Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions and Claim Status
Election without traverse to Group I in the reply filed 12 Mar 2026 is acknowledged.
Amendments to the claims filed 12 Mar 2026 are acknowledged. Claims 1-11 and 13-347 are canceled, claims 348-368 are newly added, and claims 363 and 367-368 are withdrawn by applicant. Claims 12 and 348-368 are pending. Claims 12, 348-362, and 364-366 are under consideration.
Information Disclosure Statement
It appears that in the IDS filed 11 Jan 2024 that the U.S. Patent Document citation no. 1 should be US 8,569,573 B2, not US 8,569,572 B2.
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.
Claim 365 is rejected under 35 U.S.C. § 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Regarding claim 365, the phrase "at least about" renders the claim indefinite because it is unclear whether the limitation encompasses transplantation of less than ten million hepatocytes. From the specification, the term "about" encompasses ± 5%. The phrase "at least" indicates an absolute minimum of not less than 10 million, whereas the term "about" indicates that the claim encompasses an amount between 9.5 million and 10 million. Therefore, the lower limit is unclear. See MPEP § 2173.05(b).
Claim Rejections - 35 USC § 112
The following is a quotation 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.
Claim 12, 348-362, and 364-366 are rejected under 35 U.S.C. § 112(a) because the specification, while being enabling for
A genetically modified mouse comprising inactivated endogenous Rag2, Il2rg, and Fah genes,
wherein the genetically modified mouse comprises transplanted human hepatocytes,
wherein the genetically modified mouse is modified to express human IL-6, human OSM, or humanized IL-6, or the transplanted human hepatocytes are modified to express mouse IL-R, rat IL-6R, or constitutively active GP130, and
wherein the transplanted human hepatocytes have reduced lipid droplet accumulation compared to wild-type transplanted human hepatocytes in control genetically modified mouse comprising inactivated endogenous Rag2, Il2rg, and Fah genes,
does not reasonably provide enablement for
species of non-human animals other than mice,
modifications to restore IL-6/IL-6R or GP130 signaling pathway activity by expression of other IL-6 family ligands, expression of OSMR, or other approaches to activate the IL-6 family receptors IL-6 or GP130, or
modifications to restore IL-6/IL-6R or GP130 signaling pathway activity downstream of the IL-6 family receptors IL-6R or GP130.
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 or use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). The court in Wands states, “Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word ‘undue’, not ‘experimentation.’” (Wands, 8 USPQ2d 1404). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighing many factual considerations." (Wands, 8 USPQ2d 1404). When determining whether a specification meets the enablement requirement, some of the factors to be analyzed are: (1) the breadth of the claims, (2) the nature of the invention, (3) the state of the prior 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 provided by the inventor, (7) the existence of working examples, and (8) whether the quantity of any necessary experimentation to make and use the invention based on the content of the disclosure is undue (Wands). While all of these factors are considered, those sufficient for establishing a prima facie case are discussed below.
Nature of the invention
The invention pertains to a transgenic non-human animal with an FRG genetic background with a humanized liver and modifications to the interleukin 6 (IL-6)/ interleukin 6 receptor (IL-6R) and/or interleukin-6 receptor-associated membrane glycoprotein 130 (GP130) signaling pathways to correct for cross species incompatibility between IL-6 ligands and receptors that without correction results in lipid droplet accumulation in the transplanted human hepatocytes.
The genetic background of the animal is based on the FRG mouse. Inactivation of endogenous recombination activating 2 (Rag2) and interleukin 2 receptor gamma (Il2rg) (The R and G in FRG, respectively) renders the animal immunodeficient to allow transplantation of xenogeneic cells. Inactivation of endogenous fumarylacetoacetate hydrolase (Fah, the F in FRG) induces hepatoxicity in endogenous liver cells, which allows for transplanted hepatocytes with intact Fah to overtake and repopulate the host liver.
Breadth of the claims
Issue 1: With regard to claims 12, 348-360, and 364-366, the claims are broad with respect to species of non-human animal. Under the broadest reasonable interpretation, the claims cover any non-human animal with endogenous wild-type Rag2, Il2rg, and Fah genes.
Issue 2: With regard to claims 12, 348-350, 354-355, 360-362, and 364-366, the claims are broad with respect to what modifications may be used to restore IL-6/IL-6R and/or GP 130 signaling pathway activity. This activity may be influenced by the addition of transgenes to the host or transplanted hepatocyte genomes, modification to how endogenous genes are expressed, transduction of genes not incorporated into the genome, modification to translation of pathway members, modification of localization or degradation of pathway members, addition of small molecules or other chemical to change activity of signaling pathway proteins directly, or other mechanisms.
Issue 3: With regard to claims 12, 348-350, 354-355, 360-362, and 364-366, the claims are broad with respect to what aspect of IL-6/IL-6R and/or GP 130 signaling pathway activity is restored by the modification. In addition to modifications to the IL-6/IL-6R, OSM/OSMR, or GP130 proteins themselves, the breadth of the claim includes modifications to the activity of any protein or other molecule downstream of the IL-6, OSMR, or GP130 receptors.
Existence of working examples
Regarding Issue 1, the specification provides working examples for FRG and FSRG mouse and rat with humanized liver (Examples 1-6, par. 260-271 and Methods, par. 272). The specification does not provide working examples for other species of non-human animal.
Regarding Issue 2, the specification provides working examples of modifications with respect to IL-6/IL-6R or GP130 signaling pathway in the genetically modified mouse or rat or in the transplanted human hepatocytes.
Some humanized liver mice and rats were transplanted with human hepatocytes expressing rodent IL-6R (Example 2, par. 262-263 and Fig. 2C and 2E). Cross species compatibility between IL-6 and IL-6R is required for reduction or prevention of lipid droplet accumulation in transplanted human hepatocytes (Fig. 2C , 2E, and 5A). Some transplanted human hepatocytes were modified to express a constitutively active GP130 with Y186-Y190 deletion (CA-GP130) via lentiviral transduction, which also resulted in reduced lipid droplet accumulation (Example 3, par. 264 and Fig. 2F-2G).
Some humanized liver mice or rats were treated with ectopic human IL-6 or OSM via adenovirus transduction (Examples 4-5, par. 266-268, Fig 5A-5B and 14D) or transplantation of human Kupffer cells expressing IL-6 (Example 6, par. 269). Some humanized liver mice were treated with ectopic human oncostatin M (OSM) via adenovirus transduction (Example 5, par. 268). Some humanized liver mice were genetically modified to express humanized non-human IL-6 (Example 4 par. 267). In each of these examples, expression of a human or humanized IL-6 or human OSM reduced lipid droplet accumulation in the transplanted human hepatocytes compared to control mice. However, wild-type mouse IL-6 is insufficient to reduce lipid droplet accumulation in unmodified transplanted human hepatocytes in humanized liver mice (Example 4, par. 267 and Fig. 4).
The working examples provided are drawn only to expression of human or humanized non-human IL-6, human OSM, mouse or rat IL-6R, and CA-GP130 as modifications to restore IL-6/IL-6R or GP130 signaling. The working examples do not include expression of rodent OSMR in transplanted human hepatocytes, ectopic expression of other human IL-6 family ligands, or other any other mechanism of inducing IL-6/IL-6R or GP130 signaling pathway activity. Furthermore, the working examples show that the phenotype of reduced lipid droplet accumulation in the transplanted human hepatocytes does not occur when mouse or rat IL-6 are overexpressed; therefore, modifications that reduce lipid droplet accumulation are limited to restoring species compatibility between IL-6 ligands and receptors.
Regarding Issue 3, humanized liver mice and rats treated with exogenous IL-6 and comprising a compatible IL-6R (human IL-6 and human IL-6R, or rodent IL-6 and rodent IL-6R) or treated with exogenous OSM show phosphorylation of signal transducer and activator of transcription 3 (STAT3) (Example 2, par. 262 and Fig. 2A-B) and expression of STAT3 target genes (Example 5, par. 268, Fig. 11C, 12C, and 14C). STAT3 is widely known in the art as a downstream effector of IL-6R and GP130. These findings show a correlation between STAT3 activation and lipid accumulation, but it is unclear whether phosphorylation and activation of STAT3 merely serve as biomarkers of IL-6/IL-6R OR GP130 signaling pathway activity, or if STAT3 activation contributes to, or is required for, the observed phenotype of reduced lipid accumulation. Furthermore, the specification does not show data regarding changes in downstream effectors of IL-6R or GP130 other than STAT3 or contemplate how the working examples to modify IL-6/IL-6R or GP130 may affect other downstream effectors. Therefore, the specification does not provide working or prophetic examples of modifications to IL-6/IL-6R or GP130 signaling pathway activity downstream of the IL-6 family receptors IL-6R or GP130.
Amount of direction provided by the inventor
Regarding Issue 1, the specification provides examples of non-human animal species that may be used as hosts for transplanting human hepatocytes (par. 136 and 166), but does not provide for ways to overcome barriers in the art when practicing the invention in non-human animal species other than mice and rats.
Regarding Issue 2, the specification provides examples of modifications to restore IL-6/IL-6R or GP130 signaling pathway activity, including lists of ligands for IL-6R and GP130 (par. 156 and 177), but does not describe whether or how the modes of action of the ligands may differ those provided in the working examples or how to adapt the invention when using a different ligand. Furthermore, the specification does not contemplate other modifications to restore IL-6/IL-6R signaling pathway activity, such as small molecule activators or inhibitors.
Regarding Issue 3, the specification notes that that OSM/OSMR signaling activates STAT3 downstream (par. 148) and that GP130 homodimerization results in activation of the Janus kinase (JAK)- mitogen activated protein kinase (MAPK) and JAK-STAT3 signaling pathways (par. 153). However, these statements appear to be generic background information what is known in the art about these signaling pathways. The specification does not contemplate activation of other downstream signal pathways or which, if either, of the JAK-MAPK or JAK-STAT3 pathways are associated with the phenotype of lipid accumulation in transplanted human hepatocytes. Therefore, the specification does not provide direction by which a skilled artisan could predict whether modification of downstream effectors of the IL-6/IL-6R or GP130 signaling pathways would result in restoration of IL-6/IL-6R/GP130 signaling pathway activity or in the observed phenotype of reduced lipid accumulation in transplanted human hepatocytes.
State of the prior art and Level of predictability in the art
Regarding Issue 1, FRG non-human animals with transplanted human hepatocytes has been reported for mice and rats. Zhang (L. Zhang, et al., Adv Sci, 2021, cited in IDS filed 11 Jan 2024) teaches difficulty in generating an FRG rat with transplanted human hepatocytes. Administration of nitisinone (also referred to as NTBC in the art) alleviates hepatoxicity due to the absence of Fah, and withdrawal of nitisinone allows users of the invention to precisely control timing and degree of hepatotoxicity to optimize engraftment of transplanted hepatocytes. Zhang teaches that rats have a much higher mortality rate after withdrawal of nitisinone compared to mice and require a materially different protocol for inducing hepatoxicity when transplanting human hepatocytes (Introduction p. 2 and Results section 2.4 p. 5). Thus, transplantation of human hepatocytes into rats is present in the art, the art also teaches difficulty in doing so. Therefore, there is a high level of unpredictability when practicing the claimed invention in rats.
Zhao (H. Zhao, et al., Front Immunol, 09 Aug 2022) teaches challenges in generating an FRG pig model. Zhao reports generation of miniature pigs with an FRG background by somatic cell nuclear transfer shows that FAH deficiency in pigs results in in utero fetal death, a phenotype not observed in mice (Results p. 5 and Table 2). With nitisinone supplementation to overcome FAH deficiency, infertility or in utero mortality was observed in 11 of 12 recipient sows, and live birth was observed in 2 of 8 fetuses in the only sow to give live birth (Results p. 5 and Table 3). Of the two live births, neonates were not maintained on nitisinone after D3 and died within one month (Discussion p. 9). Zhao further notes that pigs with only inactivation of Rag2 also have very high mortality rates (Discussion p. 9).
Furthermore, Ren (J. Ren, et al., Cell Biosci, Mar 2022) teaches that generation of FRG pigs is more difficult than pigs with inactivation of Fah and Il2rg alone (FG pigs) (Abstract and Discussion p. 7). Ren successfully generated FG pigs and transplanted human hepatocytes that engrafted (Results pp. 2-3). Ren further generated FRG pigs, but like Zhao, observed high mortality rates, as well as very low birth weight and short survival time insufficient to allow for transplantation of human hepatocytes (Results p. 5 and Discussion p. 7). Thus, modification of FG pigs to further inactivate endogenous Rag2 presents an additional challenge to overcome in the art.
While FRG pigs have been generated as of the earliest effective filing date of the claimed invention of 02 Sep 2022, barriers to overcome high mortality rate and very short lifespan have not been overcome to allow use FRG pigs as hosts to transplant human hepatocytes. Findings by Zhao and Ren further illustrate challenges in making the invention as claimed, as species have varying degree of sensitivity to inactivation of Fah and Rag2. Zhao and Ren suggest that FRG pigs can be transplanted human hepatocytes, but not provide suggestions for how to overcome barriers to achieve transplantation.
Generation of animals with inactivation of endogenous Fah, Rag2, or Il2rg has been reported in in other species in isolation or in inactivation of two of the genes, but inactivation of all three genes has only been reported in mice, rats, and pigs. Furthermore, the art does not teach expression of IL-6, IL-6R, OSM, OSMR, or GP130 via genome modification, viral transfection, or transplant of Kupffer cells in FRG animals.
Regarding Issue 2, the art is silent with regard to the metabolic phenotype of FRG rats and mice transplanted with human hepatocytes. Furthermore, the role of IL-6 in liver lipid pathology and physiology remains controversial and context-dependent (Kang, p. 1009) (S. Kang, Immunity, 2019). IL-6 has been found to be both a positive and a negative regulator of lipid metabolism, and the various roles of environmental and dietary factors influencing disparate observations have not been resolved (Kang p. 1009). Furthermore, the IL-6 signaling family comprises 12 members, which stimulate the receptors IL-6R and GP130 through tissue- and cell-specific modes of action that result in different metabolic phenotypes (Giraldez pp. 787-789 and Fig. 5) (M.D. Giraldez, Nat Rev Gastroenterol Hepatol, 2021, hereafter Giraldez, cited in IDS filed 11 Jan 2024).
Therefore, due to the complex and controversial role of IL-6 family in liver lipid metabolism, a skilled artisan would be unable to predict success in making modifications to restore IL-6/IL-6R or GP130 signaling pathway activity to reduce lipid droplet accumulation in transplanted human hepatocytes by expression of other IL-6 family ligands or receptors not provided in the working examples or other modifications to activate IL-6R or GP130.
Regarding Issue 3, Giraldez teaches that because all IL-6 family cytokines signal through the GP130, GP130-mediated signaling is highly pleiotropic and plastic (p. 787). The IL-6 family signaling pathways affect multiple intracellular signaling pathways, including the MAPK, phosphoinositide 3-kinase (PI3K)- protein kinase B (Akt), yes-associated protein (YAP), Notch, and STAT3 signaling pathways (Giraldez Fig. 4 and Kang pp. 1008-1009), representing hundreds of proteins as downstream effectors regulating numerous cell activities. Giraldez further teaches that the mechanisms by which regulation of signaling pathway activities downstream of IL-6 family cytokines is achieved is unknown (p. 788).
Therefore, the numerous downstream effectors influenced by IL-6 family cytokines and incomplete characterization regarding how IL-6/IL-6R and GP130 regulate their activity, a skilled artisan would be unable to predict success in making modifications to restore IL-6/IL-6R or GP130 signaling pathway activity to reduce lipid droplet accumulation in transplanted human hepatocytes by modifying the activity of downstream effectors.
Conclusion
The specification provides several working examples that are narrowly enabling for the invention as claimed. However, the breadth of the claims encompasses numerous non-enabled embodiments and neither the specification nor the art provide sufficient direction for a skilled artisan to make and use the full scope of the claimed invention. A skilled artisan would have to overcome high mortality rate when generating FRG animals other than mice to practice transplantation of human hepatocytes, and the specification does not provide direction for how to do so. Furthermore, the specification only provides working examples for select members of the IL-6/IL-R and GP130 signaling pathway, and different members are known to act through different mechanisms. The specification does not provide a mechanism by which IL-6/IL-6R or GP130 signaling pathway downstream effectors results in reduced lipid droplet accumulation that a skilled artisan might use to predict success in using alternative approaches to restore IL-6/IL-6R or GP130 signaling pathway activity and achieve reduced lipid droplet accumulation in transplanted human hepatocytes. Furthermore, the art does not provide sufficient direction to overcome lack of direction from the specification due to the highly pleiotropic nature of those pathways and inconsistent observations in the art. A skilled artisan would have to untangle the contradictory findings with respect to the role of IL-6 family signaling in liver lipid metabolism to predict success in applying other approaches to restoring IL-6/IL-6R or GP130 signaling pathway activity in transplanted human hepatocytes. This constellation of challenges represents an undue burden of experimentation to a skilled artisan attempting to make or use the claimed invention.
Conclusion
No claim is allowed.
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/Eric B Wright/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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