opening element (UCOE) between the divergent promoters. Kwon successfully reduces to practice that up to five transgenes can be expressed under a single promoter, including four of the above recited transgenes of ‘797 (CD55 (hDAF), CD39, TFPI, and A20). Therefore, it would have been obvious that the nucleic acid of ‘797 could be configured to include three polycistronic cassettes encoding at least two and up to five transgenes for the twelve transgenes of ‘797 as Ayares and Hwang successfully reduce to practice that two transgenes can be expressed under the control of the same promoter within a polycistronic cassette as part of a larger multicistronic cassette comprising more than one promoter and Kwon successfully reduces to practice that up to five transgenes can be expressed under a single promoter. Furthermore, it would have been obvious to use the divergent miniCMV promoter and PGK promoter as this was known to generate increased expression compared to both promoters alone and Hwang shows that divergent promoters can drive expression of at least two transgenes under the control of the same promoter.
Furthermore, Hwang successfully reduces to practice that nucleic acids can include three cistronic cassettes. As shown in Figures 1-2, the three cistronic cassettes include divergent promoters for the first and second cistron and a constitutively active promoter encoding a separate transgene for the third cistron and one polycistronic cassette encoding two transgenes. Additionally, Kwon successfully reduced to practice that five human transgenes (CD55 (hDAF), CD39, TFPI, C1 inhibitor, and A20 (TNFAIP3)) could be incorporated into the GGTA1 locus of a pig. Therefore, it would have been obvious that the twelve transgene multicistronic nucleic acids of ‘797 could have the claimed polycistronic construction of two divergent constitutive promoters and a third constitutive promoter as Ayares and Hwang successfully reduce to practice that two transgenes can be expressed under the control of the same promoter within a polycistronic cassette as part of a larger multicistronic cassette comprising more than one promoter and Kwon teaches that five human transgenes can be expressed under the control of the same promoter and knocked in at the GGTA1 locus of a pig for expression.
‘797 directly claims CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, A20, PD-L1, HO-1, and CD47 as the transgenes within their nucleic acid. As such, it would have been obvious that one could make a nucleic acid comprising three polycistronic cassettes (with their own constitutive promoters) encoding two or more of these transgenes each and wherein the first and second promoters are divergent as Ayares directly contemplates encoding six or more transgenes in a single nucleic acid and Ayares directly identifies CD39, CD46, CD55, CD47, A20, HO-1, THBD, CD59, TFPI, HLA-E, B2M, PD-L1, FasL, and EPCR as potential transgenes within their nucleic acid (paragraphs 0134, 0207-211, 0289, and 0508), Amendola reduces to practice bicistronic cassettes with divergent, constitutively active promoters, and Hwang reduces to practice the three polycistronic cassettes wherein the third cistron has a constitutive promoter encoding transgenes and the divergent first and second promoters.
Furthermore, in regard to choosing to use three promoters, Ayares provides multiple embodiments of “at least two promoters”, and “at least four promoters” ([0095, 0172], see also claims 16 & 17 of Ayares); thus, three promoters would have been immediately obvious choice considering the preferred embodiments of Ayares and would embrace the teaching that the nucleic acid can comprise at least four transgenes, wherein the at least four transgenes are incorporated and expressed at a single locus under the control of at least two promoters, wherein at least two of the promoters are constitutive. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claims 169 and 186, as stated supra, Hwang uses a ubiquitous chromatin opening element (UCOE) between the divergent promoters (Figure 1). As this was shown to be successful in expressing the transgenes under the control of the divergent promoters (Figure 3), it would have been obvious to include this as part of the combined nucleic acid molecule of ‘797, Ayares, Kwan, and Hwang.
Regarding claim 182, ‘797 is silent as to the types of promoters used.
However, Ayares teaches that the multicistronic nucleic acids can comprise constitutive promoters for both polycistronic cassettes (paragraph 0417). Ayares directly identifies CAG as one possible constitutive promoter (Figure 4 and paragraph 0095).
Ayares does not identify Ef1α as a possible constitutive promoter, only identifying that other constitutive promoters outside of CAG could be used (paragraph 0095). However, Ef1α is a commonly used constitutive promoter that would have been readily envisioned as another potential constitutive promoter that could be used. Therefore, it would have been obvious to one of ordinary skill in the art that one polycistronic cassette could be under the control of a CAG promoter (as identified by Ayares) and another cassette could be under control of the constitutively active EF1α promoter (another constitutive promoter, which is contemplated by Ayares).
Regarding claim 185, the placement of CD55 within the first polycistronic cassette would have been readily contemplated as a known option as there are a limited number of possible combination of transgenes in each polycistronic cassette.
Regarding claim 187, as there are twelve transgenes and three polycistronic cassettes for the combined teachings of ‘797, Ayares, Amendola, Kwon, and Hwang, it would necessarily require at least one of the polycistronic cassettes comprise at least three transgenes.
Regarding claim 188, ‘797 claims wherein one or more nucleic acid sequence(s) encoding said twelve polypeptides encoded by the twelve human transgenes is inserted within a non-orthologous loci of a porcine ortholog (claim 73). Although not specifically stated, insertion of the non-endogenous genes in the porcine loci would require delivery of the nucleic acid to the cell.
Regarding claim 190, ‘797 claims wherein said isolated transgenic porcine islet cells comprise a frameshift mutation in GGTA, B4GALNT2, CMAH, or a combination thereof, resulting in premature termination of translation, thereby ablating activity of GGTA, B4GALNT2, CMAH, or a combination thereof (claim 72).
Regarding claims 192, 194, 198, and 201, ‘797 claims wherein the pharmaceutical composition comprises an isolated porcine pancreas (i.e. isolated from a genetically modified pig) comprising said isolated transgenic porcine islet cells (claim 87).
This is a provisional nonstatutory double patenting rejection.
Claims 189, 191, 196, and 202 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 54-58 and 72-89 of copending Application No. 17/790,797 in view of United States Patent Application No. 20180249688 (Ayares), Amendola et al. (Nature Biotechnology 23: 108-116. 2005), Kwon et al. (Transgenic Res 26:153–163. 2017), and Hwang et al. (Lipids 53: 933-945. 2018) as applied to claim 189 above and further in view of Dieckhoff et al. (Xenotransplantation 15: 36-45. 2008). This is a new rejection made in response to the new claims.
The teachings of ‘797, Ayares, Amendola, Kwon, and Hwang are as discussed above. ‘797 claims a method for treating an insulin resistant condition or an insulin- deficient condition in a non-porcine mammal in need thereof, comprising administering to said non-porcine mammal a pharmaceutical composition comprising a therapeutically effective dose of isolated, genetically modified, transgenic porcine islet cells (i.e. xenotransplantation of a porcine cell to a non-porcine mammal).
The combined teachings of ‘797, Ayares, Amendola, Kwon, and Hwang do not teach using porcine cells modified to be free of porcine endogenous retroviruses.
However, Dieckhoff teaches that specified pathogen-free breeding of pigs can prevent transmission of most porcine microbes. However, porcine endogenous retroviruses (PERVs) are integrated in the porcine genome, and can be released as infectious particles from normal pig cells and can infect human cells in vitro. Retroviruses are tumorigenic and may cause immunodeficiencies in infected hosts. The risk of PERV infection has to be carefully assessed specifically in view of the fact that the human immunodeficiency viruses HIV-1 and HIV-2, causing AIDS in humans, are the result of multiple trans-species transmissions of retroviruses from non-human primates. Results of their study demonstrate a strong and long lasting inhibition of PERV mRNA expression in primary porcine fibroblasts using a lentiviral vector system expressing a PERV-specific shRNA and use in somatic cell nuclear transfer. This is the first report demonstrating shRNA mediated reduction of PERV expression in shRNA-transgenic pigs in vivo. The strategy applied here could lead to microbiological safer porcine xenotransplants and is therefore of considerable medical importance (page 3, paragraphs 1-2 and page 7, paragraph 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the pig cell of the combined teachings of ‘797, Ayares, Amendola, Kwon, and Hwang by modifying the porcine cell to be free of porcine endogenous retroviruses 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 ‘797 teaches using their porcine cells for xenotransplantation and Dieckhoff teaches that porcine endogenous retroviruses (PERVs) are integrated in the porcine genome, and can be released as infectious particles from normal pig cells and can infect human cells in vitro. Retroviruses are tumorigenic and may cause immunodeficiencies in infected hosts. Dieckhoff teaches that results of their study demonstrate a strong and long lasting inhibition of PERV mRNA expression in primary porcine fibroblasts using a lentiviral vector system expressing a PERV-specific shRNA and use in somatic cell nuclear transfer. The strategy applied here could lead to microbiological safer porcine xenotransplants and is therefore of considerable medical importance. Therefore, it would have been obvious to use porcine cells free of porcine endogenous retroviruses. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
This is a provisional nonstatutory double patenting rejection.
Claims 189, 192, 198, and 200 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 54-58 and 72-89 of copending Application No. 17/790,797 in view of United States Patent Application No. 20180249688 (Ayares), Amendola et al. (Nature Biotechnology 23: 108-116. 2005), Kwon et al. (Transgenic Res 26:153–163. 2017), and Hwang et al. (Lipids 53: 933-945. 2018) as applied to claim 189 above and further in view of Kulick et al. (The Journal of Thoracic and Cardiovascular Surgery 119: 690-699. 2000). This is a new rejection made in response to the new claims.
The teachings of ‘797, Ayares, Amendola, Kwon, and Hwang are as discussed above. ‘797 claims a method for treating an insulin resistant condition or an insulin- deficient condition in a non-porcine mammal in need thereof, comprising administering to said non-porcine mammal a pharmaceutical composition comprising a therapeutically effective dose of isolated, genetically modified, transgenic porcine islet cells (i.e. xenotransplantation of a porcine cell to a non-porcine mammal).
The combined teachings of ‘797, Ayares, Amendola, Kwon, and Hwang do not teach wherein the genetically modified pig is a Yorkshire pig.
However, Kulick teaches that they used Yorkshire swine for generating transgenic pigs expressing human transgenes and identify the potential for xenotransplantation (whole document).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the genetically modified pig could be a Yorkshire pig as Kulick already teaches that this type of pig has been previously used for genetic modification with human transgenes and has been considered for xenotransplantation. Therefore, it would have been obvious it could have also been used to generate the pig of the combined teachings of Ayares, Amendola, Hwang, and Kwon. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 159, 161-163, 165, 169, 181-182, and 185-190, 192-195, and 198-201 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 155 and 173-210 of copending Application No. 18/327,565 in view of Amendola et al. (Nature Biotechnology 23: 108-116. 2005) and Hwang et al. (Lipids 53: 933-945. 2018). This is a new rejection made in response to Applicant’s amendments to claim 159 and new claims 188-202. Applicant’s traversal has been fully considered but is considered moot in response to the new rejection.
Regarding claims 159, 162-163, 181, 187, and 189, ‘565 claims a genetically modified porcine cell comprising a nucleic acid molecule, comprising (a) a first polycistronic cassette, comprising: (i) an EPCR transgene, and (ii) a THBD transgene; (b) a second polycistronic cassette, comprising: (i) an A20 transgene, and (ii) an HO1 transgene; and(c) a third polycistronic cassette, comprising: (i) a CD46 transgene, (ii) a CD55 transgene, and (iii) a CD47 transgene (claim 138) and wherein the molecule is integrated at a single genomic locus (claim 155). ‘565 claims the first, second, and third polycistronic cassettes comprise a promoter, wherein the promoter comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 126-145, 167-168, 178-179, 231-238, or 250 (claim 194).
‘565 also claims a genetically modified porcine cell comprising a nucleic acid molecule, comprising (a) a first polycistronic cassette, comprising: (i) a TFPI transgene, (ii) an EPCR transgene, and (iii) a THBD transgene; (b) a second polycistronic cassette, comprising: (i) a transgene encoding a B2M HLA-E fusion; and (ii) a CD47 transgene; and (c) a third polycistronic cassette, comprising: (i) a CD46 transgene, and (ii) a CD55 transgene (claim 183).
‘565 is silent as to the use of divergent constitutive promoters.
Amendola teaches a bidirectional promoter system (i.e. divergent promoters) as part of a lentiviral vector comprising a miniCMV promoter driving expression of a first gene and a PGK or UBI-C promoter driving expression of a second gene in the opposite direction (see Figure 1). As can be seen in Figure 1, the combination of the miniCMV and PGK divergent promoters amplifies the expression of the gene driven by the miniCMV promoter while maintaining similar expression of the gene driven by the PGK or UBI-C promoter (Figure 1 and page 108, column 2, paragraph 3-page 110, column 2, paragraph 2).
Hwang teaches that nucleic acids can comprise three cistronic cassettes encoding separate transgenes (Figure 1). As can be seen in Figure 1, the first cistron encodes rt-TA under the control of a CMV promoter, the second cistron encodes pSCD1 and GFP under the control of a Tre3G promoter, and the third cistron encodes puromycin under the control of a PGK promoter. Furthermore, as can be seen in Figure 1, the first and second promoters are divergent promoters encoding genes in opposite directions with a ubiquitous chromosome opening element (UCOE) between the divergent promoters. As can be seen in Figure 3, the bidirectional plasmid of Figure 1 resulted in expression of the SCD1 gene of interest.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the nucleic acid of ‘565 to incorporate divergent constitutive promoters for the first and second promoter and a third constitutive promoter 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 Amendola successfully reduces top practice that two divergent, constitutive promoters can be incorporated within a nucleic acid construct to drive expression of an upstream gene (under the control of the first constitutive promoter) and a downstream gene (under the control of the second constitutive promoter) and that the combination of the miniCMV promoter and PGK promoter leads to improved expression of the upstream gene. Hwang successfully reduces to practice that nucleic acids can include three cistronic cassettes encoding separate transgenes, and that the first and second promoters can be divergent promoters encoding genes in opposite directions with a ubiquitous chromosome opening element (UCOE) between the divergent promoters, including one cassette driven by one of the divergent promoters encoding two transgenes. Therefore, it would have been obvious that the nucleic acid of ‘565 could be modified to make the promoters of the first and second polycistrons divergent constitutive promoters and the third promoter constitutive as Amendola identifies that the divergent miniCMV promoter and PGK promoter known to generate increased expression compared to both promoters alone and Hwang shows that divergent promoters can drive expression of at least two transgenes under the control of the same promoter and that divergent promoters could also be with a third constitutive promoter. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 161, as ‘565 claims seven or eight transgenes, this would be less than twelve transgenes (claims 155 and 183).
Regarding claim 165, ‘565 claims the nucleic acid molecule of claim 138, wherein the nucleic acid sequence encodes a 2A polypeptide (claim 200).
Regarding claims 169 and 186, as stated supra, Hwang uses a ubiquitous chromatin opening element (UCOE) between the divergent promoters (Figure 1). As this was shown to be successful in expressing the transgenes under the control of the divergent promoters (Figure 3), it would have been obvious to include this as part of the combined nucleic acid molecule of ‘565 and Hwang.
Regarding claim 182, ‘565 claims the first, second, and third polycistronic cassettes comprise a promoter, wherein the promoter comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 126-145, 167-168, 178-179, 231-238, or 250 (claim 141). Table B of the specification identifies CAG and EF1α as being represented by some of the SEQ ID NOs. Therefore, the first promoter could be EF1α and the second promoter could be CAG.
Regarding claim 185, although ‘565 identifies CD55 as being part of a third polycistronic cassette, there is no specific order associated with the first, second, and third polycistronic cassettes of ‘565 or the claims of the instant application. Therefore, the naming of the first, second, and third polycistronic cassettes are considered arbitrary. As ‘565 identifies CD55 as being a part of one of the polycistrionic cassettes (the third polycistronic cassette), one could consider it as part of the first polycistronic cassette.
Regarding claim 188, ‘565 claims a genetically modified cell wherein the nucleic acid is integrated at a single genomic locus (claims 155 and 183). Although not specifically stated, integration of the non-endogenous genes in the porcine loci would require delivery of the nucleic acid to the cell. Therefore, the method of claim 188 of the instant application would have been obvious to generate the cell of ‘565.
Regarding claim 190, ‘565 claims wherein the genetically modified porcine cell lacks expression of an endogenous glycoprotein alpha-1,3- galactosyltransferase (GGTA1) gene, an endogenous 3-1,4-N-acetyl-galactosaminyltransferase 2 (p4GALNT2) gene, and an endogenous cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene (claim 205).
Regarding claims 192-195 and 198-201, ‘565 claims a genetically modified organ comprising the genetically modified porcine cell of claim 155 or 183 wherein the genetically modified organ is a liver, a heart, or a kidney and a genetically modified pig comprising the genetically modified porcine cell of claim 155 or 183 wherein the genetically modified pig is a genetically modified Yucatan pig or a genetically modified Yorkshire pig (claims 207-210).
This is a provisional nonstatutory double patenting rejection.
Claims 189, 191, 196-197, and 202 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims155 and 173-210 of copending Application No. 18/327,565 in view of Amendola et al. (Nature Biotechnology 23: 108-116. 2005) and Hwang et al. (Lipids 53: 933-945. 2018) as applied to claim 189 above and further in view of United States Patent Application No. 20180249688 (Ayares) and Dieckhoff et al. (Xenotransplantation 15: 36-45. 2008). This is a new rejection made in response to the new claims.
The teachings of ‘565, Amendola, and Hwang are as discussed above. ‘565 claims that each of the transgenes are human (claim 206).
The combined teachings of ‘565, Amendola, and Hwang do not teach using porcine cells modified to be free of porcine endogenous retroviruses.
However, Ayares teaches producing transgenic porcine animals by inserting a bicistronic nucleic acid encoding CD46 and CD55 (DAF) (Figure 16) and multicistronic nucleic acids encoding a combination of four transgenes encoding thrombomodulin (TBM), CD39, EPCR, CD55 (DAF), A20, TFPI, CD47, and HO-1 with either an ICAM-2 promoter or CAG promoter (different promoters) and a gene encoding a 2A peptide between genes within the same polycistronic cassette (i.e. under control of the same promoter) (paragraph 0518 and Figure 4).
Ayares teaches nucleic acids encoding CD46 and CD55 and encoding at least five additional transgenes. The additional transgenes are selected from an anticoagulant (TFPI, EPCR, CD39), an immunosuppressant (B2M, PD-L1, FasL, CD47, HLA-E), a cytoprotective gene (A20 and HO-1) or combination thereof (paragraphs 0276, 0279, 0289, 0298, 0317, and 0335-0337).
Ayares teaches that their transgenic porcine animals (pigs) are a useful source of organs, organ fragments, tissues or cells for xenotransplantation (paragraphs 0085-0096).
Dieckhoff teaches that specified pathogen-free breeding of pigs can prevent transmission of most porcine microbes. However, porcine endogenous retroviruses (PERVs) are integrated in the porcine genome, and can be released as infectious particles from normal pig cells and can infect human cells in vitro. Retroviruses are tumorigenic and may cause immunodeficiencies in infected hosts. The risk of PERV infection has to be carefully assessed specifically in view of the fact that the human immunodeficiency viruses HIV-1 and HIV-2, causing AIDS in humans, are the result of multiple trans-species transmissions of retroviruses from non-human primates. Results of their study demonstrate a strong and long lasting inhibition of PERV mRNA expression in primary porcine fibroblasts using a lentiviral vector system expressing a PERV-specific shRNA and use in somatic cell nuclear transfer. This is the first report demonstrating shRNA mediated reduction of PERV expression in shRNA-transgenic pigs in vivo. The strategy applied here could lead to microbiological safer porcine xenotransplants and is therefore of considerable medical importance (page 3, paragraphs 1-2 and page 7, paragraph 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the pig cell of the combined teachings of ‘565, Amendola, and Hwang by modifying the porcine cell to be free of porcine endogenous retroviruses 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 ‘565 teaches their porcine cells comprise human transgenes and Ayares identifies that the cells expressing the human transgenes of ‘565 can be used for xenotransplantation and Dieckhoff teaches that porcine endogenous retroviruses (PERVs) are integrated in the porcine genome, and can be released as infectious particles from normal pig cells and can infect human cells in vitro. Retroviruses are tumorigenic and may cause immunodeficiencies in infected hosts. Dieckhoff teaches that results of their study demonstrate a strong and long lasting inhibition of PERV mRNA expression in primary porcine fibroblasts using a lentiviral vector system expressing a PERV-specific shRNA and use in somatic cell nuclear transfer. The strategy applied here could lead to microbiological safer porcine xenotransplants and is therefore of considerable medical importance. Therefore, it would have been obvious to use porcine cells free of porcine endogenous retroviruses. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
This is a provisional nonstatutory double patenting rejection.
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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/KEENAN A BATES/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631