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 .
Withdrawn Objections/Rejections
The objection to the drawings because the lines graphs in figure 11, figure 13, figure 20, figure 23, figure 24, figure 25, and figure 28 cannot be discerned is withdrawn. The supplemental drawing correct the issues of the objection.
The rejection of claims 54-58 and 72-89, 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention, is withdrawn. The amendments to the claims clarify or remove the issues of indefiniteness of record.
The rejection of claim(s) 54-57, 72, 75, 76, 83, 87, and 89, under 35 U.S.C. 102(a)(2) as anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Yang (US2022/0267805 A1 effectively filed 5/16/2019), is withdrawn. Applicant provided evidence of exception because Yang is co-owned with the instant application.
The rejection of claim(s) 54-57, 72, 75, 76, 83, 87, and 89, under 35 U.S.C. 102(a)(2) as anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Yang WO (WO2021/072777 A1 effectively filed 10/18/2019), is withdrawn. Applicant provided evidence of exception because Yang WO is co-owned with the instant application.
Claim Rejections - 35 USC § 112
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 54-58 and 72-89 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Claims 54-58 and 72-88, as amended or previously presented, 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 the following:
A method of reducing glucose level in an insulin resistant or deficient non-porcine mammal comprising administering 4-7 cells to the kidney capsule or liver of said non-porcine mammal, wherein said administering results in reducing blood glucose level in said non-porcine mammal, does not reasonably provide enablement for:
1) administering any genetically modified porcine cell having any genomic modifications that lead to the claimed expression of polypeptides and the substantial freedom of GGTA, B4GALNT2, and CMAH enzymatic activity; and
2) administering to any target tissue by any route of administration. 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.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make and use the claimed invention, if not, whether an artisan would require undue experimentation to make and use the claimed invention and whether working examples have been provided. When determining whether a specification meets the enablement requirements, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make and use the invention based on the content of the disclosure is “undue”.
Nature of Invention: Cell therapy comprising Xeno transplanting genetically modified porcine islet cells into a non-porcine subject to treat an insulin deficiency or insulin resistance. The genetic modification of the porcine cells is to make them biocompatible with the non-porcine subject.
Breadth of the Claims: The claims are moderately narrow in scope with areas of great breadth. The polypeptides to be expressed or suppressed are narrowly define. However the genetic make-up of the islet cells is broadly defined by the claims. The porcine islet cell is partially defined by functional attribute in that the cells are substantially free of enzymatic activity of GGTA, B4GALNT2, and CMAH. Porcine islet cell endogenously have this enzyme activity. Thus breadth encompass disrupting, downregulating, removing, inhibiting this enzyme activity by introducing any structural means into the islet cell that will cause such reduction/ablation. This includes small molecule inhibitor, anti-sense iRNA down regulation, genomic disruption by homologous recombination or random insertion using a gene editing or non-gene editing means among others that lead to transient or permanent reduction/ablation of the enzyme activity. The islet cells are further modified by introducing by the functional characteristics of expressing 7 non-porcine mammalian species polypeptides. This is structural exacted by being expressed from “a single construct if different cistrons”. This has been broadly interpreted as discussed above in the 112(b) rejection above. The B2M and HLAE are described as being expressed as a fusion, which is a functional limitation. The genetic make-up that leads to this fusion is not recited. As such, the breadth of the genetic structure is a nucleic acid encoding any configuration of a fusion of at least part of the B2M protein and at least part of the HLAE protein located in one cistronic unit of the single construct. The islet cell has an additional genetic modification that lead to the function of expressing addition non-porcine proteins. The genetic construct is defined as said single construct…wherein transgenes within each of the different cistrons are separated by skipping 2A peptide. Thus breadth of this recitation encompasses any transgene encoding any factor, protein, transcript or RNA element that directly or indirectly results in expression of the at least one polypeptide and defines the cistronic units as being separated by 2A peptide. This is also being interpreted as discussed above in the indefiniteness rejection. The breath of the additionally expressed protein of (b) and (c) are any non-porcine mammalian species of the protein. The claim does not specify the route of administration of the pharmaceutical composition comprising the porcine islet cells or the target location of the cells for transplantation. As such, the breadth encompasses any route of administration to any location in the host non-porcine mammal’s body. The claims narrowly specifies that result of the administration is to lower blood glucose levels which treats the insulin resistant or deficient condition. The claims also do not specify how one obtains the porcine islets cells that are genetically modified as claimed. As such, the breadth of the means of arriving at the cells encompasses in vivo transgenic pig production or in vitro cell construction from isolated pig islet cells that are not genetically modified.
Specification Guidance: citations from the pre-grant publication:
[0086] In some embodiments, any of the genetically modified cells, tissues or organs disclosed herein may be used to treat a subject of a different species as the genetically modified cells. In some embodiments, the disclosure provides for methods of transplanting any of the genetically modified cells, tissues or organs described herein into a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate.
[0087] The non-porcine mammalian species may be a primate species. In some embodiments, the non-porcine mammalian species is a non-human primate.
[0088] In some embodiments, the non-porcine mammalian species is Homo sapiens.
[0104] The non-porcine mammalian species may be a primate species. In some embodiments, the non-porcine mammalian species is a non-human primate. The non-human primate includes non-human living primates according to any or all of various classifications of non-human living primates, including, but not limited to, families Callitrichidae (marmosets and tamarins), Cebidae (New World monkeys), Cercopithecidae (Old World monkeys), Cheirogaleidae (dwarf lemurs and mouse lemurs), Daubentoniidae (aye-aye), Galagonidae (bushbabies and galagos), Hominidae (including great apes), Hylobatidae (gibbons and lesser apes), Indridae (indris, sifakas, and relatives), Lemuridae (true lemurs), Loridae (lorises), Megaladapidae (sportive lemurs), and Tarsiidae (tarsiers). The term “non-human primates” encompasses non-human primates and groups thereof classified according to any or all of various classifications of non-human living primates. For example, Wilson and Reeder (1993) split Megaladapidae from Lemuridae, Galagonidae from Loridae (and in spelling the latter Loridae rather than Lorisidae), and include the great apes in Hominidae. Wilson, D. E., and D. M. Reeder. 1993. Mammal Species of the World, A Taxonomic and Geographic Reference. 2nd edition. Smithsonian Institution Press, Washington. Anderson and Jones (1984) divide the order of living primates (Primates) into two suborders, the Strepsirhini and the Haplorhini. Thorington, R. W., Jr., and S. Anderson. 1984. Primates. Pp. 187-217 in Anderson, S. and J. K. Jones, Jr. (eds). Orders and Families of Recent Mammals of the World. John Wiley and Sons, N.Y. The Strepsirhines include mostly arboreal species with many primitive characteristics, but at the same time, some extreme specializations for particular modes of life, and wherein the Haplorhines are the so-called “higher” primates, further divided into two major groups, the Platyrrhini and the Catarrhini. Platyrrhines have flat noses, outwardly directed nasal openings, three premolars in upper and lower jaws, anterior upper molars with 3 or 4 major cusps, and are found only in the New World (families Cebidae and Callitrichidae). Catarrhines have paired downwardly directed nasal openings, which are close together; usually two premolars in each jaw, anterior upper molars with 4 cusps, and are found only in the Old World (Cercopithecidae, Hylobatidae, Hominidae). Most primate species live in the tropics or subtropics, although a few also inhabit temperate regions. Except for a few terrestrial species, primates are arboreal. Some species eat leaves or fruit; others are insectivorous or carnivorous. See Myers, P. 1999. “Primates” (On-line), Animal Diversity Web. Accessed Aug. 26, 2005.
[0105] In some embodiments, the non-porcine mammalian species is Homo sapiens.
[0106] The method of treating the insulin resistant or deficient condition in the non-porcine mammal in need thereof may involve the administration or transplant of any of the compositions, cells, organs, or tissues described herein. In some cases, when a cell composition is administered, the composition is centrally administered, e.g. is administered via an internal jugular vein or a hepatic portal vein of the non-porcine mammal.
[0080] In some embodiments, the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes has been modified by addition, deletion, inactivation, disruption, excision of a portion thereof, or a portion of the gene sequence has been altered.
[0081] In some embodiments, the cells, tissues, or organs of the disclosure comprise one or more mutations that inactivate one or more genes. In some embodiments, the cells, tissues, organs or animals comprise one or more mutations or epigenetic changes that result in decreased or eliminated expression of one or more genes having the one or more mutations. In some embodiments, the one or more genes is inactivated by genetically modifying the nucleic acid(s) present in the cells, tissues, organs or animals. In some embodiments, the inactivation of one or more genes is confirmed by means of an assay. In some embodiments, the assay is a reverse transcriptase PCR assay, RNA-seq, real-time PCR, or junction PCR mapping assay. In some embodiments, the assay is an enzymatic assay for the function of the gene protein or an immunoassay for a protein transcribed from the gene or a fragment of the gene.
[0082] The cells, tissues, or organs of the present disclosure can be genetically modified by any suitable method. Non-limiting examples of suitable methods for the knockout (KO), knockin (KI), and/or genomic replacement strategies disclosed and described herein include CRISPR-mediated genetic modification using Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Cas12 d, Cas12e, Cas12g, Cas12h, Cas12i, or other CRISPR endonucleases, Argonaute endonucleases, transcription activator-like (TAL) effector and nucleases (TALEN), zinc finger nucleases (ZFN), expression vectors, transposon systems (e.g., PiggyBac transposase), or any combination thereof. In some embodiments,
[0083] In some embodiments, the cells, tissues, or organs are substantially free of enzymatic activity of at least one glycosyltransferase enzyme, wherein said glycosyltransferase enzyme is GGTA, B4GALNT2, or CMAH. The cells, tissues, or organs can be substantially free of enzymatic activity of at least two glycosyltransferase enzymes selected from GGTA, B4GALNT2, and CMAH. The cells, tissues, or organs can be substantially free of enzymatic activity of three glycosyltransferase enzymes selected from GGTA, B4GALNT2, and CMAH. In some cases, the cells substantially free of enzymatic activity of at least one glycosyltransferase enzyme selected from GGTA, B4GALNT2, and CMAH are substantially free of detectable levels of a full-length copy of the glycosyltransferase enzyme protein. In some cases, the cells substantially free of enzymatic activity of at least one glycosyltransferase enzyme selected from GGTA, B4GALNT2, and CMAH are substantially free of detectable levels of a functional polypeptide fragment of the glycosyltransferase enzyme protein. In some cases, the cells substantially free of enzymatic activity of at least one glycosyltransferase enzyme selected from GGTA, B4GALNT2, and CMAH are substantially free of transcription of mRNA encoding the full-length glycosyltransferase enzyme. In some cases, the cells substantially free of enzymatic activity of at least one glycosyltransferase enzyme selected from GGTA, B4GALNT2, and CMAH are substantially free of transcription of mRNA encoding a functional fragment of the glycosyltransferase enzyme. In some cases, the cells substantially free of enzymatic activity of at least one glycosyltransferase enzyme selected from GGTA, B4GALNT2, and CMAH comprise an indel within an open reading frame of the at least one glycosyltransferase enzyme. The indel may be generated using site-directed nuclease. The indel may disrupt the open reading frame (ORF) (or in the case of a gene having multiple copies within the genome, all of the ORFs) of the at least one glycosyltransferase enzyme such that when the glycosyltransferase gene is transcribed, production of a full length or functional fragment mRNA or protein is prevented.
[0084] In some embodiments, the cells, tissues, or organs express at least two polypeptide sequences (e.g., at least two heterologous polypeptide sequences) derived from a non-porcine mammalian species, wherein said at least two polypeptide sequences comprise at least two of CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1. The cells, tissues, or organs may express at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or all of CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1. In some cases, the at least two polypeptide sequences derived from a non-porcine mammalian species comprise a full-length sequence of CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1, or a combination thereof. In some cases, the at least two polypeptide sequences derived from a non-porcine mammalian species comprise a functional fragment of CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1, or a combination thereof. In some cases, the cells, tissues, or organs expressing at least two polypeptide sequences derived from a non-porcine mammalian species express mRNA encoding a full-length sequence of CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1, or a combination thereof. In some cases, the cells, tissues, or organs expressing at least two polypeptide sequences derived from a non-porcine mammalian species express mRNA encoding a functional fragment sequence of CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1, or a combination thereof.
[0085] In some embodiments, any one of the heterologous polypeptide sequences disclosed herein is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide sequence encoded by a human gene of interest or a fragment thereof. In some embodiments, a polynucleotide sequence encoding the any one of the heterologous polypeptide sequences disclosed herein is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a human gene of interest or a fragment thereof. In some examples, the human gene of interest disclosed herein may comprise one or more members (e.g., two or more members) selected from: CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, and HO-1.
While the specification contemplates the subject being treated as any non-porcine mammal, the specification more narrowly focuses on primates and humans to be treated with the porcine cells. Also while the specification generically contemplates a broad range of genetic modifications, the specification solely provides specific guidance to using gene editing to knockout GGTA, B4GALNT2 and CMAL and introducing nucleic acids encoding the claimed polypeptides in a single genetic expression construct, introduced into a porcine SCNT unit and producing a transgene pig comprising the constructs in all of its cells. Further while the specification contemplates any route of administration any “centrally administered”, the specification solely provides more specific guidance to administered via an internal jugular vein or a hepatic portal vein of the non-porcine mammal. As such, the specification does not provide specific guidance, thus does not enable, the great breadth of the claims as recited.
Working Examples:
Example 1
[0112] CRISPR-Cas9 mediated NHEJ was used to functionally knock out the three major carbohydrate-producing glycosyltransferase/glycosylhydrolase genes GGTA1, CMAH, and B4GALNT2 in pig primary fibroblasts from Bama minipigs. Twelve human transgenes (CD46, CD55, CD59, CD39, CD47, A20, PD-L1, HLA-E, B2M, THBD, TFPI, HO-1) were then integrated into a single multi-transgene cassette in the pig genome via PiggyBAC transposon-mediated random integration to generate 3KO/12TG cells designated “4-7”, which were used to generate pigs via somatic-cell nuclear transfer (SCNT). Wild-type porcine ear fibroblasts were first electroporated with both: a) CRISPR-Cas9 reagents targeting the GGTA, CMAH, and B4GALNT2 genes; and b) payload plasmids bearing (i) a PiggyBac transposase cassette (ii) a transgenic construct comprising one or more of the 12 human transgenes. The transgenes were arranged into 4 different cistrons with desired ubiquitous or tissue-specific promoters. The transgenes within each cistron were separated with ribosomal skipping 2A peptides to ensure expression in a similar molar ratio. Furthermore, a combination of cis-elements such as ubiquitous chromatin opening elements (UCOEs) were introduced to prevent transgene silencing and insulators with strong polyadenylation sites and terminators to minimize the interaction among transgenes and between transgenes and the flanking chromosome. Single-cell clones of the fibroblasts were generated and screened by fragment analysis/whole genome sequencing to identify clones with the desired genomic modifications, and a clone bearing the desired modifications was then used as a donor to produce a live pig by SCNT.
Example 2
[0127] Transgenic male Bama minipigs (produced as in Example 1) were anesthetized and subjects to laparotomy and exsanguination at 0-7 days post birth. Pancreases were excised and were cut into small fragments under sterile conditions with a scalpel. Pancreatic fragments were subjected to collagenase V digestion (1 mg/ml) and transferred to a gas-permeable culture bag (OriGen PermaLife™ Cell Culture bags) and held at 22-24 dC for transport to the culture lab. Islets were cultured in bags or petri dishes in either EGM-2 medium (EGM-2 with FGF-B, VEGF, R3-IGF, ascorbic acid, hEGF, heparin, D-glucose, nicotinamide, 10% porcine serum, 50 μM IBMX, 120 μM amikacine, and 60 μM ampicillin), EGM-2 medium plus corticosteroid (EGM-2 plus 1 μM methylprednisolone), or Ham's F-10 medium for 7 days. Islet equivalents (IEQ) were measured over the 7 days in culture and graphed (see FIGS. 14, 15, and 16). EGM-2 medium with corticosteroid was associated with improved yields of islets among the 3 conditions.
[0128] In some cases, subsequent to mechanical or enzymatic digestion, pancreatic fragments may be purified by sedimentation (e.g., ficoll gradient sedimentation). In other cases, such purification step via sedimentation may not or need not be required. In such cases, the pancreatic fragments may be cultured (e.g., in culture dishes for about 7 days) before transplantation, during which time non-islet cells (e.g., exocrine cells) may die off.
Example 4—Transplantation of Islets into Recipient Mice
[0133] To test the functionality of the 4-7 porcine transgenic islet cells upon xenotransplantation, an STZ-based mouse diabetes islet adoptive transfer model was established. Exemplary blood glucose for mice using this model procedure is depicted in FIG. 19. This model uses a toxin (streptozotocin, STZ) to kill islet cells in immunodeficient mice, causing dramatic increases in blood glucose levels. Transplantation of islet cells results in normalization of blood glucose levels by ˜60 days post-transplant.
[0134] For assessing 4-7 islet cell efficacy in treating diabetes, diabetes was first induced in n=12 NCD (NOD-Prkdc.sup.em26cd52Il2rg.sup.em26cd22/NjuCrl) mice by treatment with a single dose of streptozotocin (STZ, 125 mg/kg) followed by a 3-day washout period, leaving 3 untreated age-matched mice as a control. The untreated mice and 3 of the STZ treated mice were then subjected to a sham transplantation operation, whereas 3 of the STZ mice received wild-type porcine islets (3000IEQ) isolated as in FIG. 9/Example 2 and 3 of the STZ mice received 4-7 transgenic porcine islets (3000IEQ) isolated as in FIG. 9/Example 2. Where islets were transplanted, they were transplanted under the left kidney capsule. Briefly, the 4-7 islet cells were mixed or dispersed in a solution (e.g., a buffer) and injected (e.g., slowly injected) under the kidney capsule via a syringe and soft tubes.
[0135] FIG. 20 shows blood glucose of NCG mice (as a T1D rodent model) receiving islet-like cell clusters (NICC) comprising the subject 4-7 porcine transgenic islet cells provided herein. NICC comprising wild-type pig islet cells were used as a control. Various amounts of NICC were transplanted to the NCG mice: 4000 IEQ, 2000 IEQ, and 1000 IEQ. Data indicates that the 4-7 porcine transgenic islet cells exhibited a similar efficacy in controlling the increased blood glucose level in mice, as compared to WT pig islet cells. The 4-7 porcine transgenic islet cells became functional (e.g., in controlling blood glucose level) in vivo at about two weeks after transplantation.
[0136] In some cases, abnormal growth of transplanted porcine cells may be monitored for a longer time (e.g., longer than 5, 6, 7, 8, 9, 10, 11, 12 months, or longer). In some cases, human adult islet cells may be used as a positive control, e.g., at a clinical human adult islet treatment dose. In some cases, non-obese diabetic (NOD) T1D mice model may be used as a secondary in vivo model. In some cases, porcine transgenic islet cells may be administered via intraportal vein injection to test its compatibility and safety.
Example 5—Transplantation of Islets into Recipient Monkeys
[0137] To test the functionality of the 4-7 porcine transgenic islet cells upon xenotransplantation to primates, an STZ-based NHP diabetes islet adoptive transfer (using intraportal islet cell transplantation) model was established. 4-7 islets and WT isolated as in FIG. 9/Example 2 were transplanted to Cynomolgus monkeys via percutaneous transhepatic portal catheterization guided by ultrasound. A scheme for these experiments is presented in FIG. 21.
[0138] The immunosuppression protocol used for transplant of porcine cells was as follows:
[0139] ATG was given IV on days −7 d (±2 d), −6 d (±2 d), −4 d (±2 d) at a dose of 5 mg/kg, and an additional dose of ATG was administered on ˜1 d if lymphocyte depletion to <5% of baseline level in the blood was achieved.
[0140] Anti-CD40 was given IV on −4 d (±1 d), 0 d, 4 d, 7 d, 10 d, 14 d and then weekly at a first dose of 50 mg/kg and 30 mg/kg then after.
[0141] Anti-CD20 monoclonal antibody Rituximab was given IV on 0 d (±2 d) at a dose 375 mg/m2, to be repeated up to every three months if B cell count rises above 5% of baseline.
[0142] Rapamycin and Tacrolimus were started on −3 d (±1 d) per oral at start doses of 0.3 mg/kg QD and 0.02 mg/kg BID, respectively, and adjusted according to the plasma concentration.
[0143] Ganciclovir was given IM starting from −7 d (±2 d) at a dose of 5 mg/kg.
[0144] Prophylactic use of Chlortrimeton 0.4 mg/kg IM, and Methylprednisolone 10 mg/kg IV was administered before ATG, anti-CD40 and anti-CD20 administration to prevent infusion reactions.
[0145] Supportive administration of insulin to the STZ induced animals to support health was provided as follows:
[0146] Glargine insulin was administered QD and was administered initially at 2 U QD. The dose was increased 2 U when FBG was >150 mg/dl, and was decreased decrease 2 U when FBG was <100 mg/dl.
[0147] Insulin was administered BID, in the morning and evening according to the recorded blood glucose level of the animal. For morning doses, <200 mg/dl received no insulin, 200-350 mg/dl received 4 U insulin, 350-400 mg/dl received 6 U insulin, 400-600 mg/dl received 8 U insulin, and >600 mg/dl received 10 U insulin. For evening doses, <300 mg/dl received no insulin, 300-350 mg/dl received 4 U insulin, 350-400 mg/dl received 6 U insulin, 400-600 mg/dl received 8 U insulin, and >600 mg/dl received 10 U insulin.
[0148] A pilot experiment using the STZ diabetes induction protocol on a monkey (MB-1) is shown in FIG. 22, where the animal is managed according to the scheme in FIG. 21. The animal was assessed for blood glucose, C-peptide, and insulin following administration of 50% dextrose 1 ml/kg iv to measure the functional output of the transplanted cells; the data in FIG. 22 indicates that the diabetes induction protocol was successful due to the increase in blood glucose and decrease in C-peptide and insulin following STZ treatment. Using this protocol, further animals MA-1, MA-2, MB-2, MC-1, MD-1, and ME-1 were induced with diabetes and transplanted with grafts according to Table 2 below. The animals were monitored for white blood cell count, lymphocyte count, CD4+ cell types, CD8+ cell types, B cells, NK cells, and Rapamycin levels following transplantation (FIGS. 23, 24, and 25).
[0149] Animals MTA-1 and MA-2 were later analyzed for immunohistochemistry of liver biopsy 12 hr and 1 month after transplant for presence of the transplanted 4-7 islets. Liver biopsy was performed and stained for hematoxylin/eosin (in the case of MA-1 and MA-2) and (in the case of MA2) the neuroepithelial marker chromogranin A which stains islet cells, indicating the presence of islet-like structures and engraftment of the 4-7 cells into the animal liver (see FIG. 26).
[0150] Immunohistochemistry, blood glucose, C-peptide (both monkey and porcine), and insulin levels will continue to be monitored in all of the animals in Table 2 to assess the function of the graft in non-human primates over a longer period of time.
Thus the working examples provide much narrower guidance to two diabetes animals models, an induced insulin deficient mouse and an induced insulin deficient primate. In the mouse model, the genetically modified porcine beta cell named 4-7 cell was transplanted under the kidney capsule and demonstrated a decrease in blood glucose. Similarly the immunocompromised primate model had the 4-7 cell transplanted into the liver which resulted in a reduction of glucose levels. However, the working examples do not provide additional guidance to any other routes of administration, any other mammalian models other than mice and primate, any porcine islet cells other than the 4-7 cell that successfully leads to a reduction in glucose levels. As such, the working examples also do not provide specific enablement guidance to the full breadth of the claims.
State of the Art:
Regarding gene editing in genetically modified pig using non-targeted mutagenesis such as transposons, Jin et al. (Animal Res One Health 2023;1;242-258) report, “The non‐targeted mutagenesis has several limitations: (1) Foreign sequences could be randomly inserted into any locus, which could be destructive to the stability of the host genome, thus resulting in a confounded or abnormal phenotype; (2) Transgenic expression levels are difficult to control because of different insertion locations and uncertain integration copies of foreign genes; (3) Transgenic expression levels may gradually decrease due to dilution or loss of copies over multiple generations of breeding and may even be silent due to epigenetic modification of the inserted locus.” See page 243, paragraph bridging col 1 and 2.
The instant invention uses one specific genetically modified porcine beta cell that has been made using transposons to introduce the claimed single construct. Jin teaches that this occurs by random integration which lead to unpredictable stability of the introduce expression construct and unpredictable expression of the proteins of the introduced construct. As such, the ability to reproduce the 4-7 cell disclosed by the specification as capable of improving glucose levels is highly unpredictable.
Aggarwal et al (Current Opinion in Endocrine and metabolic Research 2022, 24:100354, pages 1-6) describes the state of the art of porcine islets from treating diabetes in humans. “There is a need to thwart the hyperacute xenogeneic immune response, leading to the subsequent requirement of heavy immunosuppressive medication, which remains a barrier to clinical implementation…. Early events of rejection are characterized by infiltration of immunosuppressive medication, 2) encapsulation of porcine islets, which reduces the requirement of immunosuppression, and 3) genetically modified islets which invoke a reduced immunogenic response following transplant...innate immune cells, such as macrophages and neutrophils…. There are three primary strategies to facilitate engraftment and prolonged function of porcine islets: 1) transplantation of free porcine islets with immunosuppressive medication, 2) encapsulation of porcine islets, which reduces the requirement of
immunosuppression, and 3) genetically modified islets which invoke a reduced immunogenic response following transplant….” P. 2-3.
As such, Aggarwal reports that the state of the art for porcine islet transplantation into diabetics is unpredictable and host many obstacles. Aggarwal further addresses using genetic modification of porcine islet cells via transgenesis to overcome these obstacles. Aggarwal teaches multiple genetically modified pigs with modified proteins known to be involved in immune rejection of the porcine cells. Aggarwal refers to the eGenesis and the inventors work to produce genetically modified pigs to serve as donors for islets for human transplantation (page 4). Aggarwal reports that while many advances and breakthroughs are being made for the use of these pigs for islets donors in xenotransplantation (p. 4), no predictable therapeutic use of these cells are reported for actual alleviation of symptoms of diabetes yet. Aggarwal further states, “Efficacy demonstrated in small and large animals, tolerability and safety demonstrated in early clinical trials, the advent of genetic manipulation to reduce immunogenicity and increase compatibility, and patient acceptance, warrant further clinical investigation of
xenogeneic islet transplantation.” P 4, conclusion. Thus while advances were being made further research was necessarily to take the development of potential pig donor cell to application as a predictable therapeutic.
Thus the state of the art teaches a high degree of unpredictability and obstacles to the instantly claimed invention. Particularly the art suggests that the reliable production of a transgenic cells that has predictable expression of the added transgenes in the pig cells is unpredictable as is their use in a xenotransplantation. Further the art suggests that the actual alleviation of symptoms of diabetes has not been achieved at the time of the invention and that mere survival of the islets cells in the non-porcine host is unpredictable. The instant invention has provided genetically modified porcine islet cell species that was able to increase blood glucose in a mouse and primate diabetes model. However, given the unpredictable nature of this art and the breadth of the claimed invention, one species example of cell, 4-7 cell with one genetic multi-gene modification configuration, does not provide adequate enabling guidance to the use of the great breadth of genetically modified porcine islet cells produced by any means to predictable treat insulin resistance/deficiency by lowering glucose levels. As such, the breadth of the claimed invention is not enabled.
Therefore at the time of filing the skilled artisan would need to perform an undue amount of experimentation without a predictable degree of success to implement the invention as claimed.
Response to Arguments
Applicant's arguments filed 12/16/2025 have been fully considered but they are not persuasive.
Applicant submits that the specification teaches “single-cell clones of the fibroblast were generated and screened by fragment analysis/whole genome sequence to identify clones bearing desired modifications was then used as a donor to produce a live pig by SCNT”. Applicant submits that these teaching provide enablement for reliable production of the cells.
In response, Applicant is misunderstanding the issue of enablement and regarding the unpredictability of cell production. The rejection is not suggesting that a means of introducing the desired genes is unpredictable, but rather that expression patterns and reliable expression of those gene when they are introduced by random integration is unpredictable. Further, producing a cell that expresses at a particular expression profile that is therapeutic.
Applicant submits the specification provides enablement for “treating an insulin resistant condition or an insulin-deficient condition in a non-porcine mammal. Applicant refers to [0133], “transplantation of islet cells results in normalization of blood glucose levels by 60 days post-transplant” in diabetic mice induce by STZ. Further, this was reliably demonstrated in at least 3 diabetic mice, as shown in Fig 19. Further, Fig 20 shows, “blood glucose of NCG mice receiving STZ followed by islet transplant with WT porcine islet cells, 4-7 islet cells, and sham operation over a period of 126 days and demonstrates that “4-7 porcine transgenic islet cells became functional (i.e. in controlling blood glucose levels) in vivo at about two weeks after transplantation”.
In response, Applicant is reminded that the rejection is a scope of enablement rejection and the example described by Applicant has been acknowledged as enabled by the specification. However, the breadth of the claims are broader including a genus transgenic pancreatic islet cells that are generically contemplated by the specification but not described in a specific manner that overcome prior art described unpredictability transgene expression profiles associated with random integration of introduced transgenes, as well as immune response to xenogeneic cells, among other unpredictability described in the rejection of record. Applicant has not provide sufficient evidence to the part of the specification that provide guidance to overcome these art-described unpredictabilites. As such, the breadth of the claims still lack adequate written description, as amended.
The following new rejection is necessitated by the amendments to the claims:
Claim Rejections - 35 USC § 112
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 54-58 and 72-88 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.
When determining if a newly added recitation has adequate written description, the specification and claims, as originally filed, are examined to determine if the new recitation has explicit or implicit description.
Independent claim 54 recites, “wherein the administering comprises central administration”. A search of the originally filed claims and specification fail to provide a literal recitation for this wherein clause. As such, the specification and claims fails to provide explicit written description for this newly added recitation.
The closest description found is as follows (citation from the pre-grant publication):
[0106] The method of treating the insulin resistant or deficient condition in the non-porcine mammal in need thereof may involve the administration or transplant of any of the compositions, cells, organs, or tissues described herein. In some cases, when a cell composition is administered, the composition is centrally administered, e.g. is administered via an internal jugular vein or a hepatic portal vein of the non-porcine mammal.
While the specification does state “centrally administered”, this recitation is not defined by the specification is inconsistent with terminology used to describe routes of administrations. The specification provides two species examples of the genus, “administering via an internal jugular vein or a hepatic portal vein”. “Internal jugular vein” administration is considered in the art to be a system route of administration. In contrast, a hepatic portal vein is a type of local administration to the liver and pancreas. As such, one of ordinary skill would not be able to envision the what the members of the genus “central administration” would include or exclude, given the term is not defined and appears to be outside of common use of the term known in the art. As such, the term also fails to have adequate implicit written description as well.
Since the term “central administration” lacks written description, the new recitation constitutes new matter.
Claims 55-58 and 72-88 are dependent upon claim 54 and thus also recite the new matter.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 54-58 and 72-88 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.
The term “central administration” in claim 54 is a relative term which renders the claim indefinite. The term “central administration” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
“Central administration” indicates that the administration is “central” in reference to some other parameter. The specification does not define that other parameter. As such, the metes and bounds of the term “central administration” (i.e. central to regards to what?) are not apparent.
Claims 55-58 and 72-88 are dependent upon claim 54 and thus also recite the indefinite subject matter.
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MARCIA STEPHENS NOBLE whose telephone number is (571)272-5545. The examiner can normally be reached M-F 9-5:30.
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MARCIA S. NOBLE
Primary Examiner
Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632