Ethical organizations for transplantation

Abstract

This disclosure relates to organisms genetically engineered to have reduced higher brain structure, tissues obtained from such organisms, and methods for creating organisms in which the capacity to experience pain is severely reduced or completely absent, for the purpose of supplying tissues and organs free from cruelty. This approach ensures a lack of sensory perception in the animal from which the tissue is intended, thereby eliminating pain and suffering in the animal involved. This strategy provides an approach to generating tissues (including organs for transplantation) through the creation of organisms incapable of developing higher brain function.

Description

Technical Field

[0001] 1. Cross - References to Related Applications This application claims priority and benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 521,069, filed on June 14, 2023; U.S. Provisional Application No. 63 / 521,072, filed on June 14, 2023; and U.S. Provisional Application No. 63 / 580,954, filed on September 6, 2023, which are hereby incorporated by reference in their entirety.

[0002] 2. Sequence Listing This application includes a sequence listing submitted electronically via Patent Center, which is hereby incorporated by reference in its entirety. The above XML copy (creation date: XX month, 2024) is named 58242 WO_sequencelisting.xml and has a size of X,XXX,XXX bytes.

Background Art

[0003] 3. Background of the Invention There is a need for alternative means to the current practices used in raising, slaughtering, and processing animals for the procurement of meat and other tissues (including complex organs) that do not cause pain and suffering to the animals involved.

Summary of the Invention

Means for Solving the Problems

[0004] 4. Gist of the Invention An approach that eliminates pain and suffering in the animal from which tissue is to be harvested by ensuring a lack of sensation in the animal. This strategy provides an approach to generating tissue (including organs for transplantation) through the creation of an organism incapable of developing higher brain functions. In some embodiments, the manipulated organism has sufficient nerve tissue to maintain basic organ function but lacks any capacity for conscious awareness. In other embodiments, lower brain functions are also reduced or eliminated so that the tissue and organ are maintained alive through artificial means.

[0005] Manipulating the bodies of organisms lacking a brain, or part thereof, allows for tissue harvesting while addressing the critical challenges of pain and suffering and eliminating the potential for conscious awareness. The limited theoretical considerations in the scientific literature surrounding the methodologies for creating the types of products explicitly described herein, the framework of considerations surrounding the types of gene editing disclosed and specific gene targets, and the highlighted ethical and conceptual barriers, hinder the consideration and development of products to meet this prominent, unmet need in cell and tissue therapy.

[0006] Non-meat animal products (e.g., hides, casings, and even organs for transplantation) can similarly be produced without pain and suffering. Furthermore, methane and other emissions can be easily captured by eliminating the need for conventional livestock farming and related land use, and by maintaining animals without higher brain function in a controlled environment; operating animals without higher brain function for ethical meat and tissue production can therefore reduce environmental impacts (e.g., greenhouse gas emissions and deforestation). [Brief explanation of the drawing]

[0007] 5. Brief description of the drawing These and other features, aspects, and advantages of the present invention will be better understood in connection with the following detailed description and the accompanying drawings.

[0008] [Figure 1] Figures 1A–1D show histological comparisons between genetically modified embryos from the same litter and their wild-type (WT) counterparts (both simultaneously implanted in recipient females). Figure 1A shows a cross-section of the entire brain of a genetically modified embryo in which the genes NDE1 and DCX have been inactivated using CRISPR technology. Figure 1B shows a cross-section of the cortex of a genetically modified embryo in which the genes NDE1 and DCX have been inactivated using CRISPR technology. Figure 1C shows a cross-section of the entire brain of a WT embryo. Figure 1D shows a cross-section of the cortex of a WT embryo.

[0009] [Figure 2] Figure 2 provides a complete diagram of embryos that have undergone CRISPR-mediated genetic modification targeting the NDE1 and DCX genes at embryonic day 13 (E14).

[0010] [Figure 3A-3D]Figures 3A–3H show immunohistochemical comparisons between genetically modified embryos with targeted knockouts of IL1RAPL1 and CHRNA7, and their wild-type counterparts. Figure 3A shows a whole E14 mouse brain derived from a wild-type embryo, stained for IL1RAPL1. The dark staining diffused throughout the brain indicates overall IL1RAPL1 expression. Figure 3B is a magnified view of the cortex, derived from the same wild-type brain in frame A. Here, the surface of the cortex shows even darker staining, which indicates higher IL1RAPL1 expression, as highlighted by the arrows. Figure 3C shows a whole brain from a knockout embryo stained for IL1RAPL1. The brain appears even brighter, indicating a lack of IL1RAPL1 expression. In addition, dysplasia features and enlarged ventricles are evident, along with a marked reduction in the brain parenchyma. Figure 3D provides a magnified view of the cortex from a knockout embryo in frame C. This frame shows that staining for IL1RAPL1 is absent in conjunction with the knockout of this gene. Figure 3E shows another section of the same wild-type brain from frames A and B, but stained for CHRNA7. This frame shows the presence of diffused CHRNA7, with some regions showing more concentrated expression as indicated by the arrows. Figure 3F shows a cortical expansion from the same wild-type brain in frame E. The outer cortex shows clear, dark staining, indicating high levels of CHRNA7 expression. Figure 3G shows another section of the same knockout brain in frames C and D, stained for CHRNA7. The brain section above repeats the dysmorphic features of frame C and does not show CHRNA7 staining. Figure 3H shows a cortical expansion from the knockout brain in frame G, showing the complete absence of CHRNA7 staining. [Figure 3E-3H]Figures 3A–3H show immunohistochemical comparisons between genetically modified embryos with targeted knockouts of IL1RAPL1 and CHRNA7, and their wild-type counterparts. Figure 3A shows a whole E14 mouse brain derived from a wild-type embryo, stained for IL1RAPL1. The dark staining diffused throughout the brain indicates overall IL1RAPL1 expression. Figure 3B is a magnified view of the cortex, derived from the same wild-type brain in frame A. Here, the surface of the cortex shows even darker staining, which indicates higher IL1RAPL1 expression, as highlighted by the arrows. Figure 3C shows a whole brain from a knockout embryo stained for IL1RAPL1. The brain appears even brighter, indicating a lack of IL1RAPL1 expression. In addition, dysplasia features and enlarged ventricles are evident, along with a marked reduction in the brain parenchyma. Figure 3D provides a magnified view of the cortex from a knockout embryo in frame C. This frame shows that staining for IL1RAPL1 is absent in conjunction with the knockout of this gene. Figure 3E shows another section of the same wild-type brain from frames A and B, but stained for CHRNA7. This frame shows the presence of diffused CHRNA7, with some regions showing more concentrated expression as indicated by the arrows. Figure 3F shows a cortical expansion from the same wild-type brain in frame E. The outer cortex shows clear, dark staining, indicating high levels of CHRNA7 expression. Figure 3G shows another section of the same knockout brain in frames C and D, stained for CHRNA7. The brain section above repeats the dysmorphic features of frame C and does not show CHRNA7 staining. Figure 3H shows a cortical expansion from the knockout brain in frame G, showing the complete absence of CHRNA7 staining.

[0011] [Figure 4] Figure 4 illustrates the expected additive effect on phenotypic reduction of brain tissue from the multiplication of various mutations in the manipulated organisms of this disclosure.

[0012] [Figure 5]Figure 5 illustrates an engineered animal transitioning from a natural uterus to an artificially assisted system under ideal conditions.

[0013] [Figure 6] Figure 6 shows the knockout states of three different female mouse specimens treated with CRISPR targeting GRIN2b and MFSD2a. All fetuses were viable and developing up to the measurement point. Lane 0 was the control; specimen 1 migrated through lanes 1 and 2, specimen 2 migrated through lanes 3 and 4, and specimen 3 migrated through lanes 5 and 6.

[0014] [Figure 7] Figure 7 shows the knockout state of two genes, NDE1(N) and MFSD2a(M), via excision in five different fetal specimens. All fetuses were viable and developing up to the measurement point. N1 and M1 represent the homozygous wild-type morphology for each gene. The shorter band for N and M indicates the knockout state.

[0015] [Figure 8] Figure 8 shows the knockout states of three different fetal specimens treated with CRISPR targeting CHRNB2 and IL1RAPL1. Specimen G1-01 was homozygous for the wild-type gene and was run in lanes 1 and 2. Specimen G1-04 was homozygous for the knockout gene and was run in lanes 3 and 4. Specimen G1-02 was run in lanes 5 and 6.

[0016] [Figure 9] Figure 9 shows microscopic images of Group 4 at 96 hours from Table 37, which are groups of mouse embryos treated with CRISPR to knock out HTR6, NDE1, and MFSD2A. [Modes for carrying out the invention]

[0017] 6. Detailed Description of the Invention 6.1. Genetically engineered organisms In a first aspect, genetically engineered organisms are provided. The genomic modification prevents higher brain development during embryo and fetal maturation.

[0018] 6.1.1. Genomic modification to prevent higher brain development In representative embodiments, the mutations are not engineered into genes in which naturally occurring mutations that cause complete or nearly complete anencephaly have been identified; in representative embodiments, the organisms are wild type at these loci. These genes are known to broadly affect the functions of centrosomes, centromeres, microtubules or cell cycle checkpoint proteins (e.g., CDK6, MCPH1, CENPJ, and WDR62). Targeting such broadly expressed factors to prevent brain formation can lead to incomplete or inappropriate development, subsequent degeneration, and / or altered function of the skeletal muscle system and other peripheral organs, limiting the usefulness of the organism for either meat production or organ generation. In addition, the anencephaly phenotype caused by mutations in such genes is sporadic and there is significant variation in the phenotypic outcome (i.e., the resulting brain development) from the same single genetic change.

[0019] Thus, in representative embodiments, a more controlled and specific genomic modification strategy is employed. In various embodiments, the genes targeted for modification are primarily specific to neural tissue or have highly enriched expression in neural tissue and have a restricted impact on non-neural tissue.

[0020] Naturally occurring mutations and animal models suggest that a range of reduced brain development is possible in addition to the complete deletion of the cerebrum that occurs in association with neural tube closure failure resulting in anencephaly. While neural tube failure is typically multifactorial and does not reliably occur, certain genetic mutations that can cause reduced brain development have consistent outcomes in humans and animals. In some embodiments, the genes are selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1 (LIS1), RELN, and TUBB3, or any established "microcephaly primary hereditary" (MCPH) genes (tier 1 genes).

[0021] While individual mutations are reproducible, they are unlikely to completely restrict higher brain development on their own. Therefore, in some embodiments, modifications are made to multiple genes. This multiplicity allows for strategic combinations of genetic alterations to further restrict brain development and neuronal survival, leading to the deletion of all or any components of the cerebrum. Using these critical gene deletions in combination with other gene deletions or other interventions ensures a significant and reliable prevention of higher nervous system development. None of the gene combinations described herein are likely to be found in nature, because each described gene is highly conserved, inactivation in a homozygous state is extremely rare, and each is individually important for the overall survival and fitness of the animal.

[0022] In some embodiments in which multiple genes are modified, at least one of the multiple genes to be modified is selected from CIT, DCX, FTCD, GRIK3, GRIN2A, LHX1, LHX2, GRIN2B, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1(LIS1), RELN, and TUBB3, or any established MCPH gene (tier 1).

[0023] In certain embodiments in which multiple genes are modified, at least one of these multiple genes is a gene that affects neuronal signaling and prevents the transmission or perception of pain signals. It is beneficial to modify genes that alter pain perception or the ability to feel pain, either by themselves or in combination with other gene modifications that restrict neuronal development. In some embodiments, such genes are selected from NTRK1, PRDM12, genes related to monoamine neurotransmitter synthesis and production, and genes related to serotonergic or dopaminergic neuronal development or function.

[0024] The genes for 5HT receptors (including HTR6 and GPR26), dopamine receptors (DRD3), GABA receptors (GABRA6), glutamate receptors (GRM2, GRM4, SLC1A2), interleukin-1 receptor family (IL1RAPL1), transcription factors (e.g., SOX1, TBR1, VAX1), potassium channels (KCNK4), and PRDM12 (tier 2) are also usefully modified in combination gene modification strategies.

[0025] One such combination is shown in Figure 4. In some embodiments, modifications are made in multiple genes selected from NTRK1, MFSD2A, and GRIN2B. NTRK1 can be modified to prevent any sensation of pain. Certain DNA modifications in NTRK1 can also result in progressive neuronal loss, which is quite sufficient to affect overall brain function. In addition, these mutations can also make surviving neurons more susceptible to apoptosis, resulting in increased effectiveness that further intentional or unintentional stressors trigger the prevention of neuronal mass formation. MFSD2A can be modified to inhibit the proper transport of certain fatty acids, such as docosahexaenoic acid (DHA), thereby preventing proper neurogenesis, which results in significantly reduced brain development. Modifications to GRIN2B (NMDA receptor) produce neuron-specific effects that can range from mild to severe developmental impairment. The specific modified forms of GRIN2B that can be used may depend on the most advantageous synergistic effects achieved through experimentation.

[0026] In some embodiments, modifications are made to genes that regulate stem cell maintenance and expansion, as well as genes that are otherwise related to the formation and proper function of neurons and their supporting cells.

[0027] The above genetic modifications can be performed on germ cells, fertilized eggs, early-stage embryos, or any other cell type that can be induced to revert to an embryo-like state.

[0028] In some embodiments, the genetic modification reduces the expression of the encoded protein. In certain embodiments, the modification is a complete knockout, eliminating the expression of the encoded protein.

[0029] In some embodiments, the above modification involves the introduction of an indel into the coding sequence, resulting in a frameshift and shortening accompanied by a shortened protein with reduced function, no function, and / or a reduced functional half-life. In certain embodiments, the indel is introduced by non-homologous end joining after a double-strand break influenced by an RNA-guided DNA nuclease such as CRISPR-Cas9.

[0030] In some embodiments, the above modifications alter the primary amino acid sequence of a protein either by inserting a modified gene or by editing an endogenous gene (e.g., by PRIME editing). In certain embodiments, the protein sequence may be mutated to cause aggregation, or specific amino acid sequences may be added or removed to induce misfolding and aggregation of the protein. Induction of misfolding and aggregation through mutations in the protein sequence or through the addition or removal of specific amino acid sequences may contribute to cellular stress and apoptosis. These changes to the protein may disrupt normal cellular processes and lead to cell death.

[0031] The manipulation may involve insertion, deletion, or other modification of the target gene or corresponding regulatory element. The modification may be homozygous or heterozygous.

[0032] 6.1.2. Further genome modification for the generation of interspecies transplant organs In some embodiments, the manipulated livestock are produced to provide organs for xenotransplantation. In certain of these embodiments, further genomic modifications may be performed to reduce immunogenicity after xenotransplantation into a human host.

[0033] In a particular embodiment, the cells are from a pig, and the further manipulation is the knockout of at least one gene selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, the further manipulation is the knockout of multiple genes selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, the animal cells are further manipulated to express at least one human transgene selected from CD39, CD46, CD47, CD55, EPCR, TFPI, THBD, HO-1, vWF, and HLA-E.

[0034] In certain embodiments, the tissue to be transplanted is cartilage, heart valves, heart, kidney, pancreatic islets, or lungs.

[0035] 6.1.3. Maturation due to pregnancy In a series of embodiments, the genetically modified embryo is implanted in a surrogate uterus, where it receives nourishment and support as it grows and develops. This embodiment allows for the spontaneous development of the genetically modified embryo in the uterus and takes advantage of the benefits provided by the maternal environment. Life support is provided after natural or induced labor.

[0036] In a series of embodiments, the genetically modified embryo is grown to maturity outside of a natural uterine environment using artificial means. The genetically modified embryo receives nutrients from an artificial intrauterine environment for maintenance as it grows and develops. This embodiment enables the development of a genetically modified embryo to grow within an artificial environment. Further life support may or may not be provided after exiting the artificial intrauterine environment.

[0037] As is the case in natural pregnancy cases where the fetus lacks a large portion of its brain, the fetus may not survive long enough to be maintained to full term in surrogate or artificial pregnancy. In various embodiments, engineered organisms, particularly those with numerous genetic modifications to limit brain development, have a greater need for support at earlier developmental stages. The transition to an support system may have to occur before viability due to insufficient brain and spinal cord development. Figure 5 illustrates the transition of an engineered animal from a natural or artificial uterus to an artificial support system under ideal conditions.

[0038] In some embodiments, the development of nerve tissue can be monitored using methods such as ultrasound or other means.

[0039] In some embodiments, modifications may be made to genes that make neurons more susceptible to apoptosis in response to changes in temperature, nutrient availability, oxygen availability, low-molecular-weight inhibitors, or other toxin exposure compared to normally functioning neurons. These conditions may be regulated during embryonic and fetal maturation to further reduce the development and survival of nerve tissue.

[0040] In some embodiments, factors that enhance the stability or resistance of developing tissues to stressors or apoptosis are introduced prophylactically or in response to changing parameters. These may include antioxidants, anti-apoptotic small molecules, and growth factors. Automated sampling and monitoring systems may be implemented to precisely adjust interventions in response to toxin levels, nutrient uptake, nerve tissue status, and basic vital signs.

[0041] 6.2. Manipulated Organizations In another context, a genetically modified tissue is provided. The tissue comprises a plurality of cells attached to a three-dimensional structure, wherein the cells differentiate from a single zygote into at least two differentiated cell types, wherein the cells generally contain at least one genomic modification which is neuronal signal reduction, neuronal depletion, and neuronal destruction. In some embodiments, the cells contain a plurality of genomic modifications in which at least one of the following neuronal signal reduction, neuronal depletion, and neuronal destruction is collectively effective.

[0042] In some embodiments, the cells are mammalian cells. In certain embodiments, the cells include at least one genomic modification that reduces the expression of at least one gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1(LIS1), RELN, and TUBB3. In some embodiments, the cells include at least one genomic modification that reduces the function of proteins encoded by at least one gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1(LIS1), RELN, and TUBB3.

[0043] In a particular embodiment, the cells include genomic modifications in MFSD2A and NDE1.

[0044] In some embodiments, the genome modification involves one or more mutations in the primary amino acid sequence of the protein. In certain embodiments, the one or more mutations cause the protein to malfold and aggregate.

[0045] In some embodiments, the cells further include at least one genomic modification that reduces the expression of at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0046] In some embodiments, the cells further include at least one genomic modification that reduces the function of proteins encoded by at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0047] In certain embodiments, the genome modification includes one or more mutations in the primary amino acid sequence of the further modified protein. In certain embodiments, the one or more mutations cause the protein to malfold and aggregate.

[0048] In some embodiments, the cells are homozygous with respect to at least one of the genomic modifications described above. In some embodiments, the cells are heterozygous with respect to at least one of the genomic modifications described above. In some embodiments, the tissue is located in the uterus. In some embodiments, the tissue is located in ex vivo.

[0049] In some embodiments, the mammalian cells are derived from the families Bovidae, Parvorder Catarrhini, or Suidae.

[0050] 6.2.1. Organs for transplantation In some embodiments, the cells of the above tissue are further manipulated to reduce immunogenicity after transplantation into a human host. In certain embodiments, the further manipulation is the knockout of at least one gene selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In certain embodiments, the further manipulation is the knockout of multiple genes selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, the cells are from a pig.

[0051] In some embodiments, the cells of the above tissue are further manipulated to express at least one human transgene selected from CD39, CD46, CD47, CD55, EPCR, TFPI, THBD, HO-1, vWF, and HLA-E.

[0052] In some embodiments, the tissue is cartilage, heart valves, heart, kidney, pancreatic islets, or lungs.

[0053] 6.3. Definition Unless otherwise defined, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art to which the present invention pertains. The genes selected herein are defined as follows: the selected genes, as well as any known genes or protein products thereof that have a high degree of sequence homology regionally or across gene / proteins, are interchangeable with the selected genes and any functional equivalents and / or products thereof that are reasonably expected to produce similar results in the context of the selected genes and their use.

[0054] 6.4. Additional Embodiments A genetically engineered tissue is provided, comprising, in one aspect, a plurality of cells closely attached to a three-dimensional structure, wherein the cells differentiate from a single zygote into a plurality of differentiated cell types, wherein the cells generally comprise at least one genomic modification which is at least one of the following: neuronal signal reduction, neuronal depletion, and neuronal destruction. In various embodiments, the cells comprise a plurality of genomic modifications which collectively enable at least one of the following: neuronal signal reduction, neuronal depletion, and neuronal destruction. In some embodiments of the genetically engineered tissue, the cells are mammalian cells.

[0055] In various embodiments of the genetically modified tissue described above, the cells include at least one genomic modification that reduces the expression of at least one gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1(LIS1), RELN, and TUBB3. In some embodiments, the cells include at least one genomic modification that reduces the function of at least one protein encoded by a gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1(LIS1), RELN, and TUBB3. In some embodiments, the genomic modification includes one or more mutations in the primary amino acid sequence of the protein. In some embodiments, the one or more mutations cause misfolding and aggregation of the protein. In some embodiments, the cells include genomic modifications in MFSD2A and NDE1.

[0056] In some embodiments of the genetically modified tissue described above, the cells further include at least one genomic modification that reduces the expression of at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0057] In some embodiments of the genetically modified tissue described above, the cells further include at least one genomic modification that reduces the function of proteins encoded by at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0058] In some further embodiments of the genetically modified tissue described above, the genome modification involves one or more mutations in the primary amino acid sequence of the further modified protein. In some embodiments, the one or more mutations cause the protein to malfold and aggregate.

[0059] In various embodiments of the genetically modified tissue described above, the cells are homozygous with respect to at least one genomic alteration. In some embodiments, the cells are heterozygous with respect to at least one genomic alteration.

[0060] In some embodiments, the mammalian cells are derived from the families Bovidae, Parvorder Catarrhini, or Suidae.

[0061] In some embodiments, the tissue is located in the uterus. In some embodiments, the tissue is located in exovivo.

[0062] In some embodiments, the cells are derived from the species of cattle, sheep, goats, pigs, or rabbits.

[0063] In some embodiments, the cells are derived from Old World monkey species.

[0064] In some embodiments of the genetically modified tissue described above, the tissue is non-human, and the cells are further manipulated to reduce immunogenicity after xenotransplantation into a human host. In some embodiments, the cells are porcine, and the further manipulation is the knockout of at least one gene selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, the further manipulation is the knockout of multiple genes selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, the cells are further manipulated to express at least one human transgene selected from CD39, CD46, CD47, CD55, EPCR, TFPI, THBD, HO-1, vWF, and HLA-E. In some embodiments, the tissue is cartilage, heart valve, heart, kidney, pancreatic islet, or lung.

[0065] In another aspect, a method is provided herein for generating a genetically engineered tissue comprising a plurality of cells closely attached to a three-dimensional structure, wherein the cells are differentiated from a single zygote into at least two differentiated cell types, wherein the cells generally comprise at least one genomic modification which is at least one of the following: neuronal signal reduction, neuronal depletion, and neuronal destruction, wherein the method comprises the steps of ex vivo preparing an embryo in which the cells generally comprise at least one genomic modification which is at least one of the following: neuronal signal reduction, neuronal depletion, and neuronal destruction, and transplanting the embryo into a appropriately prepared host surrogate uterus or artificial system.

[0066] In some embodiments of the above method, the method further includes a subsequent step of transferring the embryo from the host uterus to a life support system after a period of intrauterine growth and maturation shorter than the full-term gestation period. In some embodiments, after full-term pregnancy in uterus, the method further includes a step of giving birth to a full-term animal.

[0067] In various embodiments of the above method, the method further includes the subsequent steps of slaughtering the animal and collecting the tissue.

[0068] In another aspect, an engineered organism is provided which includes cells having at least one genomic modification that is at least one of the following: neuronal signal reduction, neuronal reduction, neuronal depletion, and neuronal destruction. In various embodiments, the cells of the engineered organism include multiple genomic modifications in which at least one of the following: neuronal signal reduction, neuronal reduction, neuronal depletion, and neuronal destruction is collectively effective.

[0069] In various embodiments of the manipulated organism described above, the cells of the organism include at least one genomic modification that reduces the expression of at least one gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1(LIS1), RELN, and TUBB3.

[0070] In various embodiments of the manipulated organism described above, the cells include at least one genomic modification that reduces the function of at least one protein encoded by a gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1(LIS1), RELN, and TUBB3.

[0071] In various embodiments of the manipulated organism described above, the cells of the organism further include at least one genomic modification that reduces the expression of at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0072] In various embodiments of the manipulated organism described above, the cells further include at least one genomic modification that reduces the function of proteins encoded by at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0073] In various embodiments of the manipulated organism described above, the cells of the organism include at least one genomic modification that reduces the expression of at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0074] In various embodiments of the manipulated organism described above, the cells include at least one genomic modification that reduces the function of proteins encoded by at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0075] In various embodiments of the manipulated organism described above, the cells include genomic modifications in MFSD2A and NDE1.

[0076] 6.5. Other Interpretive Conventions Scope: Throughout this disclosure, various aspects of the invention are presented in range form. The range includes the endpoints described. It should be understood that the range form is for convenience and brevity only and should not be interpreted as an irrevocable limitation to the scope of the invention. Therefore, the range description should be considered to have all possible subranges specifically disclosed and the individual numerical values ​​within those ranges. For example, a range description such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and the individual numerical values ​​within those ranges (e.g., 1, 2, 3, 4, 5, 5.3, and 6). This applies regardless of the width of the range.

[0077] In this disclosure, “comprises,” “comprising,” “containing,” “having,” “includes,” “including,” and their linguistic variations have the meanings set forth in U.S. Patent Law and permit the presence of further elements beyond those expressly stated.

[0078] Unless otherwise stated or evident from the context, the term "or" as used herein is understood to be inclusive.

[0079] Unless otherwise stated or evident from the context, the terms “a,” “an,” and “the” are understood to be singular or plural as used herein. That is, the articles “a” and “an” are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the articles. For example, “an element” means one element or more than one element.

[0080] Unless otherwise stated or made clear from the context, the term “about” as used herein is understood to mean within the normal range of acceptance in the art. Unless otherwise specified, “about” is intended to mean ±10% of the stated value. Where a percentage is given with respect to the amount of a component or substance in a composition, that percentage should be understood to be a percentage by weight unless otherwise stated or made clear from the context. [Examples]

[0081] 7. Examples 7.1. Zygote genome editing This example demonstrates a methodology for successfully editing selected genes in zygotes using the Cas9 nuclease system. Protospacer adjacent motif (PAM) site recognition by the Cas9 nuclease is clearly essential for its use as an effective genome editing tool, and is provided along with the guide RNA (gRNA) target, gRNA sequence, and polymerase chain reaction (PCR) primers used in the utilization of the Cas9 nuclease system for each gene target. Tables 1–42.

[0082] Methods and Materials: Guide RNA (gRNA) was incubated with CAS9 in OPTI-MEM for a minimum of 30 minutes in various combinations. The resulting ribonucleoparticles (RNPs) were electroporated into mouse zygotes using an ECM830 square electroporator at their pronuclear stage. Typically, this was done at a voltage of 30V, with 5 pulses, a pulse width of 3 minutes, and an interval of 100 milliseconds (ms). Gene: NDE [Table 1] [Table 2] [Table 3] Gene: MFSD2a [Table 4] [Table 5] [Table 6] Gene: GRIN2B [Table 7] [Table 8] [Table 9] Gene: DCX [Table 10] [Table 11] [Table 12] Gene: I11RAPL1 [Table 13] [Table 14] [Table 15] Gene: CHRNB2 [Table 16] [Table 17] [Table 18] Gene: LHX1 [Table 19] [Table 20] [Table 21] Gene: LHX2 [Table 22] [Table 23] [Table 24] Gene: OTX1 [Table 25] [Table 26] [Table 27] Gene: OTX2 [Table 28] [Table 29] [Table 30] Gene: HTR6 [Table 31] [Table 32] [Table 33] Gene: CHRNA7 [Table 34] [Table 35] [Table 36] [Table 37]

[0083] 7.2. Confirmation of gene knockout (KO) Gene knockout was subsequently confirmed through histological studies (Figures 1-3) and PCR results (Figures 6-8).

[0084] Comparative analysis of genetically modified embryos and wild-type embryos by microscopy and PCR. Table 37 shows the results of a large-scale screening of gene knockouts in mouse embryos. Figure 9 shows a microscopic image of Group 4 from Table 37 at 96 hours, as an example of the method used to count these genes, with red dots marking those counted by the inventors as healthy blastocysts. The inventors employed CRISPR-Cas9 technology to knock out specific genes in mouse embryos. The inventors' objective was to investigate the effects of these knockouts on embryonic development and to demonstrate similar viability and ability to form viable fetuses. Seven different conditions were tested (each targeting a specific combination of genes). The embryos were observed with respect to developmental progress, with particular focus on the blastocyst stage during hatching.

[0085] Our observations revealed that all treated embryos exhibited delayed development (common to embryos subjected to CRISPR and electroporation). Despite this delay, most conditions, with the exception of condition 5 (which accounted for only 5.88%), produced normal-looking blastocysts during incubation.

[0086] Condition 5 (which involved the largest number of guide RNAs targeting the most genes (LHX1, OTX2, MFSD2A, GRIN2B)) showed a significant deviation from the predicted developmental progression. This condition resulted in a significant reduction in the number of blastocysts during normal hatching. The complexity of simultaneously targeting multiple genes may have contributed to this result.

[0087] The results indicate that many embryos progress through normal development. This suggests that, despite the initial developmental stage, the CRISPR-induced gene knockout does not completely hinder fetal development and generates a large number of viable embryos. The inventors performed PCR analysis on a subset of blastocysts hatching from Table 37. The inventors' results showed that at least 60% of the developed blastocysts exhibited homozygous KO alleles for each gene target.

[0088] Controls: As expected, control embryos developed normally without any genetic modifications, and the rates of blastocyst formation and hatching were very high.

[0089] Condition 2 (OTX1, OTX2, NDE): The embryos showed reduced blastocyst formation compared to the control, but were still more than sufficient to produce many viable fetuses if implanted.

[0090] Condition 3 (LHX1, LHX2, GRIN2B): The embryos showed reduced blastocyst formation compared to the control, but were still more than sufficient to produce many viable fetuses if implanted.

[0091] Condition 4 (HTR6, NDE1, MFSD2A): Embryos showed reduced blastocyst formation compared to the control, but higher than in conditions 2 and 3, and were very sufficient to produce many viable fetuses if they were transferred. Figure 9 shows a microscopic image at 96 hours as an example of this condition.

[0092] Condition 6 (GRIN2B, MFSD2A, LHX1): The embryos exhibited reduced blastocyst formation compared to the control, but were still more than sufficient to produce a large number of viable fetuses if implanted.

[0093] Condition 7 (Il1RAPL1, CHRNA7): This condition resulted in a similar rate of normal development compared to the other conditions and controls. The inventors used this condition to demonstrate fetal development and CNS disorders in Figure 3.

[0094] Table 37 shows that CRISPR-Cas9 technology can be used to knock out numerous genes critical to central nervous system development in mouse embryos, and that these embryos can progress to blastocyst development and hatching despite their initial developmental stage. In combination with Figures 1, 2, and 3, we here show that the KO combination progresses all the way to the later fetal stages and exhibits clear CNS dysfunction as presumed (Condition 7 in Table 37 is found in Figure 3 showing the development of these conditions, which was verified by PCR as a double KO of Il1RAPL1,CHRNA7), and these data show that our gene combinations can both develop normally and impair CNS development. The data collected so far shows that most conditions produce blastocysts that appear normal during hatching, however, they lack critical CNS development genes and thus produce CNS defects as shown in Figures 1, 2, and 3.

[0095] The more complex gene targeting in condition 5 highlights the potential challenges and effects of multiple gene knockout, but still results in a lower percentage of viable blastocysts.

[0096] Histological comparative analysis of brains from genetically modified and wild-type embryos. The results shown in Figures 1A–1D illustrate a histological comparison between genetically modified embryos from the same litter and their wild-type counterparts (both simultaneously transplanted into the recipient female). The left panel shows the genetically modified embryos, while the right panel shows the wild-type embryos. More specifically, Figure 1A shows the entire brain of the genetically modified embryo (where the genes NDE1 and DCX were inactivated using CRISPR technology). This panel highlights significant morphological differences, including dysplasia, as well as areas of active degeneration in the cortex and other brain regions, as indicated by the arrows. Clearly, there was a developmental abnormality characterized by significant ventricular enlargement and inadequate ventricular separation at this developmental stage. The cerebellum appeared to be completely absent during the developmental process. Figure 1B shows a magnified view of the cerebral cortex in the genetically modified embryo. This detailed view highlights the degree of cortical abnormalities and degenerative changes, further complementing the observation of ventricular enlargement and malformations. Figure 1C shows the entire brain of a wild-type sibling embryo at a similar developmental stage for comparison. The morphology, particularly the structure of the ventricles and the overall structural pattern of the brain, was in striking contrast to that of the genetically modified embryo, where enlarged ventricles and impaired ventricular separation were observed. Figure 1D provides a magnified view of the cerebral cortex in a wild-type embryo illustrating normal cortical development. This panel serves as a control and shows representative cortical structures that are not present in the genetically modified counterpart.

[0097] Visualization of the entire embryo of a genetically modified embryo at embryonic day 14 (E14). The results shown in Figure 2 were obtained from a whole embryo at embryonic day 14 (E14). Clearly, despite the significant cerebral alterations detailed above and in Figures 1A-1D, the external morphology of the embryo appears unremarkable and is consistent with a representative developmental milestone for this stage. A key finding at the time of collection was the presence of a vigorous heartbeat, indicating the embryo's vitality. This observation was significant because it highlighted the local nature of the cerebral changes without apparent impact on the overall embryonic viability.

[0098] It is important to emphasize that genetic modifications primarily affected brain development, as extensively analyzed in Figures 1A–1D. These figures highlight the contrast between the widespread brain abnormalities and, in other words, normal external development of the embryos described above. It is illustrated that significant internal changes in brain morphology may not be externally apparent at this developmental stage.

[0099] Clearly, despite the significant cerebral abnormalities in the genetically modified embryos, their overall embryonic development was broadly normal, including a vigorous heartbeat at harvest and an unremarkable appearance of other major organs. Figure 2.

[0100] Detailed immunohistochemical analysis of IL1RAPL1 and CHRNA7 in genetically modified mouse brains and wild-type E14 mouse brains. The results showed immunohistochemical comparisons between genetically modified embryos with IL1RAPL1 and CHRNA7-targeted knockouts. Figure 3 highlights the significant effects of IL1RAPL1, CHRNB2, and CHRNA7 knockouts on brain morphology and function. Figures 3A–3H. Combinations of these three knockouts significantly altered brain structure. Basic brain structure and development were impaired. Acetylcholine signaling was also disrupted. As a result, brain function and cognition were predicted to be significantly affected, while other tissue and organ development were not significantly impaired.

[0101] PCR results indicate that numerous knockout combinations can still result in fetuses that potentially develop into late-stage gestation (Figures 6-8). Histological results show that both expression deficiencies and structural abnormalities were confirmed in animals along with the knockout variants of the target genes.

[0102] 7.3. Transfer of KO embryos to recipient targets The resulting knockout (KO) embryos were then subjected to Embryomax for 72–96 hours in groups that had grown to a size between 20 and 120.(登録商標) The embryos were group cultured in KSOM culture medium or a similar mouse embryo support medium under mineral oil in an incubator at 37.0°C and 5% CO2. The conditions were adjusted to maintain a pH between 7.2 and 7.4.

[0103] Embryos at the blastocyst stage were transferred to approximately 2.5 DPC pseudopregnancy female CD1 (ARC) mice with a target transfer weight of approximately 30 g. The target age of the recipient mice was >8 weeks old. Synchronization was induced with either 2.5 IU of pregnant mare serum gonadotropin (PMSG), followed 48 hours later by 2.5 IU–5 IU of human chorionic gonadotropin (HCG) or 50 μl of hyperova, followed 48 hours later by 5 IU of HCG. Approximately 7 hours after HCG administration, pseudopregnancy was induced in properly cycled females via mechanical and vibration-based stimulation by inserting a smooth plastic rod (which could be in contact with an electric toothbrush module or trimmer) into the vagina for 30 seconds.

[0104] Next, the embryos were directly implanted into the uterus of recipient females at or after 2.5 days of receptiveness via the vagina using a non-surgical embryo transfer (NSET) device (Paratechs). Figures 6 and 7 illustrate the methods used to process the histological images referenced below. The same processing was performed on all wild-type and knockout embryos to be compared with each other.

[0105] 7.4. Effects of genetic knockout of combinations on cognition in animal models The data shown in Figures 1A–3H provided compelling evidence of the significant impact of specific genetic knockouts on brain development, and, by extension, on overall cognition in mice. Collectively, these figures demonstrate that knockouts of the aforementioned combinations of brain-related and specific genes have a more significant impact than single-gene alterations.

[0106] In Figures 1A–1D, the inventors observed cerebral changes in embryos with CRISPR-mediated knockouts of the NDE1 and DCX genes. Abnormal brain morphology, particularly ventricular enlargement and impaired ventricular separation, suggested significant developmental disruption. These structural abnormalities, especially in areas critical to cognitive processes, suggested the potential for substantial cognitive deficits in these animals.

[0107] Figure 2 complemented these findings by showing that overall embryonic development appeared broadly normal despite significant internal brain changes. This observation was crucial because it highlighted the specificity of the genetic modification in affecting brain development while leaving other developmental processes relatively unaffected. Organs and tissues remained functional and intact.

[0108] Figures 3A–3H illustrate the effects of knocking out IL1RAPL1 and CHRNA7. The absence of IL1RAPL1 and CHRNA7 expression in the knockout model (as evidenced by the clear staining pattern) highlights the crucial role these genes play in normal brain function. Given the known associations between IL1RAPL1 and intellectual disability, and between CHRNA7 and neuronal acetylcholine receptor function, their absence strongly suggests potential impairment in cognitive abilities. The failure of CHRNB2 staining, while not visually documented, adds another layer to our understanding of the complex gene interactions influencing brain development.

[0109] Collectively, these results suggest that the combined effect of multiple gene knockouts on brain morphology and function is greater than the sum of the individual knockouts. This synergistic effect was particularly evident in areas critical to cognition. This suggests that animals with these genetic modifications are more likely to experience significant and pronounced cognitive impairments.

[0110] 7.5. Organ development in KO mouse fetuses In our exploration of the developmental effects of genetic modification, a particularly intriguing observation was the near-normal development of organs and tissues despite significant abnormalities in brain morphology and function. Figure 2 shows an E14 embryo exhibiting this phenomenon, despite the prominent cerebral abnormalities induced by genetic knockout, as detailed in Figures 1A-1B of this animal, and possessing a clear and active heartbeat, an indicator of overall embryonic vitality. This finding was noteworthy in that it suggests a degree of developmental compartmentalization, where severe genetic modification can result in specific brain malformations without disrupting the general developmental pathways of other organs and tissues. The presence of a normal heartbeat, along with representative morphological features in major organs, reinforced this idea. It also raised intriguing questions about the flexibility and adaptability of embryonic development in the face of targeted genetic disruption, particularly those affecting brain development. These observations, when comparing the brain changes and significantly delayed brain growth from Figures 1A–1D and 3A–3H with the otherwise unremarkable overall development in Figure 2, reveal a complex interaction between genetic regulation, organ specificity, and developmental robustness.

[0111] 8. Indication of Equivalents and References While the present invention has been described and illustrated in detail with reference to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made therein without departing from the spirit and scope of the invention.

[0112] All references, published patents, and patent applications cited herein are incorporated herein by reference in their entirety for the sole purpose of this specification.

Claims

1. A genetically modified tissue containing multiple cells closely attached to a three-dimensional structure, Here, the aforementioned cells are those that have differentiated from a single zygote into multiple differentiated cell types. Here, the cells generally include at least one genomic modification, which is at least one of the following: neuronal signal reduction, neuronal depletion, and neuronal destruction.

2. The tissue according to claim 1, wherein the cells include a plurality of genomic modifications that are collectively effective in at least one of the following: reduction of neuronal signaling, neuronal depletion, and neuronal destruction.

3. The tissue according to claim 1 or claim 2, wherein the cells are mammalian cells.

4. The tissue according to claim 3, wherein the cells include at least one genomic modification that reduces the expression of at least one gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1 (LIS1), RELN, and TUBB3.

5. The tissue according to claim 3, wherein the cells include at least one genomic modification that reduces the function of at least one protein encoded by a gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1 (LIS1), RELN, and TUBB3.

6. The tissue according to claim 5, wherein the genome modification comprises one or more mutations in the primary amino acid sequence of the protein.

7. The tissue according to claim 6, wherein the one or more mutations cause the incorrect folding and aggregation of the protein.

8. The cells are the tissue according to any one of claims 4 to 7, wherein the cells include genomic modifications in MFSD2A and NDE1.

9. The tissue according to any one of claims 4 to 8, wherein the cells further comprise at least one genomic modification that reduces the expression of at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

10. The tissue according to any one of claims 4 to 8, wherein the cells further comprise at least one genomic modification that reduces the function of the proteins encoded by at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

11. The tissue according to claim 10, wherein the genome modification comprises one or more mutations in the primary amino acid sequence of the further modified protein.

12. The tissue according to claim 11, wherein the one or more mutations cause the incorrect folding and aggregation of the protein.

13. The tissue according to any one of claims 1 to 12, wherein the cells are homozygous with respect to at least one genomic alteration.

14. The tissue according to any one of claims 1 to 12, wherein the cells are heterozygous with respect to at least one genomic alteration.

15. The tissue according to any one of claims 3 to 14, wherein the mammalian cells are derived from the family Bovidae or Suidae.

16. The tissue is the tissue described in any one of claims 3 to 15, located within the uterus.

17. The tissue described above is located in exovivo, according to any one of claims 3 to 15.

18. The tissue according to claim 17, wherein the cells are derived from the species of cattle, sheep, goats, pigs, or rabbits.

19. The non-human tissue according to claim 15, wherein the cells are further manipulated to reduce immunogenicity after xenotransplantation into a human host.

20. The non-human tissue according to claim 19, wherein the cells are from a pig, and the further operation is the knockout of at least one gene selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA.

21. The non-human tissue according to claim 20, wherein the further operation is the knockout of a plurality of genes selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA.

22. The non-human tissue according to claim 20 or 21, wherein the cells are further manipulated to express at least one human transgene selected from CD39, CD46, CD47, CD55, EPCR, TFPI, THBD, HO-1, vWF, and HLA-E.

23. The non-human tissue according to any one of claims 19 to 22, wherein the tissue is cartilage, heart valves, heart, kidney, pancreatic islets, or lungs.

24. A method for generating a genetically engineered tissue comprising multiple cells attached to a three-dimensional structure, wherein the cells are differentiated from a single zygote into at least two differentiated cell types, and the cells generally include at least one genomic modification which is neuronal signal reduction, neuronal depletion and / or neuronal destruction, and the method is A step of preparing an embryo exovivo, wherein the cells generally include at least one genomic modification which is neuronal signal reduction, neuronal depletion and / or neuronal destruction, and The process of transferring the aforementioned embryo into the uterus of a properly prepared host surrogate, A method of including.

25. The method according to claim 24, further comprising the subsequent step of transferring the embryo from the host uterus to a life support device after a period of intrauterine growth and maturation shorter than the full-term pregnancy period.

26. The method according to claim 24, further comprising the subsequent step of giving birth to a full-term animal after a full-term pregnancy in the uterus.

27. The method according to claim 25 or 26, further comprising the subsequent steps of slaughtering the animal and collecting the tissue.

28. An engineered organism comprising cells having one or more genome modifications, which are at least one of the following: reduced neuronal signaling, neuronal depletion, and neuronal destruction.

29. The manipulated organism according to claim 28, wherein the cells of the organism include a plurality of genome modifications which collectively result in neuronal signal reduction, neuronal depletion, and neuronal destruction.

30. A tissue according to any one of claims 4 to 15 or 28 to 29 or a combination thereof, wherein the tissue is produced for meat production and / or for the production of organs for transplantation.

31. An organism according to any one of claims 4 to 15 or 28 to 29 or a combination thereof, wherein the organism is selected, through the use of the enumerated combination of genes, to have its capacity to experience pain reduced to the greatest extent possible by lacking cognitive and / or pain receptors, or to be unable to experience pain at all.

32. The tissue according to claim 30, wherein the cells include one or more genomic modifications that reduce the expression of at least one gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1 (LIS1), RELN, and TUBB3.

33. The tissue according to claim 30, wherein the cells include one or more genomic modifications that reduce the function of at least one protein encoded by a gene selected from CIT, DCX, FTCD, GRIK3, GRIN2A, GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, OTX2, PAFAH1B1 (LIS1), RELN, and TUBB3.

34. The tissue according to claim 1 or 3, wherein the cells include one or more genomic modifications that reduce the expression of at least two genes selected from GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, and OTX2.

35. The tissue according to claim 1 or 3, wherein the cells include one or more genomic modifications that reduce the function of at least two genes selected from GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, and OTX2.

36. The tissue according to claim 1 or 3, wherein the cells include a genomic modification that reduces the function of both LHX1 and LHX2, as well as at least one genomic modification selected from GRIN2B, MFSD2A, NDE1, NTRK1, OTX1, and OTX2.

37. The tissue according to claim 1 or 3, wherein the cells include a genomic modification that reduces the function of both OTX1 and OTX2, as well as at least one genomic modification selected from GRIN2B, LHX1, LHX2, MFSD2A, NDE1, and NTRK1.

38. The tissue according to claim 1 or 3, wherein the cells include genomic modifications that reduce the functions of OTX1, OTX2, and NDE1.

39. The tissue according to claim 1 or 3, wherein the cells include genomic modifications that reduce the function of LHX1, LHX2, and GRIN2B.

40. The tissue according to claim 1 or 3, wherein the cells include genomic modifications that reduce the function of HTR6, NDE1, and MFSD2A.

41. The tissue according to claim 1 or 3, wherein the cells include genomic modifications that reduce the function of LHX1, OTX2, MFSD2A, and GRIN2B.

42. The tissue according to claim 1 or 3, wherein the cells include genomic modifications that reduce the function of GRIN2B, MFSD2A, and LHX1.

43. The tissue according to claim 1 or 3, wherein the cells include genomic modifications that reduce the function of both Il1RAPL1 and CHRNA7.

44. The tissue according to claim 1 or 3, wherein the cells include genomic modifications that reduce the function of both NDE1 and DCX.

45. The tissue according to claim 1 or 3, wherein the cells include genomic modifications that reduce the function of both NDE1 and MFSD2A.

46. The tissue according to any one of claims 28 to 45, wherein the cells further comprise at least one genomic modification that reduces the expression or function of proteins encoded by at least one gene encoding a nicotine receptor, optionally CHRNA7 or CHRNB2; a 5HT receptor, optionally HTR6 or GPR26; a dopamine receptor, optionally DRD3; a GABA receptor, optionally GABRA6; a glutamate receptor, optionally GRM2, GRM4, or SLC1A2; an interleukin-1 receptor family member, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1, or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

47. The cells are derived from humans, and the organism is an engineered organism according to claims 4 to 15.

48. The cells are derived from humans, and the organism is an engineered organism according to claims 28 to 45.

49. The mammalian cells are derived from Parvorder Catarrhini, according to any one of claims 3 to 14.

50. The manipulated organism according to claims 32 to 49, wherein the cell begins as any single cell capable of undergoing normal embryonic development.

51. The cells are tissues according to any one of claims 1 to 50, which are generated in an artificial system.

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