Ethical tissue for transplantation

Abstract

The present disclosure relates to organisms genetically engineered to have reduced advanced brain structures, tissues obtained from said organisms and methods of making such organisms for the purpose of providing humane tissues and organs from organisms with severely reduced ability to suffer or complete lack of ability to experience pain.

Description

1. Cross-reference of related applications

[0001] This application claims priority and interest in U.S. Provisional Application No. 63 / 521,069, filed June 14, 2023; U.S. Provisional Application No. 63 / 580,072, filed June 14, 2023; and U.S. Provisional Application No. 63 / 580,954, filed September 6, 2023, the entire contents of which are incorporated herein by reference.

[0002] 2. Sequence List This application contains a sequence list that has been electronically submitted through the Patent Center, the entire contents of which are incorporated herein by reference. The XML copy was created on August 12, 2024, named 58242 WO_CRF_SequenceListing.xml, and is 88,092 bytes in size.

[0003] 3. Background of the Invention There is a need for an alternative to current practices for raising, slaughtering, and processing animals to harvest meat and other tissues, including complex organs, that does not cause pain and suffering to the animals involved.

[0004] 4. Invention Overview A method for ensuring the absence of consciousness in animals used to harvest tissue to eliminate pain and suffering in the animals involved. This strategy provides a method for generating tissues (including organs for transplantation) by producing organisms incapable of developing higher brain functions. In some embodiments, the engineered organism has sufficient neural tissue to maintain basic organ function but lacks any conscious cognitive abilities. In other embodiments, lower-level brain functions are also reduced or eliminated, and the tissues and organs are kept viable by artificial means.

[0005] Engineering an organism's body without a brain or a part of it allows for the harvesting of tissues, while addressing critical issues of pain and suffering and eliminating the possibility of conscious cognition. Limited theoretical discussion in the scientific literature surrounding the methods leading to the types of products described herein, the framework of discussion surrounding the disclosed types of gene editing and specific gene targets, and prominent ethical and conceptual barriers have contributed to the discussion and development of products that meet this significant unmet need in cell and tissue therapy.

[0006] Non-meat animal products such as leather, casings, and even organs for transplantation can also be produced without pain and suffering. Furthermore, by eliminating the need for traditional livestock farming and associated land use, and by keeping animals without higher brain functions in controlled environments, methane and other emissions can be easily captured; therefore, engineered animals without higher brain functions used for ethical meat production and tissue production can reduce environmental impacts such as greenhouse gas emissions and deforestation. 5. Description of the attached drawings These and other features, aspects, and advantages of the invention will become more readily understood with reference to the following description and accompanying drawings, wherein: Figure 1A-1D Histological comparisons were presented between genetically modified embryos and their wild-type (WT) counterparts from the same litter, both of which were simultaneously implanted into female recipients. Figure 1A Figure 1 depicts a cross-section of the whole brain of a genetically modified embryo, in which genes NDE1 and DCX have been inactivated using CRISPR technology. Figure 1B depicts a cross-section of the cortex of a genetically modified embryo, in which genes NDE1 and DCX have been inactivated using CRISPR technology. Figure 1C A cross-section of the whole brain of a WT embryo is depicted. Figure ID depicts a cross-section of the cortex of a WT embryo.

[0008] Figure 2 Provides a complete view of embryos undergoing CRISPR-mediated genetic modifications targeting the NDE1 and DCX genes, as at gestational age 13 (E14).

[0009] Figures 3A-3H This is an immunohistochemical comparison between genetically modified embryos with targeted knockout of IL1RAPL1 and CHRNA7 and their wild-type counterparts. Figure 3A The brain of a whole E14 mouse derived from a wild-type embryo is shown, stained for IL1RAPL1. Diffuse, deep staining throughout the brain indicates overall expression of IL1RAPL1. Figure 3B This is a magnified view of the cortex from the same wild-type brain, as shown in box A. Here, the surface of the cortex shows even deeper staining, indicating higher IL1RAPL1 expression, as highlighted by the arrows. Figure 3C The whole brain of a knockout embryo stained with IL1RAPL1 is shown. The brain appears much paler, indicating a lack of IL1RAPL1 expression. Additionally, malformation features and enlarged ventricles are prominent, accompanied by a significant reduction in brain material. Figure 3D A magnified view of the cortex from the knockout embryo is provided in box C. This box shows the absence of IL1RAPL1 staining, consistent with the knockout of this gene. Figure 3EAnother slice of the same wild-type brain from boxes A and B was characterized, but stained for CHRNA7. The box shows diffuse presence of CHRNA7, with several regions showing denser expression, as indicated by the arrows. Figure 3F A magnified view of the cortex from the same wild-type brain is shown in box E. The outer cortex shows clear, dark staining, indicating high levels of CHRNA7 expression. Figure 3G Another slice of the knockout brain, identical to those in boxes C and D, is shown, stained for CHRNA7. The brain slice replicates the malformation features of box C and does not show CHRNA7 staining. Figure 3H The magnified view of the cortex from the knockout brain in box G demonstrates the complete absence of CHRNA7 staining.

[0010] Figure 4 This demonstrates the expected additive effect of multiple mutations on the reduction of brain tissue phenotypes in engineered organisms disclosed herein.

[0011] Figure 5 The study demonstrates the migration of engineered animals from their natural uterus to an artificial support system at an ideal time.

[0012] Figure 6 The knockout status of three different mouse fetal samples treated with CRISPR targeting GRIN2b and MFSD2a is shown. All fetuses survived and developed to the measurement point. Lane 0 is the control, sample 1 was run in lanes 1 and 2, sample 2 was run in lanes 3 and 4, and sample 3 was run in lanes 5 and 6.

[0013] Figure 7 The knockout status of two genes, NDE1 (N) and MFSD2A (M), by excision was shown in five different fetal samples. All fetuses survived and developed to the measurement point. N1 and M1 show the homozygous wild-type form of each gene. The shorter bands of N and M indicate the knockout status.

[0014] Figure 8 The knockout status of three different fetal samples treated with CRISPR targeting CHRNB2 and IL1RAPL1 is shown. Sample GL-01 was homozygous for the wild-type gene and ran in lanes 1 and 2, sample GL-04 was homozygous for the knockout gene and ran in lanes 3 and 4, and sample GL-02 ran in lanes 5 and 6.

[0015] Figure 9 The microscopic images of Group 4 from Table 37 at 96 hours are shown. This group of mouse embryos was treated with CRISPR to knock out HTR6, NDE1, and MFSD2A.

[0016] 6. Detailed Description of the Invention 6.1. Genetically engineered organisms In the first aspect, genetically engineered organisms are provided. Genome modifications prevent higher brain development during embryonic and fetal maturation.

[0017] 6.1.1. Genome modifications that prevent higher brain development In a typical implementation, the mutation is not engineered into a spontaneously mutated gene that has been identified as causing complete or near-complete anencephaly; in a typical implementation, the organism is wild-type at these loci. These genes are known to broadly affect the function of centromeres, centromeres, microtubules, or cell cycle checkpoint proteins (e.g., CDK6, MCPH1, CENPJ, and WDR62). Targeting such widely expressed factors to prevent brain formation can lead to incomplete or inappropriate development of the musculoskeletal system and other peripheral organs, subsequent degeneration, and / or altered function, thereby limiting the organism's utility for meat or organ production. Furthermore, the anencephalic phenotype resulting from mutations in these genes is sporadic, exhibiting significant variations in phenotypic outcomes (i.e., eventual brain development) arising from the same single-gene alteration.

[0018] Therefore, in typical implementations, a more controlled and specific genome modification strategy is employed. In various implementations, the genes targeted for modification are largely specific to or highly enriched in neural tissue, with limited effects on non-neural tissues.

[0019] Naturally occurring mutations and animal models have shown that a range of reduced brain development states are possible, in addition to complete brain loss in cases of anencephaly due to neural tube failure. While neural tube failure is often multifactorial and does not reliably occur, certain gene mutations that can lead to reduced brain development have consistent outcomes in both humans and animals. In some implementations, 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 inheritance" (MCPH) gene (tier 1 gene).

[0020] While individual mutations are reproducible, they are unlikely on their own to completely restrict higher brain formation. Therefore, in some implementations, multiple genes are modified. This multiplexing enables combinations of strategies to deploy genetic alterations to further restrict brain development and neuronal survival, resulting in the complete or partial absence of the brain. Combining these key gene deletions with other gene deletions or interventions ensures a deep and reliable inhibition of higher nervous system formation. Any of the disclosed gene combinations is extremely unlikely to be found in nature because each described gene is highly conserved, rarely inactivated in its homozygous state, and is individually important for the overall survival and health of the animal.

[0021] In some embodiments where 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).

[0022] In some embodiments where multiple genes are modified, at least one of the genes is a gene that affects neuronal signaling, blocks the transmission or perception of pain signals. It is advantageous to modify the gene itself, altering the ability to perceive or feel pain, or in combination with other gene alterations that restrict neural development. In some embodiments, such genes are selected from NTRK1, PRDM12, and genes associated with the synthesis and production of monoamine neurotransmitters and the development or function of serotonergic or dopaminergic neurons.

[0023] Genes of 5HT receptors (including HTR6, GPR26), dopamine receptors (DRD3), GABA receptors (GABRA6), glutamate receptors (GRM2, GRM4, SLC1A2), interleukin-1 receptor family (IL1RAPL1), transcription factors (such as SOX1, TBR1, VAX1), potassium channels (KCNK4), and PRDM12 (tier 2) are also favorablely modified in combinatorial gene modification strategies.

[0024] One such combination is as follows Figure 4As shown. In some embodiments, modifications are made to multiple genes selected from NTRK1, MFSD2A, and GRIN2B. NTRK1 can be altered to prevent any sensation of pain. Certain DNA alterations in NTRK1 can also lead to progressive neuronal loss, significant enough to affect global brain function. Additionally, these mutations can sensitize surviving neurons to apoptosis, leading to enhanced efficacy in preventing the development of neural clusters under further intentional or unintentional stressors. MFSD2A can be altered to inhibit the proper transport of certain fatty acids such as docosahexaenoic acid (DHA), thereby preventing proper neuronal formation, which leads to a significant reduction in brain development. GRIN2B (NMDA receptor) alterations result in neuron-specific effects ranging from mild to severe developmental impairment. The specific form of alteration to GRIN2B used can depend on the most favorable synergistic effect achieved experimentally.

[0025] In some implementations, genes that regulate stem cell maintenance and expansion and / or additional genes related to the formation and proper function of neurons and their supporting cells are modified.

[0026] Genetic modification can be performed on germ cells, fertilized eggs, early embryos, or any other cell type that can be induced to revert to an embryo-like state.

[0027] In some implementations, genetic modification reduces the expression of the encoded protein. In other implementations, the modification is a complete knockout, thereby eliminating the expression of the encoded protein.

[0028] In some embodiments, the modification involves introducing an indel into the coding sequence, resulting in frameshifting and truncation, wherein the truncated protein has reduced function, no function, and / or a reduced functional half-life. In some embodiments, the indel is introduced via non-homologous end joining following a double-strand break influenced by an RNA-directed DNA nuclease such as CRISPR-Cas9.

[0029] In some implementations, modification alters the primary amino acid sequence of a protein by inserting a modified gene or by editing an endogenous gene (e.g., via PRIME editing). In some implementations, the protein sequence is mutated to cause it to aggregate, or specific amino acid sequences may be added or removed to induce protein misfolding and aggregation. Mutations in the protein sequence or the induction of misfolding and aggregation by adding or removing specific amino acid sequences can contribute to cellular stress and apoptosis. These changes to proteins can disrupt normal cellular processes and lead to cell death.

[0030] The manipulation may include insertion, deletion, or other modifications to the gene of interest or the corresponding regulatory element. Modifications may be homozygous or heterozygous.

[0031] 6.1.2. Further genomic modifications for xenotransplant organ production In some implementations, engineered livestock are produced to provide organs for xenotransplantation. In some of these implementations, further genomic modifications may be performed to reduce immunogenicity after xenotransplantation into a human host.

[0032] In some embodiments, the cells are porcine, and further engineered by knocking out at least one gene selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, further engineering involves knocking out multiple genes selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, the animal cells are further engineered to express at least one human transgene selected from CD39, CD46, CD47, CD55, EPCR, TFPI, THBD, HO-1, vWF, and HLA-E.

[0033] In a particular implementation, the tissue to be transplanted is cartilage, heart valve, heart, kidney, pancreas, or lung.

[0034] 6.1.3. Pregnancy maturity In one implementation series, a genetically modified embryo is implanted into the surrogate mother's uterus, where it receives nutrition and support as it grows and develops. This implementation allows the genetically modified embryo to develop naturally within the uterus, thus taking advantage of the benefits provided by the maternal environment. Life support is provided after natural birth or induced birth.

[0035] In one implementation series, genetically modified embryos are grown entirely artificially outside the natural uterine environment. The genetically modified embryos receive nutrition from an artificial uterine environment used for support as they grow and develop. This implementation allows the genetically modified embryos to develop and grow in an artificial environment. After removal from the artificial uterine environment, additional life support may or may not be provided.

[0036] Just as in natural pregnancies where the fetus lacks most of its brain, the fetus may not survive long enough to reach full term in surrogacy or assisted reproduction. In various implementations, engineered organisms, particularly those with multiple genetic alterations to restrict brain development, will have a greater need for support, and this is especially true in the early stages of development. Due to insufficient brain and spinal cord development, migration to a support system may have to occur before survival. Figure 5 The study demonstrates engineered animal migration from a natural or artificial uterus to a human-like support system at an ideal time.

[0037] In some implementations, methods such as ultrasound or other means can be used to monitor the development of neural tissue.

[0038] In some implementations, genes are further modified to make neurons more susceptible to apoptosis in response to changes in temperature, nutrient availability, oxygen availability, small molecule inhibitors, or exposure to other toxins, compared to normally functioning neurons. These conditions can be modulated during embryonic and fetal maturation to further reduce neural tissue development and survival.

[0039] In some implementations, factors that enhance the stability or resistance of developing tissues to stressors or apoptosis are introduced preventively or in response to altered parameters. These may include antioxidants, anti-apoptotic small molecules, and growth factors. Automated sampling and monitoring systems can be implemented to precisely tailor the intervention in response to toxin levels, nutrient uptake, the state of neural tissue, and essential vital signs.

[0040] 6.2. Engineering Organization On the other hand, genetically engineered tissues are provided. The tissue comprises multiple cells aggregated into a three-dimensional structure, wherein the cells differentiate from a single fertilized egg into at least two differentiated cell types, and wherein the cells typically contain at least one genomic alteration, said genomic alteration being reduced neuronal signaling, neuronal depletion, and neuronal destruction. In some embodiments, the cells contain multiple genomic alterations, said genomic alterations being substantially common to at least one of reduced neuronal signaling, neuronal depletion, and neuronal destruction.

[0041] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell contains at least one genomic alteration 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 cell contains at least one genomic alteration that reduces the function of a protein 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.

[0042] In some implementations, the cells contain genomic alterations in MFSD2A and NDE1.

[0043] In some implementations, genomic alterations include one or more mutations in the primary amino acid sequence of a protein. In particular implementations, one or more mutations cause the protein to misfold and aggregate.

[0044] In some embodiments, the cell also contains at least one genomic alteration that reduces the expression of at least one gene encoding the 5HT receptor, optionally HTR6 or GPR26; the dopamine receptor, optionally DRD3; the GABA receptor, optionally GABRA6; the glutamate receptor, optionally GRM2, GRM4 or SLC1A2; a member of the interleukin-1 receptor family, optionally IL1RAPL1; a transcription factor, optionally SOX1, TBR1 or VAX1; a potassium channel, optionally KCNK4; and PRDM12.

[0045] In some embodiments, the cell also contains at least one genomic alteration that reduces the function of a protein 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.

[0046] In certain embodiments, genomic alterations include one or more mutations in the primary amino acid sequence of a further altered protein. In some embodiments, one or more mutations cause the protein to misfold and aggregate.

[0047] In some embodiments, the cells are homozygous for at least one of the described genomic alterations. In some embodiments, the cells are heterozygous for at least one of the described genomic alterations. In some embodiments, the tissue is located within the uterus. In some embodiments, the tissue is ex vivo.

[0048] In some implementations, the mammalian cells are derived from the Bovidae family, the Parvorder Catarrhini order, or the Suidae family.

[0049] 6.2.1. Organs for transplantation In some embodiments, the tissue cells are further engineered to reduce immunogenicity after transplantation into a human host. In some embodiments, further engineering involves knocking out at least one gene selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, further engineering involves knocking out multiple genes selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, the cells are porcine.

[0050] In some embodiments, the cells of the tissue are further engineered to express at least one human transgene selected from CD39, CD46, CD47, CD55, EPCR, TFPI, THBD, HO-1, vWF, and HLA-E.

[0051] In some implementations, the tissue is cartilage, heart valves, heart, kidney, pancreas, or lung.

[0052] 6.3. Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. A gene selected herein is defined as: in the context of its use, the selected gene and any known gene or its protein product having functional equivalence and / or high regional or gene / protein range sequence homology, with a reasonable expectation of similar results to the selected gene.

[0053] 6.4. Additional Implementation Methods In one aspect, a genetically engineered tissue is provided comprising a plurality of cells aggregated into a three-dimensional structure, wherein the cells differentiate from a single fertilized egg into multiple differentiated cell types, and wherein the cells typically contain at least one genomic alteration, said genomic alteration being at least one of the following: reduced neuronal signaling, neuronal depletion, and neuronal destruction. In various embodiments, the cells contain multiple genomic alterations, said genomic alterations being substantially common to at least one of the following: reduced neuronal signaling, neuronal depletion, and neuronal destruction. In some embodiments of the genetically engineered tissue, the cells are mammalian cells.

[0054] In various embodiments of genetically engineered tissues, the cells contain at least one genomic alteration 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 contain at least one genomic alteration that reduces the function of a protein 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. In some embodiments, the genomic alteration includes one or more mutations in the primary amino acid sequence of the protein. In some embodiments, one or more mutations cause misfolding and aggregation of the protein. In some embodiments, the cells contain genomic alterations in MFSD2A and NDE1.

[0055] In some embodiments of genetically engineered tissues, the cells also contain at least one genomic alteration 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.

[0056] In some embodiments of genetically engineered tissues, the cells also contain at least one genomic alteration that reduces the function of proteins encoded by at least one gene encoding a 5HT receptor, optionally HTR6 or GPR26; 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 further embodiments of genetically engineered tissues, genomic alterations include one or more mutations in the primary amino acid sequence of the further altered protein. In some embodiments, one or more mutations cause the protein to misfold and aggregate.

[0058] In various embodiments of genetically engineered tissues, the cells are homozygous for at least one genomic alteration. In some embodiments, the cells are heterozygous for at least one genomic alteration.

[0059] In some implementations, the mammalian cells are derived from the Bovidae, Cassiopeia, or Suidae families.

[0060] In some embodiments, the tissue is located within the uterus. In other embodiments, the tissue is ex vivo.

[0061] In some implementations, the cells are derived from cattle, sheep, goats, pigs, or rabbits.

[0062] In some implementations, the cells are derived from Old World monkey species.

[0063] In some embodiments of genetically engineered tissue, the tissue is non-human, and the cells are further engineered to reduce immunogenicity after xenografting into a human host. In some embodiments, the cells are porcine, and further engineering involves knocking out at least one gene selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, further engineering involves knocking out multiple genes selected from GGTA1, CMAH, B5GALNT2, β2M, and CIITA. In some embodiments, the cells are further engineered 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, a heart valve, a heart, a kidney, pancreas, or a lung.

[0064] On the other hand, a method is provided for generating genetically engineered tissue comprising multiple cells aggregated into a three-dimensional structure, wherein the cells differentiate from a single fertilized egg into at least two differentiated cell types, and wherein the cells typically contain at least one genomic alteration, which is at least one of the following: reduced neuronal signaling, neuronal depletion, and neuronal destruction, wherein the method comprises the ex vivo preparation of an embryo, wherein the cells typically contain at least one genomic alteration, which is at least one of the following: reduced neuronal signaling, neuronal depletion, and neuronal destruction, and the implantation of the embryo into the uterus of a suitably prepared host surrogate or an artificial system.

[0065] In some embodiments of the method, the method further includes the subsequent step of transferring the embryo from the host uterus to an artificial life support after a period of intrauterine growth and maturation that is less than the full-term gestation period. In some embodiments, the method further includes the delivery of a full-term animal after complete intrauterine pregnancy.

[0066] In various embodiments of the method, the method also includes the subsequent steps of euthanizing the animal and harvesting the tissue.

[0067] On the other hand, an engineered organism is provided comprising cells having at least one genomic alteration, said genomic alteration being at least one of the following: reduced neuronal signaling, reduced neuronal activity, neuronal depletion, and neuronal damage. In various embodiments, the cells of the engineered organism contain multiple genomic alterations that substantially collectively achieve at least one of the following: reduced neuronal signaling, reduced neuronal activity, neuronal depletion, and neuronal damage.

[0068] In various embodiments of the engineered organism, the cells of the organism contain at least one genomic alteration 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.

[0069] In various embodiments of the engineered organism, the cell contains at least one genomic alteration 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.

[0070] In various embodiments of the engineered organism, the cells of the organism also contain at least one genomic alteration 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.

[0071] In various embodiments of the engineered organism, the cell also contains at least one genomic alteration 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.

[0072] In various embodiments of engineered organisms, the cells of the organism contain at least one genomic alteration 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.

[0073] In various embodiments of the engineered organism, the cell contains at least one genomic alteration 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.

[0074] In various embodiments of engineered organisms, the cells contain genomic alterations in MFSD2A and NDE1.

[0075] 6.5. Other Interpretive Conventions Scope: Throughout this disclosure, various aspects of the invention are presented in the form of scope. Scope includes the enumerated endpoints. It should be understood that the description in scope form is merely for convenience and brevity and should not be construed as an immutable limitation on the scope of the invention. Therefore, the description of scope should be considered as having specifically disclosed all possible sub-scopes and individual numerical values ​​within those scopes. For example, a description of a scope such as 1 to 6 should be considered as specifically disclosing sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within those scopes, such as 1, 2, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0076] In this disclosure, the words “comprising,” “containing,” “having,” “including,” “including,” and their linguistic variations have the meanings given to them under U.S. patent law, allowing for the presence of additional components beyond those expressly listed.

[0077] Unless otherwise specified or obvious from the context, the term “or” as used herein shall be understood as inclusive.

[0078] Unless otherwise specified or obvious from the context, the terms “a,” “one,” and “the” are to be understood as singular or plural, as used herein. That is, the articles “a” and “one” are used in this document to refer to one or more (i.e., at least one) grammatical object of the article. For example, “one element” means one element or more.

[0079] Unless otherwise specified or obvious from the context, the term "about" as used herein shall be understood to be within the normal tolerance range of the art. Unless otherwise stated, "about" means ±10% of the value. When percentages are provided relative to the amount of a component or material in a composition, percentages shall be understood to be weight-based percentages unless the context otherwise indicates or understands.

[0080] 7. Example 7.1. Fertilized egg genome editing This embodiment demonstrates a method for successfully editing selected genes in fertilized eggs using the Cas9 nuclease system. Protospacer neighbor motif (PAM) site recognition for Cas9 nucleases is particularly critical for development as an effective genome editing tool, and also provides guide RNA (gRNA) targets, gRNA sequences, and polymerase chain reaction (PCR) primers used in the development of the Cas9 nuclease system for each gene target. Table 1-42.

[0081] Methods and Materials: Guide RNA (gRNA) was incubated with Cas9 in Opti-MEM for at least 30 minutes in various combinations. The resulting ribonucleoprotein particles (RNPs) were electroporated into pronuclear-stage mouse zygotes using an ECM830 square-wave electroporator. Typically, this was performed at 30 V using 5 pulses, each pulse lasting 3 minutes and spaced 100 ms apart.

[0082] Gene: NDE

[0083] Gene: MFSD2a

[0084] Gene: GRIN2B

[0085] Gene: DCX

[0086] Gene: I11RAPL1

[0087] Gene: CHRNB2

[0088] Gene: LHX1

[0089] Gene: LHX2

[0090] Gene: OTX1

[0091] Gene: OTX2

[0092] Gene: HTR6

[0093] Gene: CHRNA7

[0094] 1 = Control, 2 = OTX1, OTX2, NDE, 3 = LHX1, LHX2, GRIN2B, 4 = HTR6, NDE1, MFSD2A, 5 = LHX1, OTX2, MFSD2A, GRIN2B, 6 = GRIN2B, MFSD2A, LHX1, 7 = Il1RAPL1, CHRNA7 7.2. Confirmation of gene knockout (KO) Subsequently, histological studies (Figures 1-3) and PCR results ( Figure 6-8 This confirmed the gene knockout.

[0095] Comparative microscopic and PCR analysis of genetically modified and wild-type embryos Table 37 shows the results of large-scale screening for gene knockout in mouse embryos. Figure 9 The fourth group from Table 37 is shown as an example of how these counts were performed at 96 hours, with red dots on the embryos we counted as healthy blastocysts. We used CRISPR-Cas9 technology to knock out specific genes in mouse embryos. Our goal was to investigate the effects of these knockouts on embryonic development and to demonstrate viability and the potential for forming viable fetuses. Seven different conditions were tested, each targeting a unique combination of genes. Embryonic developmental progress was observed, with particular focus on the hatching blastocyst stage.

[0096] Our observations revealed that all treated embryos exhibited developmental delay, which is common for embryos subjected to CRISPR and electroporation. Despite this delay, most conditions resulted in hatching blastocysts with normal appearances, except for condition 5, which accounted for only 5.88%.

[0097] Condition 5 (which involves the largest number of guide RNAs targeting the most genes (LHX1, OTX2, MFSD2A, GRIN2B)) showed a significant deviation from the expected developmental progression. This condition resulted in a significant reduction in the number of normally hatched blastocysts. The complexity of simultaneously targeting multiple genes may have contributed to this outcome.

[0098] The results showed that many embryos were progressing through normal development. This indicates that, despite initial developmental delays, CRISPR-induced gene knockout does not completely halt fetal development and produces a large number of viable embryos. We performed PCR analysis on a subset of hatched blastocysts from Table 37. Our results show homozygous KO alleles for each gene target in at least 60% of the developing blastocysts.

[0099] Control: As expected, the control embryos developed normally without any genetic changes and had a very high blastocyst formation and hatching rate.

[0100] Condition 2 (OTX1, OTX2, NDE): Compared to the control, fewer embryos were formed into blastocysts, but if implanted, they would still produce a large number of viable fetuses.

[0101] Condition 3 (LHX1, LHX2, GRIN2B): Compared to the control, fewer embryos were formed into blastocysts, but if implanted, they were still sufficient to produce many viable fetuses.

[0102] Condition 4 (HTR6, NDE1, MFSD2A): Compared to the control, the number of embryos formed blastocysts was reduced, but higher than in conditions 2 and 3, and if implanted, they were still very sufficient to produce many viable fetuses. Figure 9 A microscope image taken 96 hours later is shown as an example of this condition.

[0103] Condition 6 (GRIN2B, MFSD2A, LHX1): Compared to the control, the embryos showed reduced blastocyst formation, but if implanted, they still produced a large number of viable fetuses.

[0104] Condition 7 (Il1RAPL1, CHRNA7): This condition showed a significant proportion of normal development compared to other conditions and controls, and we used this condition to demonstrate fetal development and CNS damage in Figure 3.

[0105] Table 37 demonstrates that CRISPR-Cas9 technology can be used to knock out multiple genes crucial for central nervous system development in mouse embryos, and that these embryos can progress to blastocyst development and hatch despite initial developmental delays. Combined with Figures 1, 2, and 3, where we demonstrate that the KO combination progresses all the way to late fetal stages and shows significant CNS damage as predicted (condition 7 in Table 37 is found in Figure 3 demonstrating the development of these conditions, which was PCR-validated as a dual KO of Il1RAPL1 and CHRNA7), these data demonstrate that our gene combination can either develop normally or impair CNS development. The data collected so far indicate that most conditions result in outwardly normal hatching blastocysts, which, given their lack of key CNS developmental genes, will develop CNS defects, as shown in Figures 1, 2, and 3.

[0106] The more complex gene targeting in condition 5 highlights the potential challenges and impacts of multi-gene knockout, but it still results in a smaller percentage of surviving blastocysts.

[0107] Comparative histological analysis of genetically modified and wild-type embryonic brains Figure 1A-1DThe results shown present a histological comparison between the genetically modified embryo and its wild-type counterpart from the same litter, both implanted in recipient females. The left image depicts the genetically modified embryo, while the right image depicts the wild-type embryo. More specifically, Figure 1A The entire brain of a genetically modified embryo is shown, in which the genes NDEI and DCX were inactivated using the CRISP technique. The figure highlights significant morphological differences, including malformations and areas of degenerative activity in the cortex and other brain regions, as indicated by arrows. Notably, there is significant ventricular enlargement and a developmental abnormality characterized by the inability of the ventricles to properly separate at this developmental stage. The cerebellum appears to be not in the process of formation at all. Figure 1B This image shows a magnified view of the cerebral cortex in a genetically modified embryo. This detailed view highlights the extent of cortical abnormalities and degenerative changes, and further complements observations of ventricular enlargement and malformations. Figure 1C The whole brain of a wild-type identical embryo at a similar developmental stage is shown for comparison. The morphology, particularly the ventricular structures and overall brain structure, contrasts sharply with that of a genetically modified embryo, in which enlarged ventricles and failure of ventricular separation are observed. Figure 1D A magnified view of the cerebral cortex in a wild-type embryo is provided, demonstrating normal cortical development. This figure serves as a control, showing typical cortical structures absent in the genetically modified counterpart.

[0108] Visualization of genetically modified embryos at gestational age 14 (E14). Figure 2 The results shown captured the entire embryo at gestational age 14 (E14). It is worth noting that, despite the details described above and Figure 1A-1D Significant brain changes were observed in the embryo, but the external morphology of the embryo appeared insignificant and consistent with typical developmental milestones for this stage. A key finding at harvest was the presence of an active heartbeat. This indicates embryonic viability. This observation is significant because it highlights the localized nature of the brain changes and has no significant impact on overall embryonic viability.

[0109] It is important to emphasize that genetic modifications primarily affect brain development, such as Figure 1A-1D This is a widely analyzed finding. The figure highlights the contrast between extensive brain abnormalities and the other normal external development of the embryo. This suggests that profound internal changes in brain morphology may not be externally visible at this developmental stage.

[0110] It is noteworthy that despite the significant brain abnormalities in the genetically modified embryos, their overall embryonic development was generally normal, including an active heartbeat at harvest and the unremarkable appearance of other major organs. Figure 2 .

[0111] Detailed immunohistochemical analysis of IL1RAPL1 and CHRNA7 in the brains of genetically modified and wild-type E14 mice The results demonstrated immunohistochemical comparisons between genetically modified embryos with targeted knockouts of IL1RAPL1 and CHRNA7. Figure 3 highlights the significant effects of IL1RAPL1, CHRNB2, and CHRNA7 knockout on brain morphology and function. Figures 3A-3H The combination of these three knockouts significantly alters brain structure. Basic brain structure and development are impaired. Acetylcholine signaling is also disrupted. Therefore, profound impacts on brain function and cognition are expected, while the development of other tissues and organs is not significantly impaired.

[0112] PCR results showed that multiple knockout combinations could still result in fetuses with the potential to develop into late-pregnancy fetuses. Figure 6-8 Histological results showed expression defects and structural abnormalities in animals confirmed by knockout variants of the target gene.

[0113] 7.3. Implantation of KO embryos into the target recipient The resulting knockout (KO) embryos were cultured in groups of 20 to 120 for 72–96 hours in an incubator at 37.0 °C and 5% CO2 on EmbryoMax® KSOM medium or a similar supportive mouse embryo culture medium in mineral oil. Conditions were adjusted to maintain pH between 7.2 and 7.4.

[0114] Blastocyst-stage embryos were transferred to approximately 2.5 DPC pseudopregnant female CD1 (ARC) mice with a target weight of approximately 30 g. Recipient mice were targeted to be >8 weeks old. Synchronization was induced by 2.5 IU of pregnant mare serum gonadotropin (PMSG) for 48 hours, followed by 2.5 IU–5 IU of human chorionic gonadotropin (HCG) or 50 μL of hyperova for 48 hours, followed by 5 IU of HCG. Pseudopregnancy was induced in appropriately cyclic females by mechanical and vibration-based stimulation via insertion of a smooth plastic rod into the vagina for 30 seconds, which could be in contact with an electric toothbrush module or trimmer.

[0115] Then, 2.5 days later, the embryos were transferred directly into the uterus of the recipient female in the receiving stage via the vagina using non-surgical embryo transfer (NSET) devices (Paratechs). Figure 6 and Figure 7 The methods used to process the histological images mentioned below are described. All wild-type and knockout embryos compared to each other underwent the same processing.

[0116] 7.4. Effects of combinatorial gene knockout on cognition in animal models Figure 1A-3H The data presented provide compelling evidence of the profound impact of specific gene knockouts on brain development and, consequently, on overall cognition in mice. These figures collectively demonstrate that combined knockouts of brain-related and specific genes have a more significant effect than single-gene alterations.

[0117] exist Figure 1A-1D In our study, we observed brain changes in embryos with CRISPR-mediated NDE1 and DCX gene knockout. Morphological abnormalities, particularly ventricular enlargement and ventricular separation failure, point to significant developmental disorders. These structural abnormalities, especially in regions crucial to cognitive processes, suggest the possibility of substantial cognitive deficits in these animals.

[0118] Figure 2 These findings supplement the evidence that despite profound internal brain changes, overall embryonic development appears to be generally normal. This observation is crucial because it highlights the specificity of genetic modifications in influencing brain development while leaving other developmental processes relatively unaffected. Organs and tissues remain functional and intact.

[0119] Figures 3A-3H The effects of knocking out IL1RAPL1 and CHRNA7 were demonstrated. The absence of IL1RAPL1 and CHRNA7 expression in the knockout model, as evidenced by different staining patterns, highlights the crucial roles these genes play in normal brain function. Given the known associations of IL1RAPL1 with intellectual disability and CHRNA7 with neuronal acetylcholine receptor function, their loss strongly suggests potential cognitive impairment. The failure of CHRNB2 staining (though not visually explicit) adds another layer to our understanding of the complex gene interactions influencing brain development.

[0120] In summary, these results indicate that the combined effects of multiple gene knockouts on brain morphology and function are greater than the sum of individual knockouts. This synergistic effect is particularly evident in regions crucial for cognition, suggesting that animals with these genetic modifications are likely to experience significant and profound cognitive deficits.

[0121] 7.5. Organ development in KO mouse fetuses In our exploration of the developmental effects of genetic modifications, a particularly interesting observation is the development of organs and tissues that are generally normal, despite significant abnormalities in brain morphology and function. Figure 2 This phenomenon was demonstrated by showing E14 embryos with a clearly active heartbeat (an indicator of overall embryonic viability), although... Figure 1A-1BThe gene knockout in this animal, described in detail, induces profound brain abnormalities. This finding is remarkable because it demonstrates a degree of developmental compartmentalization, where severe genetic alterations can lead to specific brain malformations without disrupting the overall developmental trajectory of other organs and tissues. The presence of a normal heartbeat and typical morphological features in major organs reinforce this concept. It also raises interesting questions about the resilience and adaptability of embryonic development in the face of targeted gene disruption, particularly those affecting brain development. Such observations (which will...) Figure 1A-1D and Figures 3A-3H Brain changes and significantly delayed brain growth Figure 2 The previously insignificant overall developmental juxtaposition reveals a complex interplay between genetic regulation, organ specificity, and developmental robustness.

[0122] 8. Equivalents and incorporation by reference Although the invention has been specifically shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention.

[0123] For all purposes, all references, patents and patent applications cited in the text of this specification are incorporated herein by reference in their entirety.

Claims

1. A genetically engineered tissue comprising: Multiple cells condensed into a three-dimensional structure, The cells described therein differentiate from a single fertilized egg into multiple differentiated cell types, and The cells described therein typically contain at least one genomic alteration, which is at least one of the following: reduced neuronal signaling, neuronal depletion, and neuronal destruction.

2. The tissue of claim 1, wherein the cells contain multiple genomic alterations, which are substantially collectively at least one of the following: reduced neuronal signaling, neuronal depletion, and neuronal destruction.

3. The tissue as claimed in claim 1 or claim 2, wherein the cells are mammalian cells.

4. The tissue of claim 3, wherein the cells contain at least one genomic alteration 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 of claim 3, wherein the cells contain at least one genomic alteration 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 of claim 5, wherein the genomic alteration comprises one or more mutations in the primary amino acid sequence of the protein.

7. The tissue of claim 6, wherein one or more mutations cause misfolding and aggregation of the protein.

8. The tissue according to any one of claims 4-7, wherein the cells contain genomic alterations in MFSD2A and NDE1.

9. The tissue according to any one of claims 4-8, wherein the cells further contain at least one genomic alteration 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 4-8, wherein the cell further comprises at least one genomic alteration that reduces the function of proteins encoded by at least one gene encoding at least one of the following: 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 of claim 10, wherein the genomic alteration comprises one or more mutations in the primary amino acid sequence of the further altered protein.

12. The tissue of claim 11, wherein the one or more mutations cause misfolding and aggregation of the protein.

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

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

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

16. The tissue as claimed in any one of claims 3-15, wherein the tissue is located within the uterus.

17. The tissue according to any one of claims 3-15, wherein the tissue is ex vivo.

18. The tissue of claim 17, wherein the cells are derived from a species of cattle, sheep, goat, pig, or rabbit.

19. The non-human tissue of claim 15, wherein the cells are further engineered to reduce immunogenicity after xenografting into a human host.

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

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

22. The non-human tissue of claim 20 or claim 21, wherein the cells are further engineered 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 as claimed in any one of claims 19-22, wherein said tissue is cartilage, heart valve, heart, kidney, pancreas, or lung.

24. A method for generating a genetically engineered tissue comprising a plurality of cells aggregated into a three-dimensional structure, wherein the cells differentiate from a single fertilized egg into at least two differentiated cell types, and wherein the cells generally contain at least one genomic alteration, said genomic alteration being reduced neuronal signaling, neuronal depletion, and / or neuronal destruction, the method comprising: Embryos are prepared in vitro, wherein the cells typically contain at least one genomic alteration characterized by reduced neuronal signaling, neuronal depletion, and / or neuronal destruction. The embryo is implanted into the uterus of a properly prepared host surrogate.

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

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

27. The method of claim 25 or 26, further comprising the subsequent steps of euthanizing the animal and harvesting the tissue.

28. An engineered organism comprising: cells having more than one genomic alteration, said genomic alteration being at least one of neuronal signal reduction, neuronal depletion, and neuronal destruction.

29. The engineered organism of claim 28, wherein the cells of the organism contain a variety of genomic alterations that commonly involve reduced neuronal signaling, neuronal depletion, and neuronal destruction.

30. The tissue as described in any one of claims 4-15 or 28-29 or a combination thereof, wherein the tissue produces organs for meat production and / or for creating organs for transplantation.

31. An organism as described in any one of claims 4-15 or 28-29 or a combination thereof, wherein the organism selects to minimize its ability to experience pain by using a combination of the listed genes or to be completely incapable of suffering due to a lack of cognitive ability and / or pain perception.

32. The tissue of claim 30, wherein the cells contain more than one genomic alteration 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.

33. The tissue of claim 30, wherein the cells contain more than one genomic alteration 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.

34. The tissue of claim 1 or claim 3, wherein the cells contain more than one genomic alteration, the genomic alteration reducing the expression of at least two genes selected from GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, and OTX2.

35. The tissue of claim 1 or claim 3, wherein the cells contain more than one genomic alteration that reduces the function of at least two genes selected from GRIN2B, LHX1, LHX2, MFSD2A, NDE1, NTRK1, OTX1, and OTX2.

36. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of both LHX1 and LHX2 and at least one genomic alteration selected from GRIN2B, MFSD2A, NDE1, NTRK1, OTX1, and OTX2.

37. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of both OTX1 and OTX2 and at least one genomic alteration selected from GRIN2B, LHX1, LHX2, MFSD2A, NDE1, and NTRK1.

38. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of OTX1, OTX2 and NDE1.

39. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of LHX1, LHX2 and GRIN2B.

40. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of HTR6, NDE1, and MFSD2A.

41. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of LHX1, OTX2, MFSD2A and GRIN2B.

42. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of GRIN2B, MFSD2A and LHX1.

43. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of both Il1RAPL1 and CHRNA7.

44. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of both NDE1 and DCX.

45. The tissue of claim 1 or claim 3, wherein the cells contain genomic alterations that reduce the function of both NDE1 and MFSD2A.

46. ​​The tissue of any one of claims 28-45, wherein the cells further contain at least one genomic alteration that reduces the expression or function of a protein encoded by at least one gene encoding a nicotinic 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 engineered organism as described in claims 4-15, wherein the cells are derived from humans.

48. The engineered organism as described in claims 28-45, wherein the cells are derived from humans.

49. The tissue according to any one of claims 3-14, wherein the mammalian cells are derived from the order Ceratospori.

50. The engineered organism as described in claims 32-49, wherein the cell is derived from any single cell capable of undergoing normal embryonic development.

51. The tissue according to any one of claims 1-50, wherein the cells are developed in an artificial system.

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