DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
The instant application is a U.S. patent application, filed 06/28/2024. Acknowledgement is made of the applicant’s claim for benefit to U.S. Provisional Application 63/511,413, which was filed 6/30/2023.
Election/Restrictions
It is noted that in the submission filed 08/07/2026, claims 2, 24 and 25 have been amended, wherein claims 24 and 25 have been amended to depend upon claim 1. Claim 26 has been new added, which depends upon claim 1 indirectly.
Applicant’s election without traverse of group I, claims 1-21 and 24-26, drawn to a method of making an at least partially recellularized liver composition, in the reply filed on 08/07/2026 is acknowledged. Accordingly, claims 1-21 and 24-26 have been considered on the merits. Claims 22 and 23 are withdrawn from consideration pursuant 37 CFR 1.142(b).
Claim Objections
Claim 3 is objected to because of the following informalities: Claim 3 recites an abbreviation “kGy”. Abbreviations should be spelled out at the first encounter in the claims. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2-3 and 17-18 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 2 recites “the anti-viral treatment comprises irradiation of the decellularized extracellular matrix” renders instant claim indefinite. Claim 2 depends upon claim 1, while claim 1 recites “anti-viral treatment” in step (a), which is before the step of obtaining a decellularized extracellular matrix. It is not clear how the “anti-viral treatment” in step (a) be conducted on the decellularized extracellular matrix obtained in step (b). Therefore the recitation of “the anti-viral treatment comprises irradiation of the decellularized extracellular matrix” in claim 2 makes the scope of the claim indefinite.
Claim 3 depends from claim 2, and thus inherits the deficiency and is rejected on the same basis.
Similarly, claim 17 recites “the anti-viral treatment comprises irradiation of the decellularized extracellular matrix with an electron beam (E-beam)” renders instant claim indefinite. Claim 17 depends upon claim 13, claim 13 recites “anti-viral treatment” in step (a), which is before the step of obtaining a decellularized extracellular matrix. Based on the same reason stated above, the recitation of “the anti-viral treatment comprises irradiation of the decellularized extracellular matrix” in claim 17 makes the scope of the claim indefinite.
Claim 18 depends from claim 17, and thus inherits the deficiency and is rejected on the same basis.
Claim Rejections - 35 USC § 112(a)
Written Desciption
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-21 and 24-26 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
From M.P.E.P. § 2163, the analysis of whether the specification complies with the written description requirement calls for the examiner to compare the scope of the claim with the scope of the description to determine whether applicant has demonstrated possession of the claimed invention from the standpoint of one of skill in the art at the time the application was filed. For inventions in emerging and unpredictable technologies, or for inventions characterized by factors not reasonably predictable which are known to one of ordinary skill in the art, more evidence is required to show possession. For claims drawn to a genus, possession may be shown (for example) through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. A “representative number of species” means that the species which are adequately described are representative of the entire genus, and is an inverse function of the skill and knowledge in the art. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to
reflect the variation within the genus. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus. See, e.g., Eli Lilly. If a representative number of adequately described species are not disclosed for a genus, the claim to that genus must be rejected as lacking adequate written description under 35 U.S.C. 112, para. 1.
Instant claims are directed to a method of making an at least partially recellularized liver composition, the method comprising: (a) treating a non-human animal liver with an anti-viral treatment; (b) perfusion decellularizing the non-human animal liver to obtain a decellularized extracellular matrix; (c) contacting the decellularized extracellular matrix with a cell composition comprising a population of human liver cells to form an at least partially recellularized liver composition. The claims recite non-human animals that include ANY animals exclusive of humans, which cover a genus of animal species including ALL members of the animal kingdom except humans, including rodents (such as mice and rats), non-human primates, large animals (such as dogs, pigs and sheep), as well as simpler organisms such as insects, zebrafish, fruit fly, worms and frogs. Even the non-human mammals are broadly including marine mammals such as dolphins, seals and whales, flying mammals such as bats. Therefore the scope of the claims is extremely broad. The issue at hand is whether or not Applicants have possession of the full scope of the genus of non-human animal liver in the method of making an at least partially recellularized liver composition, as recited in instant claims.
In the instant case, Applicants have failed to provide disclosure of species which are representative of the full scope of the genus of the animals exclusive of humans. The dependent claim 11 further limit the non-human animal as a non-human mammal. The specification only discloses porcine (i.e., see Examples) as the non-human animal liver in the method. There is no other species disclosed in instant application regarding the non-human animal liver to form a partially recellularized liver composition in instant application.
Furthermore, Applicant has failed to provide disclosure of relevant, identifying characteristics, such as the functional characteristics (be able to at least partially recellularized and have liver functionality such as glucose consumption) coupled with known or disclosed structure of non-human animal liver (liver structure after decellularization and then recellularization). While Applicants have recited the functional characteristics (e.g., metabolism such as glucose consumption by the at least partially recellularized liver), Applicants have failed to identify or show a known correlation exists between the functional characteristics changes and the liver structures and/or bioactivity in a representative species of animals. Given the genus of animals exclusive of humans cover such broad species which have structurally different liver tissue and cells, one of ordinary skill in the art, in looking to the instant specification, would not be able to determine that Applicants were in possession of the invention, as claimed, at the time the invention was made.
Accordingly, the claims are considered to lack sufficient written description and are properly rejected under 35 USC 112(a).
Scope of Enablement
Claims 1-21 and 24-26 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for using animals such as rat, mouse, porcine, sheep, and cattle, does not reasonably provide enablement for ANY animals exclusive of humans, such as an insect, as an non-human animal to produce an at least partially recellularized liver composition. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
In determining whether Applicant’s claims are enabled, it must be found that one of skill in the art at the time of invention by applicant would not have had to perform “undue experimentation” to make and/or use the invention claimed. Such a determination is not a simple factual consideration, but is a conclusion reached by weighing at least eight factors as set forth in In re Wands, 858 F.2d at 737, 8 USPQ 1400, 2d at 1404. Such factors are: (1) The breadth of the claims; (2) The nature of the invention; (3) The state of the art; (4) The level of one of ordinary skill in the art; (5) The level of predictability in the art; (6) The amount of direction and guidance provided by Applicant; (7) The existence of working examples; and (8) The quantity of experimentation needed to make and/or use the invention.
The office has analyzed the specification in direct accordance to the factors outlines in Jn re Wands. MPEP 2164.04 states: “[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection.” These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform “undue experimentation” to make and/or use the invention and therefore, applicant’s claims are not enabled.
Nature of the Invention:
The claims are directed to a method of making an at least partially recellularized liver composition, the method comprising: (a) treating a non-human animal liver with an anti-viral treatment; (b) perfusion decellularizing the non-human animal liver to obtain a decellularized extracellular matrix; (c) contacting the decellularized extracellular matrix with a cell composition comprising a population of human liver cells to form an at least partially recellularized liver composition. Claim 11 further limits the non-human animal is a non-human mammal.
Breadth of the claims:
The claims broadly embrace a genus of non-human animals that includes ANY animals exclusive of humans, which cover a genus of animals including any member of the animal kingdom, including rodents (such as mice and rats), non-human primates, large animal models (such as dogs, pigs and sheep), as well as simpler organisms such as insects, zebrafish, fruit fly, worms and frogs. Even the Non-human mammals are broadly include marine mammals such as dolphins, seals and whales, flying mammals such as bats. Thus the scope of the claims is extremely broad.
Guidance of the Specification and The Existence of Working Examples:
The specification does not provide disclosure regarding the species of non-human animal as a liver donor to make an at least partially recellularized liver composition. The Examples only use porcine as the liver donor for all the experiments, functional test and analysis. There is no other species of the non-human animal liver donor disclosed in instant application.
State of the Art and Predictability of the Art and the Amount of Experimentation Necessary:
The state of the art regarding using non-human animal liver in the method of making an at least partially recellularized liver composition is summarized by references including Wang et al. (Cells Tissues Organs 2017;204: 12.5—136, cited in IDS), Dai et al. (Front Bioeng Biotechnol. 2022 Feb 10;10:831477) and Toprakhisar et al. (Cells. 2023 Jan 13;12(2):301). Wang et al. review methods for the generation of a functional hepatic replacement, and highlights the most recent advances made in the field of liver decellularization and recellularization (Abstract). Wang et al. teach representative liver decellularization techniques (see i.e., Table 1), which using procine, rat and mouse in the method of liver decellularization. Dai et al. review the liver, hepatic ECM, decellularization, recellularization, and recent advances in liver engineering (see p1-p2). Dai et al. teach decellularized scaffolds from xenogeneic animals (such as pigs, sheep, and cattle) serve as a reservoir for growth factors, cytokines, and signaling molecules that are critical and indispensable for cellular growth, proliferation, differentiation, and neovascularization (see p1). Toprakhisar et al. review recent advancements in relation to liver whole-organ engineering (Abstract). Toprakhisar et al. teach decellularized mouse or rat livers (p8, parag 1) and decellularized porcine liver (p10, parag 3). None of the references teach using non-human animals such as insects, worms, or non-human mammals such as bats or dolphins in the method of making an at least partially recellularized liver composition. Therefore, in view of the state of the art regarding non-human animal liver for decellularization and recellularization, the lack of specific guidance in the specification for providing a representative species of non-human animals as the liver donors in the claimed method, regarding the breadth of the claims, it would have required undue experimentation to make the breadth of non-human animals as claimed.
In conclusion, in view of breadth of the claims and absence of a strong showing by Applicant, in the way of specific guidance and direction, and/or working examples demonstrating the same, such invention as claimed by Applicant is not enabled commensurate with full scope. An artisan of skill would have required undue experimentation to practice the invention without reasonable expectation of success.
Claim Interpretation
As stated above, claims 2 and 17 are indefinite. In the interest of compacted prosecution, claims 2 and 17 are interpretated as the anti-viral treatment comprises irradiation of the non-human animal liver with an electron beam (E-beam).
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.
Claims 1-11, 13, 16-20 and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Ross et al. (US 2021/0322642 A1, published 10/21/2021, cited in IDS), evidenced by ChEBI (online 8/17/2021), in view of Makowski et al. (Allografts, published in 2018) and Baptista et al. (Hepatology. 2011 Feb;53(2):604-17, cited in IDS), evidenced by Schmelzer et al. (J Exp Med. 2007 Aug 6;204(8):1973-87).
Ross et al. teach recellularized livers prepared from decellularized liver extracellular matrices, as well as methods of recellularizing livers from decellularized liver extracellular matrices (Abstract).
Regarding claim 1, Ross et al. teach methods of preparing at least partially recellularized livers (parag 30). Methods comprise providing a perfusion decellularized extracellular matrix of a decellularized mammalian liver in media, introducing a first solution that comprises a population of
endothelial cells to a perfusion decellularized extracellular matrix (parag 0013). Ross et al. also teach in Example 11, whole livers were decellularized by perfusion with a series of detergent containing buffers to generate the extracellular matrix scaffolds, seeded with human umbilical vein endothelial cells (HUVECs) and primary hepatocytes, wherein the primary hepatocytes are from porcine. In sum, the teachings above teach a method of making an at least partially recellularized liver composition, the method comprising: (provide a non-human animal, i.e., porcine), (b) perfusion decellularizing the non-human animal liver to obtain a decellularized extracellular matrix; (c) contacting the decellularized extracellular matrix with a cell composition comprising a population of (porcine) liver cells (as well as endothelial cells, i.e., human umbilical endothelial cells) to form an at least partially recellularized liver composition.
Instant claim differs from Ross et al. that: (1) Ross et al. teach a decellularized organ or tissue can be further treated with, for example, irradiation (e.g., UV, gamma) to reduce or eliminate the presence of any type of microorganism remaining on or in a decellularized organ or tissue (parag 0063), while instant claim teach (a) treating a non-human animal liver with an anti-viral treatment (before perfusion decellularizing the non-human animal liver); and (2) Ross et al. teach contacting the decellularized extracellular matrix with a cell composition comprising a population of endothelial, i.e., seeded with HUVECs and porcine primary hepatocytes (see Example 11), while instant claim teaches contacting the decellularized extracellular matrix with a cell composition comprising a population of human liver cells (i.e., human primary hepatocytes) to form an at least partially recellularized liver composition. However, such was disclosed by Makowski et al. and Baptista et al. at the time of instant application.
Makowski et al. teach two validated methods of tissue sterilization: gamma radiation and E-beam radiation (parag 1).
Baptista et al. teach the fabrication of three-dimensional, naturally derived scaffolds with an intact vascular tree. Livers from different species were perfused with detergent to selectively remove the cellular components of the tissue while preserving the extracellular matrix components and the intact vascular network (abstract).
Regarding (1), Makowski et al. teach both gamma and E-beam radiation are delivered in quantifiable doses validated to kill or inactivate specific harmful microorganisms including bacteria, viruses, and fungi (p2, parag 2). Both gamma and E-beam radiation are extremely safe sterilization methods; neither method results in any lingering radioactivity in the sterilized allograft (p3, parag 4).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ross et al.’s methods of preparing at least partially recellularized livers, and treat a non-human animal liver with an anti-viral treatment such as gamma and E-beam radiation as taught by Makowski et al.. The skilled artisan would have been motivated to use gamma and E-beam radiation to kill or inactivate specific harmful microorganisms including bacteria, viruses, and fungi in the liver without resulting in any lingering radioactivity in the sterilized tissue to obtain a sterilized tissue for further treatment. There would be a reasonable expectation of success of using gamma and E-beam radiation to the liver tissue since Makowski et al. teach the dose of gamma radiation (15-20kGy) and E-beam sterilization (9-21 KGy) (p2, parag 4 and p3, parag 2).
Regarding (2), Baptista et al. teach reseed the decellularized liver scaffolds of different species (mice, rats, ferrets, rabbits, and pigs) with human fetal liver and endothelial cells (see p606, right column-p607, left column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ross et al.’s methods of preparing at least partially recellularized livers, and conduct the recellularization of decellularized liver scaffolds with mixture of human endothelial cells and liver cells as taught by Baptista et al.. The only difference between instant claim and Ross et al.’s methods of preparing at least partially recellularized livers is instant claim seeding human liver cells (together with human endothelial cells). Given that Ross et al. teach hepatocyte function was enhanced in scaffolds seeded initially with HUVECs (parag 0108), and Baptista et al. teach reseeding the decellularized liver scaffolds of different species (mice, rats, ferrets, rabbits, and pigs) with human fetal liver and endothelial cells, one of ordinary skill in the art would have substituted Ross et al.’s co-seeding the decellularized liver scaffolds with HUVECs and porcine primary hepatocytes, and co-seeding the decellularized liver scaffolds with human fetal liver and endothelial cells according to their research interest. This simple substitution of one known element (co-seeding the decellularized liver scaffolds with human fetal liver and endothelial cells) for another known element (co-seeding the decellularized liver scaffolds with HUVECs and porcine primary hepatocytes) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding claims 2 and 3, following the discussion above, Makowski et al. teach E-beam sterilization of allografts at doses of 9-21 kGy has been shown to achieve a sterility assurance level (SAL) of 10-6, the highest level of terminal sterilization (parag 3, parag2), which is in the range of 2-50 kGy as recited in instant claims.
Regarding claim 4, Ross et al. teach in Example 3, the decellularized livers were disinfected with 1000 ppm peracetic acid (PAA) (parag 0115). The peracetic acid is a peroxy acid, which is evidenced by ChEBI (see p1, the definition in the box).
Regarding claim 5, as discussed above, Ross et al. teach a decellularized organ or tissue can be further treated with, for example, irradiation (e.g., UV, gamma) to reduce or eliminate the presence of any type of microorganism remaining on or in a decellularized organ or tissue (parag 0063), and teach the decellularized livers were disinfected with 1000 ppm peracetic acid (PAA) (parag 0115), which is a peroxy acid as evidenced by ChEBI (see p1, the definition in the box), but do not teach treating a non-human animal liver with an anti-viral treatment (E-beam). However, Makowski et al. teach both gamma and E-beam radiation are delivered in quantifiable doses validated to kill or inactivate specific harmful microorganisms including bacteria, viruses, and fungi (p2, parag 2). Both gamma and E-beam radiation are extremely safe sterilization methods; neither method results in any lingering radioactivity in the sterilized allograft (p3, parag 4).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ross et al.’s methods of preparing at least partially recellularized livers, and treat a non-human animal liver with an anti-viral treatment such as E-beam radiation as taught by Makowski et al.. The skilled artisan would have been motivated to use E-beam radiation to kill or inactivate specific harmful microorganisms including bacteria, viruses, and fungi in the liver without resulting in any lingering radioactivity in the sterilized tissue to obtain a sterilized tissue for further treatment. There would be a reasonable expectation of success of using E-beam radiation to the liver tissue since Makowski et al. teach the dose of E-beam sterilization (9-21 KGy) (p2, parag 4 and p3, parag 2).
Regarding claim 6, following the discussion above, Ross et al. teach a population of endothelial cells can be a population of human umbilical vein endothelial cells (HUVEC) (parag 0015) can be used in the method of preparing at least partially recellularized livers. For instance, Ross et al. teach in Example 11, whole livers were decellularized by perfusion with a series of detergent containing buffers to generate the extracellular matrix scaffolds, seeded with human umbilical vein endothelial cells (HUVECs) and primary hepatocytes.
Regarding claim 7, as discussed above, Ross et al. also teach in Example 11, whole livers were decellularized by perfusion with a series of detergent containing buffers to generate the extracellular matrix scaffolds, seeded with human umbilical vein endothelial cells (HUVECs) and primary hepatocytes, wherein the primary hepatocytes are from porcine. Ross et al. teach hepatocyte function was enhanced in scaffolds seeded initially with HUVECs (parag 0108). Baptista et al. teach reseed the decellularized liver scaffolds of different species (mice, rats, ferrets, rabbits, and pigs) with human fetal liver and endothelial cells (see p606, right column-p607, left column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ross et al.’s methods of preparing at least partially recellularized livers, and conduct the recellularization of decellularized liver scaffolds with mixture of human endothelial cells (i.e., HUVECs) and human liver cells as taught by Baptista et al.. The only difference between instant claim and Ross et al.’s methods of preparing at least partially recellularized livers is instant claim seeding human HUVECs and human liver cells on the decellularized liver scaffolds. Given that Ross et al. teach hepatocyte function was enhanced in scaffolds seeded initially with HUVECs (parag 0108), and Baptista et al. teach reseeding the decellularized liver scaffolds of different species (mice, rats, ferrets, rabbits, and pigs) with human fetal liver and endothelial cells, one of ordinary skill in the art would have substituted Ross et al.’s co-seeding the decellularized liver scaffolds with HUVECs and porcine primary hepatocytes, and co-seeding the decellularized liver scaffolds with human fetal liver and endothelial cells according to their research interest. This simple substitution of one known element (co-seeding the decellularized liver scaffolds with human fetal liver and endothelial cells) for another known element (co-seeding the decellularized liver scaffolds with HUVECs and porcine primary hepatocytes) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding claim 8, as discussed above, Baptista et al. teach using freshly isolated human fetal liver cells (p612, right column), which are primary human liver cells.
Regarding claim 9, following the discussion above, Baptista et al. teach obtaining human fetal liver cells, as described by Schmelzer et al. (p606, right column- p607, left column), wherein Schmelzer et al. provide the evidence of using (contacting to) type IV collagenase and deoxyribonuclease for processing liver tissues which comprise liver cells. Herein the collagenase is a protease.
Regarding claim 10, Ross et al. teach co-culture grafts exhibited enhanced ammonia clearance
kinetics and higher albumin production than hepatocyte only grafts (parag 0157). Perfusion of endothelial cells produces enhanced annnonia clearance relative to perfusion of hepatocytes alone (parag 0159).
Regarding claim 11, Ross et al. teach decellularization of rat liver (Example 2) and porcine liver (Example 3). Both rats and porcine are non-human mammals.
Regarding claim 13, following the discussion above, Ross et al. teach methods of preparing at least partially recellularized livers (parag 30). Methods comprise providing a perfusion decellularized extracellular matrix of a decellularized mammalian liver in media, introducing a first solution that comprises a population of endothelial cells to a perfusion decellularized extracellular matrix (parag 0013). Ross et al. also teach in Example 11, whole livers were decellularized by perfusion with a series of detergent containing buffers to generate the extracellular matrix scaffolds, seeded with human umbilical vein endothelial cells (HUVECs) and primary hepatocytes, wherein the primary hepatocytes are from porcine (but not human). In sum, the teachings above teach a method of making an at least partially recellularized liver composition, the method comprising: (provide a non-human animal, i.e., porcine), (b) perfusion decellularizing the non-human animal liver to obtain a decellularized extracellular matrix; (c) contacting the decellularized extracellular matrix with a first cell composition comprising a population of human vascular endothelial cells; and (d) contacting the decellularized extracellular matrix with a second cell composition comprising a population of porcine (but not human) liver cells to form an at least partially recellularized liver composition.
Instant claim differs from Ross et al. that: (1) Ross et al. teach a decellularized organ or tissue can be further treated with, for example, irradiation (e.g., UV, gamma) to reduce or eliminate the presence of any type of microorganism remaining on or in a decellularized organ or tissue (parag 0063), while instant claim teaches (a) treating a non-human animal liver with an anti-viral treatment (before perfusion decellularizing the non-human animal liver); and (2) Ross et al. teach contacting the decellularized extracellular matrix with a cell composition comprising a population of endothelial, i.e., seeded with HUVECs and porcine primary hepatocytes (see Example 11), while instant claim teach contacting the decellularized extracellular matrix with a cell composition comprising a population of HUVECs and human liver cells instep (d) to form an at least partially recellularized liver composition. However, such was disclosed by Makowski et al., Faulk et al. and Baptista et al..
Regarding (1), Makowski et al. teach both gamma and E-beam radiation are delivered in quantifiable doses validated to kill or inactivate specific harmful microorganisms including bacteria, viruses, and fungi (p2, parag 2). Both gamma and E-beam radiation are extremely safe sterilization methods; neither method results in any lingering radioactivity in the sterilized allograft (p3, parag 4).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ross et al.’s methods of preparing at least partially recellularized livers, and treat a non-human animal liver with an anti-viral treatment such as gamma and E-beam radiation as taught by Makowski et al.. The skilled artisan would have been motivated to use gamma and E-beam radiation to kill or inactivate specific harmful microorganisms including bacteria, viruses, and fungi in the liver without resulting in any lingering radioactivity in the sterilized tissue to obtain a sterilized tissue for further treatment. There would be a reasonable expectation of success of using gamma and E-beam radiation to the liver tissue since Makowski et al. teach the dose of gamma radiation (15-20kGy) and E-beam sterilization (9-21 KGy) (p2, parag 4 and p3, parag 2).
Regarding (2), Baptista et al. teach reseed the decellularized liver scaffolds of different species (mice, rats, ferrets, rabbits, and pigs) with human fetal liver and endothelial cells (see p606, right column-p607, left column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ross et al.’s methods of preparing at least partially recellularized livers, and conduct the recellularization of decellularized liver scaffolds with mixture of human endothelial cells (i.e., HUVECs) and liver cells as taught by Baptista et al.. The only difference between instant claim and Ross et al.’s methods of preparing at least partially recellularized livers is instant claim seeding human liver cells together with human endothelial cells in the decellularized liver scaffolds. Given that Ross et al. teach hepatocyte function was enhanced in scaffolds seeded initially with HUVECs (parag 0108), and Baptista et al. teach reseeding the decellularized liver scaffolds of different species (mice, rats, ferrets, rabbits, and pigs) with human fetal liver and endothelial cells, one of ordinary skill in the art would have substituted Ross et al.’s co-seeding the decellularized liver scaffolds with HUVECs and porcine primary hepatocytes, and co-seeding the decellularized liver scaffolds with human fetal liver and human endothelial cells according to their research interest. This simple substitution of one known element (co-seeding the decellularized liver scaffolds with human fetal liver and endothelial cells) for another known element (co-seeding the decellularized liver scaffolds with HUVECs and porcine primary hepatocytes) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding claim 16, Ross et al. teach co-culture grafts exhibited enhanced ammonia clearance
kinetics and higher albumin production than hepatocyte only grafts (parag 0157). Perfusion of endothelial cells produces enhanced annnonia clearance relative to perfusion of hepatocytes alone (parag 0159).
Regarding claims 17 and 18, Makowski et al. teach E-beam sterilization of allografts at doses of 9-21 kGy has been shown to achieve a sterility assurance level (SAL) of 10-6, the highest level of terminal sterilization (parag 3, parag2), which is in the range of 2-50 kGy as recited in instant claims.
Regarding claim 19, Ross et al. teach in Example 3, the decellularized livers were disinfected with 1000 ppm peracetic acid (PAA) (parag 0115). The peracetic acid is a peroxy acid, which is evidenced by ChEBI (see p1, the definition in the box).
Regarding claim 20, as discussed above, Baptista et al. teach using freshly isolated human fetal liver cells (p612, right column), which are primary human liver cells.
Regarding claims 24-26, Ross et al. teach HUVEC seeding of decellularized liver (Example 14), glucose consumption rate (GCR) measured throughout the period of bioreactor culture exhibited sigmoidal kinetics and could be generally characterized by low (<20 mg/h), mid (20-45 mg/h), and high (>45 mg/h) GCR phases with evidence of primary engraftment in larger vessels and subsequent expansion and migration into the parenchymal or sinusoidal niche at mid and high GCRs (parag 0176). Figure 6C shows the glucose consumption, the range of high can be 45-90 mg/h. Ross et al. teach peak (PGCR) and end glucose consumption rate (EGCR) are good markers to predict subsequent percentile blood flow (parag 0020), also teach co-culture grafts (seeding HUVEC and hepatocytes) maintained stable flow rates when perfused with blood, suggesting significant reendothelialization and the presence of a functional vasculature. These teachings indicate that the cell composition has a glucose consumption rate of 0-90 mg/h (i.e., at least about 30 mg/hr or about 70 mg/hr), depends on the days of culturing and growing of the seeded HUVEC and hepatocytes (i.e., migration of seeded cells into the parenchymal or sinusoidal niche leads to high GCR, see figure 6C).
Claims 1-11, 13-20 and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Ross et al. (US 2021/0322642 A1, published 10/21/2021, cited in IDS), evidenced by ChEBI (online 8/17/2021), in view of Makowski et al. (Allografts, published in 2018) and Baptista et al. (Hepatology. 2011 Feb;53(2):604-17, cited in IDS), evidenced by Schmelzer et al. (J Exp Med. 2007 Aug 6;204(8):1973-87), as applied to claims 1-11, 13, 16-20 and 24-26 above, further in view of Ross et al. (WO 2021/113747, hereinafter Ross747’).
The teaching of Ross et al., Makowski et al. and Baptista et al. is set forth above.
Regarding claims 14 and 15, Ross et al., Makowski et al. and Baptista et al. do not teach the decellularized extracellular matrix is contacted with the second cell compositions when the first cell composition is characterized as having a glucose consumption rate of at least about 30 mg/hr and 10 days after contacting the decellularized extracellular matrix with the first cell composition. However, such was disclosed by Ross747’ at the time of instant invention.
Ross747’ teaches compositions and methods to decellularize an isolated organ or portion thereof (Abstract).
Regarding claims 14 and 15, Ross747’ teaches characterization of bioengineered liver (BEL) constructs seeded with primary endothelial cells and hepatocytes, it was previously reported that daily glucose consumption rates (GCR) provide a robust, non-invasive metric for monitoring cell proliferation in HUVEC-seeded bioengineered liver (BEL) constructs and is predictive of successful perfusion outcomes in vivo. BELs were cultured until a minimum GCR of 50 mg/h was observed (typically 13-16 days following HUVEC seeding) prior to infusing hepatocytes into the scaffold. Ross747’ also teaches a method comprising introducing a second exogenous population of cells into an at least partially recellularized isolated organ or portion thereof comprising a first exogenous population of engrafted cells, wherein, prior to the introduction of the second population of exogenous cells, at least a portion of the first exogenous population of engrafted cells is functional as determined by: glucose consumption at a rate of at least about 10 mg/h (i.e., parag 0005). The teaching indicates that the GCR of 10-50 mg/h is suitable for seeding the second cell population. Ross747’ also teaches seeding second cell population 10 days after HUVECs seeding (see parag 0311).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to Ross et al.’s methods of preparing at least partially recellularized livers, and use GCR of 10-50 mg/h (i.e., 30 mg/h) to decide the first exogenous population of engrafted cells is functional and second cell population can be seeded, and /or seed the second cell population 10 or 13-16 days after HUVECs seeding as taught by Ross747’. The skilled artisan would have been motivated to monitor the GCR to confirm a successful HUVECs graft (GCR of at least 10 mg/h, i.e., 30 mg/h or 50 mg/h) and then seed the second cell population (i.e., 10 or 13-16 days after HUVECs seeding). There would be a reasonable expectation of success of having GCR of 30 mg/h and/ or 10 days after HUVECs seeding for further seeding the second cell population since Ross747’ teach the standard of GCR as well as timing for seeding the second cell population. In addition, it is not inventive to find optimal workable ranges by routine experimentation. Generally, differences in concentration or temperature (or herein the glucose consumption rate for determining the timing of seeding the second cells) will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." See Jn re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Claims 1-13, 16-21 and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Ross et al. (US 2021/0322642 A1, published 10/21/2021, cited in IDS), evidenced by ChEBI (online 8/17/2021), in view of Makowski et al. (Allografts, published in 2018) and Baptista et al. (Hepatology. 2011 Feb;53(2):604-17, cited in IDS), evidenced by Schmelzer et al. (J Exp Med. 2007 Aug 6;204(8):1973-87), as applied to claims 1-11, 13, 16-20 and 24-26 above, further in view of Mazza et al. (Sci Rep. 2015 Aug 7;5:13079, cited in IDS).
The teaching of Ross et al., Makowski et al. and Baptista et al. is set forth above.
Regarding claims 12 and 21, Ross et al., Makowski et al. and Baptista et al. do not teach the non-human animal liver is frozen prior to the anti-viral treatment. However, such was disclosed by Mazza et al. at the time of instant invention.
Mazza et al. teach decellularization of human liver and repopulation with derived human liver cells (Abstract).
Regarding claims 12 and 21, Mazza et al. teach human livers were then frozen at − 80 °C for at least 24 h for the purposes of initial destruction of the various cellular compartments (p11, parag 7).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Ross et al.’s methods of preparing at least partially recellularized livers, and have the non-human animal liver frozen prior to the anti-viral treatment as taught by Mazza et al.. The only difference between instant claims and Ross et al.’s methods of preparing at least partially recellularized livers is instant claims have the non-human animal liver frozen prior to the anti-viral treatment. Given that Mazza et al. teach human livers can be frozen at − 80 °C for at least 24 h for the purposes of initial destruction of the various cellular compartments, one of ordinary skill in the art would have substituted Ross et al.’s fresh liver for frozen liver for decellularization as needed. This simple substitution of one known element (frozen liver for decellularization) for another known element (fresh liver for decellularization) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Conclusion
No claims are allowed.
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/Q.G./Examiner, Art Unit 1633
/FEREYDOUN G SAJJADI/Supervisory Patent Examiner, Art Unit 1699
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