Cells, tissues, organs, and animals having one or more modified genes for enhanced xenograft survival and tolerance and methods of use

Abstract

This disclosure relates to cells, tissues, organs, and / or animals having one or more modified genes for enhancing xenograft survival and / or tolerance. Furthermore, this disclosure relates to methods for preparing and using said cells, tissues, organs, and / or animals having one or more of said modified genes.

Description

prior art

[0001] The shortage of human organs and tissues for transplantation has become increasingly severe over the past few decades and represents one of the most important unmet medical needs. Xenotransplantation has the potential to provide an almost unlimited supply of transplanted organs for patients with chronic organ failure. Similarities in organ size and physiology, combined with genetic engineering to eliminate molecular incompatibilities, make pigs the donor of choice for kidney xenotransplantation. Preclinical studies have shown that pig kidney xenotransplantation can extend the life of nonhuman primate recipients by weeks to months (Higginbotham 2015, Iwase 2015b). However, due to the evolutionary distance between pigs and humans, pig organs trigger rejection by the human immune system in multiple forms, including (i) hyperacute rejection; (ii) acute humoral rejection, caused by thrombotic dysregulation and II (iii) thrombotic microangiopathy consisting of intravascular thrombus formation with platelet consumption and EC initiation, fibrin deposition, and thrombosis due to lack of thrombus regulation, and (iv ) chronic vascular disease. These adverse events are at least in part due to molecular incompatibility between the donor and recipient, particularly with regard to genes involved in complement, coagulation, inflammation, and immune response systems. The clinical use of xenogeneic organs (eg, pigs) is hampered by these immunological incompatibilities that have so far prevented the use of porcine cells, tissues, and vascularized porcine organs in clinical xenotransplantation.

[0002] Over the past two decades, several genetic modifications have been identified that reduce interspecies incompatibility between pigs and humans. However, these previously identified genetic modifications have not yet achieved long-term xenograft survival. Furthermore, technical limitations of large-scale genome engineering hinder the integration of these modifications in individual animals. Contents of the invention

[0003] There is a need to develop porcine cells, tissues, organs and / or porcine animals with novel combinations of genetic modifications for xenotransplantation and development of related methods.

[0004] The present disclosure provides cells, tissues, organs, and animals comprising genetic modifications that result in enhanced immunological compatibility, as well as vectors and methods for producing such cells, tissues, organs, and animals, and such cells, tissues, organs and the use of animals in xenotransplantation.

[0005] Genetic modification Improved xenograft compatibility through several modifications introduced in pigs. These modifications eliminate the expression of antigens known to stimulate host-versus-graft responses. Furthermore, overexpression of the protein helps regulate additional responses to the graft to optimize graft survival in the transplant host. Disclosed herein are cassettes expressing proteins that provide control over host responses. The cassettes may be combined in nucleic acid, also referred to herein as payload, to be introduced into the genome of the pig.

[0006] The payload can contain a coagulation cassette that expresses multiple proteins, each of which can modulate one or more aspects of the host's coagulation response. For example, proteins shown to reduce or eliminate graft-associated coagulation can include THBD and TFPI. THBD reduces blood clotting by converting thrombin from prothrombin to antithrombin. TFPI blocks the initiation of blood coagulation by inhibiting Factor Vila and prothrombinase.

[0007] The payload can contain a complement regulatory cassette expressing multiple proteins, each of which can modulate one or more aspects of the host's complement response, thereby reducing complement deposition and thereby reducing anti-graft immune response. The protein shown to reduce or eliminate complement deposition on the graft may include CD46, CD55, or CD59. CD46 is a cofactor that contributes to the inactivation of complement components C3b and C4b by serum factor I. CD55 indirectly blocks membrane attack complex formation by limiting the amplification of complement cascade convertases. Membrane attack complexes are formed on the surface of the cell membrane after complement initiation, and the membrane attack complexes cause complement-mediated cell lysis. CD59 prevents C9 from polymerizing and forming the membrane attack complex.

[0008] The payload may contain an innate immunity cassette that reduces the host's innate immune response against the graft. Proteins shown to suppress innate immune attack against the graft may include B2M, HLA-E, and CD47. B2M and HLA-E reduce natural killer (NK) cell-mediated lysis of transplanted cells. CD47 inhibits macrophage phagocytosis. Optionally, the cassette may be an innate immunity and inflammation cassette, and express additional proteins that reduce inflammation in addition to innate immune regulatory components; for example A20 and HO1. A20 reduces NF-κB activation in response to external stimuli. HO1 prevents vascular inflammation by cleaving the heme group leading to the formation of biliverdin, carbon monoxide and the release of ferrous iron.

[0009] Provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI An antiproton, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; (iii) is selected from the promoter of SEQ ID NO: 126-145 and 167-168; (iv) is selected from the promoter of SEQ ID NO: 126-145 and 167-168; . 112-125, 154-156, 159-162, 190-192 and 239-240 poly A sequences, and combinations thereof; (v) at least one having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 2A polypeptide of sequence 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD59 The second cistron of CD59, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86,108; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one polyA sequence selected from SEQ ID NO. NO: 2A polypeptides of sequences 1-10, 12-14 and 159-162; and (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- 168, 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240, and combination; and (v) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162.

[0010] In embodiments, said nucleic acid comprises at least two of said polycistronic cassettes. In embodiments, said nucleic acid comprises three of said polycistronic cassettes. In an embodiment, the nucleic acid comprises a sequence selected from SEQ ID NO: 24, 25, 29, 202, 203 and 207.

[0011] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI Antion, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one polyA sequence selected from SEQ ID NO. NO: 2A polypeptides of sequences 1-10, 12-14 and 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD59 The second cistron of CD59, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86,108; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one polyA sequence selected from SEQ ID NO. NO: 2A polypeptides of sequences 1-10, 12-14 and 159-162; and (c) an innate immunity and inflammation and apoptosis cassette comprising: (i) a first cistron encoding a B2M HLA-E fusion protein, which encodes said B2M HLA-E The nucleic acid sequence of E fusion protein is selected from SEQ ID NO: 62,66,105; (ii) the second cistron of coding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) The third cistron of encoding A20, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (iv) the fourth cistron of encoding HO1, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 64-65, 104, 182 and 188; ID NOs: 63, 109, 165, 183 and 189; (v) a promoter selected from SEQ ID NOs: 126-145, 167-168, 178-179, 231-238 and 250; (vi) a promoter selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240 poly A sequences, and combinations thereof; and (vi) at least one having a sequence selected from SEQ ID NO: 1-10, 12- 2A polypeptides of sequences 14 and 159-162.

[0012] In embodiments, said nucleic acid comprises at least two of said polycistronic cassettes. In embodiments, said nucleic acid comprises three of said polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 26 or SEQ ID NO: 204.

[0013] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI (iii) a third cistron encoding CD39, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NO: 67-70; ( iv) a promoter selected from SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; and (v) selected from SEQ ID NO. 112-125, 154-156, 159-162 , polyA sequences of 190-192 and 239-240, and combinations thereof; (vi) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD59 The second cistron of CD59, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NO:86,108; (iii) the third cistron of CD55, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184; (iv) the promoter selected from SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (v) selected from the promoter of SEQ ID NO. 112-125, 154-156, PolyA sequences of 159-162, 190-192, and 239-240, and combinations thereof; (vi) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14, and 159-162; as well as (c) an innate immunity and inflammation and apoptosis cassette comprising: (i) a first cistron encoding a B2M HLA-E fusion protein, which encodes said B2M HLA-E The nucleic acid sequence of E fusion protein is selected from SEQ ID NO: 62,66,105; (ii) the second cistron of coding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) The third cistron of encoding A20, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (iv) the fourth cistron of encoding HO1, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 64-65, 104, 182 and 188; ID NO: 63, 109, 165, 183 and 189; (v) the fifth cistron encoding PD-L1, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89-91; (vi) selected from SEQ ID NO: 89-91; Promoters of ID NOs: 126-145, 167-168, 178-179, 231-238 and 250; (vii) selected from the group consisting of SEQ ID NOs. 112-125, 154-156, 159-162, 190-192 and 239 - a polyA sequence of 240, and combinations thereof; and (vii) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14, and 159-162.

[0014] In embodiments, said nucleic acid comprises at least two of said polycistronic cassettes. In embodiments, said nucleic acid comprises three of said polycistronic cassettes. In an embodiment, the nucleic acid comprises a sequence selected from SEQ ID NO: 23, 27, 28, 201, 205 and 206.

[0015] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of encoding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of encoding PROCR Antion, wherein the nucleotide sequence of PROCR is selected from SEQ ID NO: 92, 93 and 181; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one polyA sequence selected from SEQ ID NO: 2A polypeptides of the sequence 1-10, 12-14 and 159-162; (b) inflammation and apoptosis box, said inflammation and apoptosis box comprises: (i) the first cistron of coding A20, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (ii) the second cistron encoding HO1, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; (iii) selected from SEQ ID NO: 126-145, 167 - the promoter of 168, 178-179, 231-238 and 250; and (iv) a polyA sequence selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162; and (c) a complement regulation and innate immunity cassette, said complement regulation and The innate immunity box comprises: (i) a first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) a second cistron encoding CD47, wherein CD47 The nucleic acid sequence of CD55 is selected from SEQ ID NO: 77-83 and 180; (iii) the third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84,85, 107 and 184; (iv) selected From the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (v) selected from the promoter of SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and polyA sequences of 239-240, and combinations thereof; and (vi) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14, and 159-162.

[0016] In embodiments, nucleic acids disclosed herein comprise at least two of said polycistronic cassettes. In embodiments, the nucleic acid comprises three of said polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 208.

[0017] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of encoding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of encoding PROCR An antitron, wherein the nucleotide sequence of PROCR is selected from SEQ ID NO: 92, 93 and 181; (iii) a third cistron encoding TFPI, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187 (iv) selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (v) selected from the promoter of SEQ ID NO. 112-125, 154-156, 159- PolyA sequences of 162, 190-192 and 239-240, and combinations thereof; (vi) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD55 The third cistron, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184; (iii) selected from SEQ ID NO:126-145,167-168,178-179,231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one having 2A polypeptides selected from the sequences of SEQ ID NO: 1-10, 12-14 and 159-162; and (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- 168, 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combination; and (v) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162.

[0018] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises a sequence selected from SEQ ID NO: 31-34 and 209-212.

[0019] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of encoding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of encoding PROCR Antion, wherein the nucleotide sequence of PROCR is selected from SEQ ID NO: 92, 93 and 181; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one polyA sequence selected from SEQ ID NO: 2A polypeptides of the sequence 1-10, 12-14 and 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD55 The third cistron, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184; (iii) selected from SEQ ID NO:126-145,167-168,178-179,231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one having A 2A polypeptide selected from the sequence of SEQ ID NO: 1-10, 12-14 and 159-162, and (c) an innate immunity and inflammation and apoptosis cassette comprising: (i) a first cistron encoding a B2M HLA-E fusion protein, which encodes said B2M HLA-E The nucleic acid sequence of E fusion protein is selected from SEQ ID NO: 62,66,105; (ii) the second cistron of coding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) The third cistron of encoding A20, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (iv) the fourth cistron of encoding HO1, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 64-65, 104, 182 and 188; ID NOs: 63, 109, 165, 183 and 189; (v) a promoter selected from SEQ ID NOs: 126-145, 167-168, 178-179, 231-238 and 250; and (vi) a promoter selected from SEQ ID NOs: The poly A sequence of ID NO.112-125,154-156,159-162,190-192 and 239-240, and combination thereof, and (vii) at least one has the sequence selected from SEQ ID NO:1-10,12 - 2A polypeptides of sequences 14 and 159-162.

[0020] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 37 or SEQ ID NO: 215.

[0021] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI (iii) the third cistron encoding CD39, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NO: 67-70; ( iv) a promoter selected from SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (v) a promoter selected from SEQ ID NO. 112-125, 154-156, 159-162, PolyA sequences of 190-192 and 239-240, and combinations thereof; (vi) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14, and 159-162; (b) complement regulation and inflammation and apoptosis box, described complement regulation and inflammation and apoptosis box comprise: (i) the first cistron of coding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71 -76, 185 and 200; (ii) the second cistron encoding CD59, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NO: 86, 108; (iii) the third cistron encoding CD55, wherein the nucleic acid sequence of CD55 Sequence is selected from SEQ ID NO:84,85,107 and 184; (iv) the fourth cistron of coding A20, wherein the nucleotide sequence of A20 is selected from SEQ ID NO:64-65,104,182 and 188; (v ) the fifth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; (vi) the sixth cistron encoding PD-L1, wherein the PD-L1 The nucleic acid sequence is selected from SEQ ID NO: 89-91; (vii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (viii) is selected from the promoter of SEQ ID NO . 112-125, 154-156, 159-162, 190-192 and 239-240 poly A sequences, and combinations thereof; (ix) at least one having a sequence selected from SEQ ID NO: 1-10, 12-14 and The 2A polypeptide of the sequence 159-162, and (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- 168, 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combination; and (v) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162.

[0022] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 35 or SEQ ID NO: 213.

[0023] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI Antion, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and (v) at least one polyA sequence selected from SEQ ID NO. ID NO: 2A polypeptides of the sequences of 1-10, 12-14 and 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD55 The third cistron, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184; (iii) selected from SEQ ID NO:126-145,167-168,178-179,231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) at least one having a sequence selected from SEQ ID NO. 2A polypeptides of the sequences of ID NO: 1-10, 12-14 and 159-162, and (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- The promoters of 168, 178-179, 231-238 and 250; (iv) polyA sequences selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof, and (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159-162.

[0024] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises a sequence selected from SEQ ID NO: 36, 40, 214 and 218.

[0025] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI Antion, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 and (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) at least one having a sequence selected from SEQ ID NO: 2A polypeptides of the sequence 1-10, 12-14 and 159-162; (b) complement regulation and inflammation and apoptosis box, described complement regulation and inflammation and apoptosis box comprise: (i) the first cistron of coding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71 -76, 185 and 200; (ii) the second cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; (iii) the third cistron encoding A20, Wherein the nucleic acid sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (iv) the fourth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; (v) a promoter selected from SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; and (vi) a promoter selected from SEQ ID NO. 112-125, and 154 - polyA sequences of 156, 159-162, 190-192, and combinations thereof; (vii) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14, and 159-162; and (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- The promoters of 168, 178-179, 231-238 and 250; (iv) polyA sequences selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; and (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159-162.

[0026] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises a sequence selected from SEQ ID NO: 41, 42, 219 and 220.

[0027] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI (iii) the third cistron encoding CD39, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NO: 67-70; ( iv) a promoter selected from SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (v) selected from SEQ ID NO. 112-125, and 154-156, 159-162 , polyA sequences of 190-192, and combinations thereof; (vi) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14, and 159-162, (b) complement regulation and inflammation and apoptosis box, described complement regulation and inflammation and apoptosis box comprise: (i) the first cistron of coding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71 -76, 185 and 200; (ii) the second cistron encoding CD59, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NO: 86, 108; (iii) the third cistron encoding CD55, wherein the nucleic acid sequence of CD55 Sequence is selected from SEQ ID NO:84,85,107 and 184; (iv) the fourth cistron of coding A20, wherein the nucleotide sequence of A20 is selected from SEQ ID NO:64-65,104,182 and 188; (v ) the fifth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; (vi) the sixth cistron encoding PD-L1, wherein the PD-L1 The nucleic acid sequence is selected from SEQ ID NO: 89-91; (vii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (viii) is selected from the promoter of SEQ ID NO . 112-125, and 154-156, 159-162, 190-192 poly A sequences, and combinations thereof; and (ix) at least one having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159- 2A polypeptide of sequence 162, and (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- The promoters of 168, 178-179, 231-238 and 250; (iv) polyA sequences selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; and (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159-162.

[0028] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises a sequence selected from SEQ ID NO: 38, 39, 173, 216, 217 and 226.

[0029] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI Antion, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; and (v) at least one polyA sequence selected from SEQ ID NO: 2A polypeptides of the sequence 1-10, 12-14 and 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD55 The third cistron, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184; (iii) selected from SEQ ID NO:126-145,167-168,178-179,231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) at least one having a sequence selected from SEQ ID NO. ID NO: 2A polypeptide of the sequence of 1-10, 12-14 and 159-162, (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- 168, 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof, ( v) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162; and (d) cellular immunity and coagulation box, said cellular immunity and coagulation box comprises: (i) the first cistron of encoding LEA29Y, wherein the nucleic acid sequence of LEA29Y is selected from SEQ ID NO: 87-88; (ii) encoding CD39 The second cistron, wherein the nucleotide sequence of CD39 is selected from SEQ ID NO: 67-70; (iii) selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 and (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof, and (v) at least one polyA sequence selected from SEQ ID NO. : 2A polypeptides of sequences 1-10, 12-14 and 159-162.

[0030] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises at least three of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 43 or SEQ ID NO: 221.

[0031] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI Antion, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) at least one polyA sequence selected from SEQ ID NO: 1 - 2A polypeptides of the sequences 10, 12-14 and 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD55 The third cistron, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184; (iii) selected from SEQ ID NO:126-145,167-168,178-179,231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) at least one having a sequence selected from SEQ ID NO. ID NO: 2A polypeptides of the sequences of 1-10, 12-14 and 159-162; (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- 168, 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof, ( v) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162; and (d) inflammation and apoptosis box, described inflammation and apoptosis box comprise: (i) the first cistron of coding A20, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (ii) the second cistron encoding PD-L1, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89-91; (iii) selected from SEQ ID NO: 126-145, 167-168 , 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof, and ( v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159-162.

[0032] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises at least three of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises a sequence selected from SEQ ID NO: 44, 172, 222 and 225.

[0033] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI Antion, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) at least one polyA sequence selected from SEQ ID NO: 1 - 2A polypeptides of the sequence 10, 12-14 and 159-162, (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD55 The third cistron, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184; (iii) selected from SEQ ID NO:126-145,167-168,178-179,231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) at least one having a sequence selected from SEQ ID NO. ID NO: 2A polypeptides of the sequences of 1-10, 12-14 and 159-162; (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- 168, 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof, ( v) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162, and (d) Cell apoptosis and blood coagulation box, described cellular immunity and blood coagulation box comprise: (i) the first cistron of coding XIAP, wherein the nucleotide sequence of XIAP is selected from SEQ ID NO: 110; (ii) the cistron of coding CD39 The second cistron, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NO: 106; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; and (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof, and (v) at least one polyA sequence selected from SEQ ID NO: 1- 2A polypeptides of sequences 10, 12-14 and 159-162.

[0034] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises at least three of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 45 or SEQ ID NO: 223.

[0035] Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation box, described coagulation box comprises: (i) the first cistron of coding THBD, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding TFPI Antion, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; (iii) is selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250 (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof, (v) at least one polyA sequence selected from SEQ ID NO: 1 - 2A polypeptides of the sequences 10, 12-14 and 159-162; (b) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding CD55 The third cistron, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184; (iii) selected from SEQ ID NO:126-145,167-168,178-179,231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) at least one having a sequence selected from SEQ ID NO. ID NO: 2A polypeptides of the sequences of 1-10, 12-14 and 159-162; (c) an innate immunity box, the innate immunity box comprising: (i) the first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126-145, 167- 168, 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof, ( v) at least one 2A polypeptide having a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162, and (d) cell apoptosis and cellular immunity box, described cell apoptosis and cellular immunity box comprise: (i) the first cistron of coding A20, wherein the nucleotide sequence of A20 is selected from SEQ ID NO:64-65,104 , 182 and 188; (ii) the second cistron encoding LEA29Y, wherein the nucleotide sequence of LEA29Y is selected from SEQ ID NO: 87-88; (iii) selected from SEQ ID NO: 126-145, 167-168, 178 - the promoter of 179, 231-238 and 250; and (iv) a polyA sequence selected from SEQ ID NO. 112-125, and 154-156, 159-162, 190-192, and combinations thereof, and (v ) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159-162.

[0036] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises at least three of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 46 or SEQ ID NO: 224.

[0037] In embodiments, provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation and innate immunity box, described coagulation and innate immunity box comprise: (i) the first cistron of coding THBD albumen, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) ) the second cistron of encoding CD47 protein, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) the third cistron of encoding EPCR protein, wherein the nucleic acid sequence of EPCR is selected from SEQ ID NO: 77-83 and 180; NO: 94-96, 103 and 187; (iv) be selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (v) be selected from the promoter of SEQ ID NO.112 - 125, 154-156, 159-162, 190-192 and 239-240 poly A sequences, and combinations thereof; and (vi) at least one sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159 - 2A polypeptide encoded by the sequence of 162; (b) cell apoptosis and cellular immunity box, said cell apoptosis and cellular immunity box comprising: (i) the first cistron of coding A20 protein, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (ii) the second cistron encoding HO1 protein, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; (iii) selected from SEQ ID NO: 126 - the promoter of 145, 167-168, 178-179, 231-238 and 250; (iv) a polynucleotide selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240 A sequences, and combinations thereof, and (v) at least one 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14, and 159-162; and (c) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46 protein, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding The third cistron of CD55 protein, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; (iii) selected from SEQ ID NO: 126-145, 167-168, 178-179, 231 - the promoter of 238 and 250; (iv) a polyA sequence selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and (v) at least A 2A polypeptide encoded by a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159-162.

[0038] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises at least three of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 227.

[0039] In embodiments, provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of: (a) coagulation and innate immunity box, described coagulation and innate immunity box comprise: (i) the first cistron of coding THBD albumen, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166; (ii) ) the second cistron of encoding CD47 protein, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (iii) the third cistron of encoding EPCR protein, wherein the nucleic acid sequence of EPCR is selected from SEQ ID NO: 77-83 and 180; NO: 94-96, 103 and 187; (iv) be selected from the promoter of SEQ ID NO: 126-145, 167-168, 178-179, 231-238 and 250; (v) be selected from the promoter of SEQ ID NO.112 - 125, 154-156, 159-162, 190-192 and 239-240 poly A sequences, and combinations thereof; and (vi) at least one sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159 - 2A polypeptide encoded by the sequence of 162; (b) cell apoptosis and cellular immunity box, said cell apoptosis and cellular immunity box comprising: (i) the first cistron of coding A20 protein, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (ii) the second cistron of encoding CTLA-4 protein, wherein the nucleotide sequence of CTLA-4 is selected from SEQ ID NO: 87-88, 174 and 186; (iii) is selected from SEQ ID NO : the promoter of 126-145, 167-168, 178-179, 231-238 and 250; (iv) selected from the group consisting of SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240 and combinations thereof; and (v) at least one 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162; and (c) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46 protein, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding The third cistron of CD55 protein, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; (iii) selected from SEQ ID NO: 126-145, 167-168, 178-179, 231 - the promoter of 238 and 250; (iv) a polyA sequence selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; (v) at least one A 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162.

[0040] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises at least three of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 175 or SEQ ID NO: 228.

[0041] In embodiments, provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of: (a) blood coagulation box, described blood coagulation box comprises: (i) the first cistron of encoding THBD albumen, wherein the nucleic acid sequence of encoding described THBD albumen is selected from SEQ ID NO: 97-102 and 166; (ii) encoding The second cistron of the TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein is selected from SEQ ID NO: 94-96, 103 and 187; (iii) selected from SEQ ID NO: 126-145, 167-168, 178 - the promoter of 179, 231-238 and 250; (iv) a polyA sequence selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and (v) at least one 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162; (b) an innate immunity box, the innate immunity box comprising: (i) a first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47 protein, wherein the nucleic acid sequence encoding the CD47 protein is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126 - the promoter of 145, 167-168, 178-179, 231-238 and 250; (iv) a polynucleotide selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240 A sequences, and combinations thereof; and (v) at least one 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14, and 159-162. (c) a complement regulation box, the complement regulation box comprising: (i) a first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding the CD46 protein is selected from SEQ ID NO: 71-76, 185 and 200; (ii) a second cistron encoding CD55 protein, wherein the nucleic acid sequence encoding said CD55 protein is selected from SEQ ID NO: 84, 85, 107 and 184; (iii) selected from SEQ ID NO: 126-145, 167 - the promoter of 168, 178-179, 231-238 and 250; (iv) a polyA sequence selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and (v) at least one 2A polypeptide encoded by a sequence selected from the group consisting of SEQ ID NO: 1-10, 12-14 and 159-162.

[0042] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises at least three of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 176 or SEQ ID NO: 229.

[0043] In embodiments, provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of: (a) blood coagulation box, described blood coagulation box comprises: (i) the first cistron of encoding THBD albumen, wherein the nucleic acid sequence of encoding described THBD albumen is selected from SEQ ID NO: 97-102 and 166; (ii) encoding The second cistron of the TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein is selected from SEQ ID NO: 94-96, 103 and 187; (iii) selected from SEQ ID NO: 126-145, 167-168, 178 - the promoter of 179, 231-238 and 250; (iv) a polyA sequence selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and (v) at least one 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162; (b) an innate immunity box, the innate immunity box comprising: (i) a first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47 protein, wherein the nucleic acid sequence encoding the CD47 protein is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 126 - the promoter of 145, 167-168, 178-179, 231-238 and 250; (iv) a polynucleotide selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240 A sequences, and combinations thereof; and (v) at least one 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14, and 159-162. (c) a complement regulation box, the complement regulation box comprising: (i) a first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding the CD46 protein is selected from SEQ ID NO: 71-76, 185 and 200; (ii) a second cistron encoding CD55 protein, wherein the nucleic acid sequence encoding said CD55 protein is selected from SEQ ID NO: 84, 85, 107 and 184; (iii) selected from SEQ ID NO: 126-145, 167 - the promoter of 168, 178-179, 231-238 and 250; (iv) a polyA sequence selected from SEQ ID NO. 112-125, 154-156, 159-162, 190-192 and 239-240, and Combinations thereof; and (v) at least one 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14 and 159-162; and (d) Inflammation and cell apoptosis box, described inflammation and cell apoptosis box comprise: (i) the first cistron of coding A20 protein, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (ii) the second cistron encoding HO1 protein, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; (iii) selected from SEQ ID NO: 126-145 , 167-168, 178-179, 231-238 and 250 promoters; (iv) poly A sequences selected from SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240 , and combinations thereof; and (v) at least one 2A polypeptide encoded by a sequence selected from SEQ ID NO: 1-10, 12-14, and 159-162.

[0044] In an embodiment, the aforementioned nucleic acid comprises at least two of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises at least three of the aforementioned polycistronic cassettes. In an embodiment, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In an embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 177 or SEQ ID NO: 230.

[0045] Provided herein is a nucleic acid comprising a CD46 cistron comprising or consisting of a nucleic acid sequence selected from SEQ ID NO: 72, 73, 185 and 200. Provided herein is a nucleic acid comprising a THBD cistron comprising or consisting of a nucleic acid sequence selected from any one of SEQ ID NOs: 99-102 and 166. In an embodiment, provided herein is a cell comprising a nucleic acid comprising a CD46 cistron comprising or consisting of a nucleic acid sequence selected from SEQ ID NO: 72, 73, 185 and 200. In an embodiment, provided herein is a cell comprising a nucleic acid comprising a THBD cistron comprising or consisting of a nucleic acid sequence selected from any one of SEQ ID NOs: 99-102 and 166. In an embodiment, provided herein is a tissue, organ, or animal comprising a cell comprising a nucleic acid comprising a CD46 cistron comprising a nucleic acid selected from the group consisting of SEQ ID NO: 72, 73, 185, and 200 or consisting of nucleic acid sequences. In an embodiment, provided herein is a tissue, organ or animal comprising a cell comprising a nucleic acid comprising a THBD cistron comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 99-102 and 166 sequence or consists of it.

[0046] In an embodiment, a nucleic acid described herein comprises a 5'loxP site and a 3'loxP site selected from SEQ ID NOs: 146-150 and 244. In an embodiment, a nucleic acid described herein comprises a 5' insulator site and a 3' insulator site selected from SEQ ID NOs: 49-58 and 163-164. In embodiments, the nucleic acids described herein comprise a 5' guide RNA (gRNA) target sequence and a 3' gRNA target sequence having a sequence selected from SEQ ID NO: 47-48, and combinations thereof. In an embodiment, a nucleic acid described herein comprises a ubiquitous chromatin opening element (UCOE) having the sequence of any one of SEQ ID NOs: 19-22, 157 and 193. In an embodiment, the nucleic acid described herein comprises: (i) a first inverted terminal repeat (ITR) positioned on the 5' side of the 5' polycistronic cassette and having the sequence of SEQ ID NO: 17 or 18, and (ii) a second ITR located on the 3' side of the 3' polycistronic cassette and having the sequence of SEQ ID NO: 15 or 16. In embodiments, the nucleic acids described herein comprise at least two polycistronic cassettes. In embodiments, the nucleic acids described herein comprise at least three polycistronic cassettes. In embodiments, the nucleic acids described herein comprise at least four polycistronic cassettes. In embodiments, the nucleic acids described herein comprise fluorescent proteins. In an embodiment, a nucleic acid described herein comprises a green fluorescent protein having the sequence of SEQ ID NO: 111, 242 or 246.

[0047] In embodiments, provided herein are vectors comprising any of the foregoing nucleic acids. In embodiments, provided herein are cells comprising any of the foregoing nucleic acids. In embodiments, provided herein are islet cells comprising any of the foregoing nucleic acids. In embodiments, provided herein are porcine cells comprising any of the foregoing nucleic acids. In embodiments, provided herein is an organ or tissue comprising any of the foregoing nucleic acids. In an embodiment, provided herein is an animal comprising any of the foregoing nucleic acids. In an embodiment, provided herein is a pig comprising any of the foregoing nucleic acids. In an embodiment, provided herein is a pig comprising any of the foregoing nucleic acids, wherein the pig does not contain PERV. In an embodiment, provided herein is an animal comprising any of the foregoing nucleic acids, wherein said animal comprises at least one inactivated carbohydrate antigen producing gene. In an embodiment, provided herein is an animal comprising any of the foregoing nucleic acids, wherein said animal comprises at least one inactivated carbohydrate antigen producing gene, and wherein said inactivated carbohydrate antigen producing gene is glycoprotein alpha-1 , 3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydrolase (CMAH), β-1,4-N-acetylgalactosylaminotransferase 2 (β4GALNT2) or a combination thereof.

[0048] In an embodiment, provided herein is a contiguous nucleic acid sequence having any one of SEQ ID NOs: 23 to 46, 172-177, and 201-230. In embodiments, the contiguous nucleic acid sequence is at least about 1 kb. In an embodiment, the contiguous nucleic acid sequence has a length of at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 11 kb, at least 12 kb, at least 13 kb, at least 14 kb, at least 15 kb, at least 16 kb, at least 17 kb, at least 18 kb, at least 19 kb, at least 20 kb, at least 21 kb, at least 22 kb, at least 23 kb , at least 24 kb, at least 25 kb, at least 26 kb, at least 27 kb, at least 28 kb, at least 29 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, at least 55 kb, at least 60 kb or at least 65 kb. In an embodiment, said contiguous nucleic acid sequence encodes one or more of the aforementioned polycistronic cassettes. In an embodiment, said contiguous nucleic acid sequence encodes at least two of the aforementioned polycistronic cassettes. In an embodiment, said contiguous nucleic acid sequence encodes at least three of the aforementioned polycistronic cassettes. In an embodiment, said contiguous nucleic acid sequence encodes at least four of the aforementioned polycistronic cassettes.

[0049] In embodiments, provided herein are chromosomally integrated forms having any one of SEQ ID NOs: 23-46, 172-177, and 201-230.

[0050] In an embodiment, provided herein is a contiguous nucleic acid sequence that is 95% identical to any one of SEQ ID NOs: 23-46, 172-177, and 201-230. In embodiments, provided herein are contiguous nucleic acid sequences that are at least 96% identical to any one of SEQ ID NOs: 23-46, 172-177, and 201-230. In embodiments, provided herein are contiguous nucleic acid sequences that are at least 97% identical to any one of SEQ ID NOs: 23-46, 172-177, and 201-230. In embodiments, provided herein are contiguous nucleic acid sequences that are at least 98% identical to any one of SEQ ID NOs: 23-46, 172-177, and 201-230. In an embodiment, provided herein is a contiguous nucleic acid sequence that is at least 99% identical to any one of SEQ ID NOs: 23-46, 172-177, and 201-230. In an embodiment, provided herein is a contiguous nucleic acid sequence at least 95% identical to any one of SEQ ID NOS: 23-46, 172-177 and 201-230, wherein said sequence encodes at least two polycistronic cassettes . In an embodiment, provided herein is a contiguous nucleic acid sequence at least 95% identical to any one of SEQ ID NOS: 23-46, 172-177 and 201-230, wherein said sequence encodes at least three polycistronic cassettes . In an embodiment, provided herein is a contiguous nucleic acid sequence at least 95% identical to any one of SEQ ID NOS: 23-46, 172-177 and 201-230, wherein said sequence encodes at least four polycistronic cassettes . In an embodiment, provided herein is a sequence that is at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 23 to 46, 172-177, and 201-230. nucleic acid sequence. In an embodiment, provided herein is a contiguous nucleic acid sequence encoding a polycistronic gene product polypeptide expressed by any one of said polycistronic cassettes. In an embodiment, provided herein is a polypeptide comprising one or more proteins encoded by the cistrons of any of the polycistronic cassettes described herein.

[0051] In an embodiment, provided herein is a method for genetically modifying cells, the method comprising: (i) knocking out porcine endogenous retrovirus (PERV) elements; (ii) knocking out glycoprotein α- 1,3-galactosyltransferase gene (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydrolase (CMAH), β-1,4-N-acetylgalactosyltransferase 2 ( β4GALNT2) or any combination thereof; and (iii) knocking in any one or more polycistronic cassettes as described in any one of claims 1-14. In an embodiment, step (i) is performed first, step (ii) is performed second, and step (iii) is performed last. In an embodiment, step (i) is performed first, step (iii) is performed second, and step (ii) is performed last. In an embodiment, step (ii) is performed first, step (i) is performed second, and step (iii) is performed last. In an embodiment, step (iii) is performed first, step (i) is performed second, and step (ii) is performed last. In an embodiment, step (ii) is performed first, step (iii) is performed second, and step (i) is performed last. In an embodiment, step (iii) is performed first, step (ii) is performed second, and step (i) is performed last. In embodiments, provided herein are genetically modified cells produced by any of the foregoing methods. In embodiments, provided herein are animals, organs or tissues comprising said genetically modified cells. In embodiments, the animal is a pig or a human.

[0052] In an embodiment, provided herein is a landing pad, wherein the landing pad is a nucleic acid comprising a first loxP site, a second loxP site, a promoter, and a polyA sequence. In an embodiment, the landing pad comprises an insulator sequence. In an embodiment, the landing pad comprises a fluorescent protein. In embodiments, the landing pad comprises the nucleic acid of SEQ ID NO: 248 or 249. In an embodiment, provided herein is a genetically modified cell comprising a landing pad. In an embodiment, provided herein is a tissue, organ or animal comprising a genetically modified cell comprising a landing pad.

[0053] In embodiments, provided herein are nucleic acids comprising one or more of a first loxP site, a second loxP site, and a polycistronic cassette described herein. In embodiments, provided herein are genetically modified cells comprising the foregoing nucleic acids. In an embodiment, provided herein is a tissue, organ or animal comprising the aforementioned genetically modified cells. In an embodiment, the nucleic acid comprises a sequence of any one of SEQ ID NOs: 201-230 or at least 80%, at least 85%, at least 90%, at least 91%, any one of SEQ ID NOs: 201-230. Nucleic acids that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In an embodiment, provided herein is a genetically modified cell comprising a nucleic acid of any one of SEQ ID NOs: 201-230 or at least any one of SEQ ID NOs: 201-230 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical nucleic acid. In an embodiment, provided herein is a tissue, organ or animal comprising a genetically modified cell comprising the nucleic acid of any one of SEQ ID NOs: 201-230 or in combination with SEQ ID NOs: 201-230 Any of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % or 100% identical nucleic acids. Brief description of the diagram

[0054] Figure 1A shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI, THBD and CD39 cistrons under the control of the mmEF1α1 (denoted as mEF1α1) promoter, a cistron comprising CD46, Complement regulatory cassettes for CD55 and CD59 cistrons, and innate immunity and inflammation and apoptosis cassettes containing the B2M HLA-E fusion protein, CD47, A20, HO1, and PD-L1 cistrons under the control of the CAG promoter. The genes within each cassette are separated by nucleic acid sequences encoding 2A peptides (E2A, T2A, F2A, P2A) or internal ribosome entry sites (IRES). Each cassette also contains a polyA sequence (eg, HBB, bGH or SPAActB). An exemplary nucleic acid having the cassette of Figure 1A is SEQ ID NO. 23. Figure 1B shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI and THBD under the control of the mmEF1α1 promoter, a coagulation cassette comprising CD46_LL (denoted as CD46) and The complement regulatory cassette of the CD59 cistron, and the innate immunity cassette comprising the nucleic acid encoding the B2M HLA-E fusion protein and the CD47 cistron under the control of the CAG promoter. The genes within each cassette are separated by a nucleic acid encoding a 2A peptide (P2A or F2A) or an IRES. Each cassette also contains a polyA sequence (eg, HBB, btGH or SPAActB). An exemplary nucleic acid having the cassette of FIG. 1B is SEQ ID NO. 25. Figure 1C shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI and THBD cistrons under the control of the mmEF1α1 (denoted as mEF1α1) promoter, a cistron comprising CD46 and CD59 under the control of the hEF1α1 promoter The complement regulatory cassette of the antitron, and the innate immunity and inflammation and apoptosis cassette comprising the nucleic acid encoding the B2M HLA-E fusion protein and the CD47, A20, and HO1 cistrons under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A) or IRES. Each cassette also contains a polyA sequence (eg, HBB, bGH or SPAActB). An exemplary nucleic acid having the cassette of Figure 1C is SEQ ID NO. 26. Figure 1D shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI and THBD cistrons under the control of the mmEF1α1 promoter, a cistron comprising CD46 and CD59 under the control of the hEF1α1 (denoted as hsEF1α1) promoter The complement regulatory cassette of the antitron, and the innate immunity cassette comprising the nucleic acid encoding the B2M HLA-E fusion protein and the CD47 cistron under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A) or IRES. Each cassette also contains a polyA sequence (eg, HBB, btGH or SPAActB). An exemplary nucleic acid having the cassette of FIG. 1D is SEQ ID NO. 24. Figure 2A shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI, THBD and CD39 cistrons under the control of the mmEF1α1 promoter, a cistron comprising CD46i, CD59 and CD55 under the control of the ssEF1α1 promoter The complement regulatory cassette of the CD47, A20, HO1, and PD-L1 cistrons, and the innate immunity and inflammation and apoptosis cassettes under the control of the CAG promoter comprising nucleic acids encoding B2M HLA-E fusion proteins and CD47, A20, HO1, and PD-L1 cistrons. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A) or IRES. Each cassette also contains a poly A sequence (eg, HBB, btGH, or SPA / hsActB). An exemplary nucleic acid having the cassette of Figure 2A is SEQ ID NO. 27. Figure 2B shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI, THBD and CD39 under the control of the ssUBC promoter, a complement comprising CD46i, CD59 and CD55 cistrons under the control of the ssEF1α1 promoter regulatory cassette, as well as innate immunity and inflammation and apoptosis cassettes comprising nucleic acid encoding the B2M HLA-E fusion protein and CD47, A20, HO1, and PD-L1 cistrons under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (eg, HBB, btGH, or SPA / hsActB). An exemplary nucleic acid having the cassette of Figure 2B is SEQ ID NO. 28. Figure 2C shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI and THBD under the control of the mmEF1α1 promoter, a coagulation cassette comprising CD46_LL (denoted as CD46) and The complement regulatory cassette of CD59, and the innate immunity cassette comprising the B2M HLA-E fusion protein and CD47 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A). Each cassette also contains a polyA sequence (eg, hsHBB, btGH or SPAActB). An exemplary nucleic acid having the cassette of Figure 2C is SEQ ID NO. 29. Figure 2D shows exemplary nucleic acids comprising: a coagulation cassette comprising PROCR and THBD under the control of the ssUBC promoter, an inflammation and apoptosis cassette comprising A20 and HO1 under the control of the ssEF1α1 promoter, and Complement regulation and innate immunity cassette comprising CD46i, CD55 and CD47 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (eg, hsHBB, btGH, or SPA hsActB). An exemplary nucleic acid having the cassette of FIG. 2D is SEQ ID NO. 30. Figure 2E shows an exemplary nucleic acid comprising: a coagulation cassette comprising TFPI, PROCR, and THBD (denoted as THBDda) under the control of the ssUBC promoter, under the control of the ssHSPA8 (denoted as ssHSPA8 (ATACseq)) promoter The innate immunity cassette comprising the B2M HLA-E fusion protein and CD47 under , and the complement regulatory cassette comprising CD46da1 (denoted as CD46da) and CD55 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 2E is SEQ ID NO. 31. Figure 2F shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a cassette comprising TFPI, PROCR and THBDda under the control of the ssHSPA8 promoter The coagulation cassette, and the complement regulatory cassette comprising CD46da1 (denoted as CD46da) and CD55 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 2F is SEQ ID NO. 32. Figure 2G shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a cassette comprising TFPI, PROCR and THBDda under the control of the ssHSPA8 promoter The coagulation cassette, and the complement regulatory cassette comprising CD46da1 (denoted as CD46da) and CD55 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 2G is SEQ ID NO. 33. Figure 2H shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI, PROCR and THBDda under the control of the ssUBC promoter, a B2M HLA under the control of the ssEF1α1 (denoted as ssEEF1α1 (ATACseq)) promoter -E fusion protein and the innate immunity cassette of CD47, and the complement regulatory cassette comprising CD46da1 (denoted as CD46da) and CD55 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 2H is SEQ ID NO. 34. Figure 2I shows exemplary nucleic acids comprising: a coagulation and innate immunity cassette comprising CD47-2, PROCR, and THBDda under the control of the ssUBC promoter, and a coagulation and innate immunity cassette under the control of the ssHSPA8 promoter (denoted as "ssHSPA8 (ATAC seq) ”) under the control of the apoptosis and cellular immunity cassettes containing ssA20 and ssHO1; and the complement regulation cassette containing CD46da and CD55 under the control of the CAG promoter. Genes within each cassette are separated by a nucleic acid encoding a 2A peptide (P2A, P2A*) or an IRES (e.g., IRES(FMDV) or IRES(EMCV)). Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of Figure 2I is SEQ ID NO. 174. Figure 2J shows exemplary nucleic acids comprising: a coagulation and innate immunity cassette comprising CD47-2, PROCR, and THBDda under the control of the ssUBC promoter, under the control of the ssHSPA8 promoter (denoted as "ssHSPA8 (ATAC seq) ”) under the control of the apoptosis and cellular immunity cassette comprising ssA20 and LEA29Y (indicated as “LEA29Y-Ig”); and the complement regulatory cassette comprising CD46da and CD55 under the control of the CAG promoter. Genes within each cassette are separated by a nucleic acid encoding a 2A peptide (P2A, P2A*) or an IRES (e.g., IRES(FMDV) or IRES(EMCV)). Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 2J is SEQ ID NO. 175. Figure 3A shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the mmEF1α1 (denoted as mEF1α1) promoter, an innate immune cassette comprising TFPI under the control of the hEF1α1 promoter , the coagulation cassette of THBD and CD39, and the complement regulation and inflammation and apoptosis cassette comprising CD46i, CD55, CD59, A20, HO1, and PD-L1 (denoted as PDL1) under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (P2A, P2Amut1, E2A, F2A, P2Amut2, P2Amut3, T2A) or IRES. Each cassette also contains a polyA sequence (eg, hsHBB, btGH or SPAActB). An exemplary nucleic acid having the cassette of FIG. 3A is SEQ ID NO. 35. Figure 3B shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI and THBD under the control of the mmEF1α1 promoter, a complement regulatory cassette comprising CD46i and CD55 under the control of the hEF1α1 (denoted as hsEF1α1) promoter , the innate immunity cassette comprising the B2M HLA-E fusion protein and CD47 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acid encoding 2A peptides (T2A, F2A, P2A). Each cassette also contains a polyA sequence (eg, hsHBB, btGH or SPAActB). An exemplary nucleic acid having the cassette of FIG. 3B is SEQ ID NO. 36. Figure 3C shows exemplary nucleic acids comprising: a coagulation cassette comprising PROCR and THBD under the control of the mmEF1α1 promoter, a complement regulatory cassette comprising CD46i and CD55 under the control of the hEF1α1 (denoted as hsEF1α1) promoter , the innate immunity and inflammation and apoptosis cassette comprising the B2M HLA-E fusion protein, CD47, A20 and HO1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (P2A, T2A, F2A) or IRES. Each cassette also contains a polyA sequence (eg, hsHBB, btGH or SPAActB). An exemplary nucleic acid having the cassette of FIG. 3C is SEQ ID NO. 37. Figure 4A shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, an innate immune cassette comprising TFPI, THBD and CD39 under the control of the hsHSPA8 promoter The coagulation cassette, as well as the complement regulation and inflammation and apoptosis cassette comprising CD46da1 (denoted as CD46da), CD55, CD59, A20, HO1, and PD-L1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, P2Amut2, P2Amut3, T2A, F2A) or IRES. Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 4A is SEQ ID NO. 38. Figure 4B shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, an innate immune cassette comprising TFPI, THBD and CD39 under the control of the hsHSPA8 promoter The coagulation cassette, as well as the complement regulation and inflammation and apoptosis cassette comprising CD46da2 (denoted as CD46da), CD55, CD59, A20, HO1, and PD-L1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, P2Amut2, P2Amut3, T2A, F2A) or IRES. Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of Figure 4B is SEQ ID NO. 39. Figure 4C shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a coagulation cassette comprising TFPI and THBD under the control of the hsHSPA8 promoter , and a complement regulatory cassette comprising CD46i and CD55 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acid encoding 2A peptides (E2A, P2A, F2A). Each cassette also contains a polyA sequence (eg, hsHBB, btGH, or SPA / ACTB). An exemplary nucleic acid having the cassette of FIG. 4C is SEQ ID NO. 40. Figure 4D shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a coagulation cassette comprising TFPI and THBD under the control of the hsHSPA8 promoter , and a complement regulation and inflammation and apoptosis cassette comprising CD46da1 (denoted as CD46da), CD55, A20 and HO1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 4D is SEQ ID NO. 41. Figure 4E shows exemplary nucleic acids comprising: an innate immunity cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a coagulation cassette comprising TFPI and THBD under the control of the hsHSPA8 promoter , and a complement regulatory and inflammatory and apoptotic cassette comprising CD46da2 (denoted as CD46da), CD55, A20, and HO1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, F2A) or IRES. Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 4E is SEQ ID NO. 42. Figure 4F shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 (denoted as CD47-2) under the control of the ssUBC promoter, and comprising TFPI under the control of the ssHSPA8 promoter , the coagulation cassette of THBD and CD39, and the complement regulation and inflammation and apoptosis cassette comprising CD46da2 (denoted as CD46da), CD55, CD59, A20, HO1, and PD-L1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding 2A peptides (P2A, P2Amut1, E2A, F2A, P2Amut2, PTAmut3, T2A) or IRES. Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 4F is SEQ ID NO. 173. Figure 4G shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI (denoted as "TFPI*") and THBDda under the control of the ssUBC promoter, and a coagulation cassette under the control of the ssHSPA8 promoter (denoted as "ssHSPA8 (ATAC seq )”) under the control of the innate immunity cassette comprising B2M HLA-E fusion protein and CD47-2; and the complement regulatory cassette comprising CD46da and CD55 under the control of the CAG promoter. The genes within each cassette are separated by a nucleic acid encoding a 2A peptide (P2A) or an IRES (eg, IRES(FMDV) or IRES(EMCV)). Each cassette also contains a poly A sequence (eg, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 4G is SEQ ID NO. 176. Figure 4H shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI (TFPI*) and THBDda under the control of the ssUBC promoter; The innate immunity cassette containing B2M HLA-E fusion protein and CD47-2 under the control; the complement regulatory cassette containing CD46da and CD55 under the control of the CAG promoter; and the inflammation under the control of the ssEF1α1 promoter (denoted as "ssEEF1A1") and apoptotic cassettes. Genes within each cassette are separated by a nucleic acid encoding a 2A peptide (P2A, P2A*) or an IRES (e.g., IRES(FMDV) or IRES(EMCV)). Each cassette also contains a poly A sequence (eg, hsHBB, ocHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of Figure 4H is SEQ ID NO. 177. Figure 5A shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI (ΔP-Sel) and THBDda under the control of the ssHSPA8 promoter, a B2M HLA-E fusion protein and The innate immune cassette for CD47, the cellular immune and coagulation cassette containing CTLA-4 (e.g. LEA29Y) and CD39 under the control of the ssEF1α1 promoter, and the CD46da1 (denoted as CD46da(BC1)) and CD55 under the control of the CAG promoter complement regulator box. The nucleic acid also contains GFP on the 5' side of the coagulation cassette (tagged d2GFP). The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (eg, SPA / PTCH2 pA, hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 5A is SEQ ID NO. 43. Figure 5B shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI (ΔP-Sel) and THBDda under the control of the ssHSPA8 promoter, a B2M HLA-E fusion protein and The innate immune cassette of CD47, the inflammatory and apoptotic cassette containing A20 and PD-L1 under the control of the ssEF1α1 promoter, and the complement regulation containing CD46da1 (denoted as CD46da(BC1)) and CD55 under the control of the CAG promoter box. The nucleic acid also contains GFP on the 5' side of the coagulation cassette (tagged d2GFP). The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (eg, SPA / PTCH2, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5B is SEQ ID NO. 44. Figure 5C shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI (ΔP-Sel) and THBDda under the control of the ssHSPA8 promoter, a B2M HLA-E fusion protein under the control of the ssUBC promoter and The innate immunity cassette for CD47, the apoptosis and coagulation cassette containing XIAP and CD39 under the control of the ssEF1α1 promoter, and the complement regulatory cassette containing CD46da1 (denoted as CD46da(BC1)) and CD55 under the control of the CAG promoter. The nucleic acid also contains GFP on the 5' side of the coagulation cassette (tagged d2GFP). The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (eg, SPA / PTCH2, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5C is SEQ ID NO. 45. Figure 5D shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI (ΔP-Sel) and THBDda under the control of the ssHSPA8 promoter, a B2M HLA-E fusion protein under the control of the ssUBC promoter and The innate immune cassette for CD47, the apoptotic and cellular immune cassette containing CTLA-4 (e.g. LEA29Y) and A20 under the control of the ssEF1α1 promoter, and the CD46da1 (denoted as CD46da(BC1)) under the control of the CAG promoter and the complement regulatory box of CD55. The nucleic acid also contains GFP on the 5' side of the coagulation cassette (tagged d2GFP). The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (eg, SPA / PTCH2, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5D is SEQ ID NO. 46. Figure 5E shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI (ΔP-Sel) and THBDda under the control of the ssHSPA8 promoter, a B2M HLA-E fusion protein and The innate immune cassette for CD47, the cellular immune and coagulation cassette containing LEA29Y and CD39 under the control of the ssEF1α1 promoter, and the complement regulatory cassette containing CD46da1 (denoted as CD46da(BC1)) and CD55 under the control of the CAG promoter. The nucleic acid also contains GFP on the 5' side of the coagulation cassette (tagged d2GFP). The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (eg, SPA / PTCH2 pA, hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 5A is SEQ ID NO. 43. Figure 5F shows exemplary nucleic acids comprising: a coagulation cassette comprising TFPI (ΔP-Sel) and THBDda under the control of the ssHSPA8 promoter, a B2M HLA-E fusion protein and The innate immunity cassette of CD47, the apoptosis and cellular immunity cassette comprising LEA29Y and A20 under the control of the ssEF1α1 promoter, and the complement regulatory cassette comprising CD46da1 (denoted as CD46da(BC1)) and CD55 under the control of the CAG promoter . The nucleic acid also contains GFP on the 5' side of the coagulation cassette (tagged d2GFP). The genes within each cassette are separated by nucleic acids encoding 2A peptides (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (eg, SPA / PTCH2, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5D is SEQ ID NO. 46. Figure 6 shows exemplary nucleic acids comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 (denoted as CD47-2) under the control of the ssUBC promoter, and an innate immune cassette under the control of the hsHSPA8 promoter Inflammation and apoptosis cassettes containing A20, HO1, and PD-L1, complement regulatory cassettes containing CD46da2 (expressed as CD46da(BC2)), CD55, and CD59 under the control of the CAG promoter, and ssICAM2 under the control of the promoter Clotting cassette containing TFPI (indicated as *TFPI), CD39 and THBDda. The genes within each cassette are separated by nucleic acids encoding 2A peptides (P2A, P2Amut3, T2A, F2A, P2Amut2, P2Amut1 and E2A). Each cassette also contains poly A sequences (eg, hsHBB, btGH (labeled "btGH"), btHBB and hsGH). An exemplary nucleic acid having the cassette of FIG. 6 is SEQ ID NO. 172. Fig. 7A shows the expression of polycistronic cassettes containing polycistronic cassettes of Fig. Combined arterial and portal blood flow in grafts. Fig. 7B shows the expression of polycistronic cassettes containing polycistronic cassettes of Fig. Lactic acid clearance in grafts. Figure 8A shows platelet counts over time in porcine liver xenografts treated with or without anti-GPIb fragmented murine antibody and the GPIIb / IIIa inhibitor eptifibatide (ab / etifibatide), as measured by a hemocytometer measured. Figure 8B shows platelet counts over time in porcine liver xenografts treated with or without anti-GPIb fragmented murine antibody and the GPIIb / IIIa inhibitor eptifibatide (ab / etifibatide), as measured by flow cytometry measured by cytometry. Figure 9A shows images of porcine liver xenografts. The superior and inferior hepatic vena cava (IVC) and portal vein are indicated in the image. Figure 9B shows lactate clearance in porcine liver xenografts during ex vivo normothermic perfusion of porcine liver xenografts. Figure 10A shows the mean troponin I concentration (ng / mL) over time in cardiac xenografts stored in refrigerated solution (CS) or perfused with oxygenated Steen solution with red blood cells (IM ). The following cardiac xenografts were studied: (a) cardiac xenografts knocked out for GGTA1, CMAH, and B4GALNT2 with variable expression of human complement and thrombus regulatory genes ("TKO"); (b) knockout for GGTA1 and expressing CD55 ("GTKO.hDAF"); and (c) wild-type heart xenograft "WT". Figure 10B shows the mean troponin I concentration (ng / mL) over 1 hour in cardiac xenografts stored in cold storage solution (CS) or perfused with oxygenated Steen solution with red blood cells ( IM). The following cardiac xenografts were studied: (a) cardiac xenografts knocked out for GGTA1, CMAH, and B4GALNT2 with variable expression of human complement and thrombus regulatory genes ("TKO"); (b) knockout for GGTA1 and expressing CD55 ("GTKO.hDAF"); and (c) wild-type heart xenograft "WT". Figure 10C is a graph of troponin I concentrations (ng / mL) at 1 hour and at the final time point in cardiac xenografts stored in cryogenic solution (CS) or treated with oxygenated Steen solution with red blood cells Infusion (IM). FIG. 11A is a graph showing cardiac output over time of TKO cardiac xenografts stored in cryogenic solution (CS). Figure 1 IB is a graph showing cardiac output over time in TKO cardiac xenografts perfused (IM) with oxygenated Steen solution with red blood cells. Figure 11C is a graph showing cardiac output over time of TKO cardiac xenografts stored in cryogenic solution (CS). Figure 1 ID is a graph showing cardiac output over time in TKO cardiac xenografts perfused (IM) with oxygenated Steen solution with red blood cells. Figure 12A is an exemplary polycistronic cassette comprising a polyA (pA) sequence, an untranslated region (UTR), an intron, an IRES, a 2A peptide, and three cistrons (labeled "1", "2") " and "3"). Figure 12B is an exemplary nucleic acid comprising multiple polycistronic cassettes (referred to as "transcription unit 1", "transcription unit 2", "transcription unit 3" and "transcription unit 4"). Each transcription unit contains a promoter, a polyA (pA) sequence and a cistron (labeled "1", "2", "3", "4", "5", "6", "7", " 8" and "9".) The nucleic acid also comprises an insulator sequence (labeled "INS"), a first loxP site (labeled "loxP"), and a second loxP site (labeled "lox66"). A ubiquitous chromatin opening element (UCOE) separates the second and third polycistronic cassettes. Figure 13A shows the expression by flow cytometry of nucleic acids comprising the polycistronic cassette of Figure 2D in donor cells, pig fetal cells and adult pig cells. Circled cells contain the polycistronic cassette of Figure 2D. Figure 13B shows the expression by immunohistochemistry of nucleic acids comprising the polycistronic cassette of Figure 2D in donor cells, porcine fetal cells, and adult porcine cells. Figure 14A shows that cells containing the polycistronic cassette of Figure 2D express CD55, as evidenced by binding of anti-CD55 blocking antibodies on the cell surface. Figure 14B shows that cells containing the polycistronic cassette of Figure 2D express CD46, as evidenced by binding of anti-CD46 blocking antibodies on the cell surface. 15A-15B show that in the absence of anti-CD46 and / or anti-CD55 blocking antibodies, cells containing nucleic acids containing the polycistronic cassette of FIG. 15A) and protection of complement deposition in EF cells (FIG. 15B). Figure 15C shows that EF cells that do not contain nucleic acid containing the polycistronic cassette of Figure 2D do not provide protection against complement deposition. Figures 16A-16C show that the nucleic acids described herein protect against complement deposition in islet cells. Figure 16A shows that cells comprising nucleic acids containing the polycistronic cassette of Figure 2D protect against complement deposition in islet cells in the absence of anti-CD46 and / or anti-CD55 blocking antibodies. In the absence of anti-CD46 and / or anti-CD55 blocking antibodies, cells containing nucleic acids containing the polycistronic cassette of Figure 4D conferred protection against complement deposition in islet cells, as demonstrated by a reduced percentage of C3b (Figure 16C ) and a decrease in the mean fluorescence intensity of C3b (FIG. 16B). 17A-17B show expression of CD55 and CD46 in KCDC cells transfected with nucleic acid comprising CD46da, nucleic acid comprising CD55, or nucleic acid comprising CD46da and CD55 linked by F2A peptide. Figure 17A shows CD55 expression when linked to 2A peptide. Figure 17B shows that CD46da (labeled "CD46") is expressed when linked to the 2A peptide. Figure 17C shows that nucleic acids comprising CD46da ("CD46") and CD55 reduce complement deposition. Figures 18A-18C show the expression of CD46, CD55 and CD59 in KCDC cells transfected with one of the following nucleic acids: (i) hsCD46da2; (ii) hsCD55; (iii) hsCD59; (iv) hsCD46da2-F2A-hsCD55 ; (v) hsCD46da2-F2A-hsCD59; (vi) hsCD55-P2A-hsCD59; or (viii) hsCD46da2-F2A-hsCD55-P2A-hsCD59. Figures 18A-18C show the respective expression of CD46 (Figure 18A), CD55 (Figure 18B) and CD59 (Figure 18C). Figure 18D shows that each of these nucleic acids appear to reduce complement deposition. In each of Figures 18A-18D, CD46da2 is labeled "46", CD55 is labeled "55", and CD59 is labeled "59". Figure 19A shows the expression of CD46 isoforms BC-Cyt1 ("SG_BC1"), Bc-Cy2 ("SG_BC2") and ABC1 ("SG_ABC1"). Figure 19B shows the ability of CD46 isoforms BC-Cyt1 ("SG_BC1"), Bc-Cy2 ("SG_BC2") and ABC1 ("SG_ABC1") to reduce complement deposition. Figure 20A shows that AEC, KEC, PUVEC and HUVEC cells transfected with nucleic acid comprising the polycistronic cassette of Figure 2D express CD46 and CD55 (Figure 20A). Figure 20B shows that AEC, KEC, PUVEC and HUVEC cells transfected with nucleic acid comprising the polycistronic cassette of Figure 4C express CD46 and CD55 (Figure 20B). Figure 20C shows that AEC, KEC and PUVEC cells containing nucleic acids containing the polycistronic cassette of Figure 2D reduce complement deposition in AEC, KEC and PUVEC cells. Figure 20D shows that AEC, KEC and PUVEC cells containing nucleic acids containing the polycistronic cassette of Figure 4C reduce complement deposition in AEC, KEC and PUVEC cells. Figure 21A shows that expression of nucleic acids containing the polycistronic cassette ("15S") of Figure 2D reduces complement deposition even in the presence of donor-specific alloantibodies (DSA). Figure 21B shows the concentration of DSA after transplantation. "POD" refers to the number of days after transplantation. Figure 22 shows the timeline of the experiment of Example 1. Figure 23 shows images of CD46, CD55, CD59, HLA-E, CD47, and PD-L1 expression in TKO-A and TKO-B xenografts compared to wild-type xenografts in animals of Example 1A. Figure 24 shows images of CD46, CD55, CD47, EPCR, THBD, A20 and HO1 expression in TKO-C animals of Example 1A. Figure 25 shows images of CD46, CD55, B2M, CD47, TFPI and THBD expression in TKO-D animals of Example 1A. Figure 26 shows the concentration of serum creatinine (labeled as Cr) up to 313 days after transplantation in animals of Example 1A. Figure 27 shows images of kidney tissue stained with hematoxylin and eosin or complement component C4d at 217 days ("POD217"), 230 days ("POD230") or 105 days ("POD105") post-transplantation. Animal B6 received TKO-B xenografts. Animal C9 received TKO-F xenografts. Animal D2 received TKO-G xenografts. Figure 28A shows an exemplary landing stand. The landing pad contains an insulator sequence (HS4), a loxP site (lox71 and lox2272), a polyA sequence (SPAAct1 pA and hsHBB pA), a CAG promoter, and a marker gene (d2BFP). Figure 28B shows the incorporation of the 15S5 payload of Figure 2I at the genomic safe harbor locus. Figure 28C shows an exemplary landing stand. The landing rack contains loxP sites (loxP and lox 2272), hsEF1α1, poly A sequence (SPAC2 pA) and blue fluorescent protein (BFP). The landing shelf is integrated between exon 1 and exon 2 of the safe harbor of the AAVS1 genome. 29A-29E are graphs showing blood urea nitrogen (BUN) and creatinine in five monkeys containing TKO-F xenografts. 30A-30B are graphs showing BUN and creatinine levels in two monkeys containing TKO-G xenografts. Figures 31 and 32 are graphs showing BUN and creatinine levels in two monkeys containing xenografts containing the polycistronic cassette of Figure 2H. Figure 33 shows the expression of nucleic acids comprising the polycistronic cassette of Figure 2E in fibroblasts from Large Yorkshire pigs. Figure 34 (see circled cells) shows the expression of nucleic acids comprising the polycistronic cassette of Figure 2E in fibroblasts from Yucatan pigs. Figure 35 shows the expression of nucleic acids comprising the polycistronic cassette of Figure 2D in fibroblasts from Yucatan pigs. Figure 36 (see circled cells) shows the expression of nucleic acids comprising the polycistronic cassette of Figure 2D in fibroblasts from Yucatan pigs containing a PERV knockout. Figure 37 shows the expression of A20 in islet cells containing the polycistronic cassette of Figure 4A. Figures 38A-38C show the expression of the transgene in TKO-F xenografts of Example 1A in tissues, glomeruli, and tubules. Figure 39 shows the expression of the 15S4 transgene in tissues, glomeruli and renal tubules of monkeys containing nucleic acids containing the polycistronic cassette of Figure 2H. Figure 40 shows immunohistochemical images of transgene expression in monkeys comprising nucleic acids containing the polycistronic cassette of Figure 2H. Figures 41A-41D show the comparative performance of the polycistronic cassette of Figure 2H ("15S4") or Figure 2E ("15S1") in monkeys containing kidney xenografts. Figure 41A shows transgene expression by immunohistochemistry. Figures 41B-41D ​​show transgene expression in whole tissue (Figure 41B), glomeruli (Figure 41C) and renal tubules (Figure 41D). Figure 42A shows the expression of the 15S1 transgene in pig fetal kidneys relative to expression in wild-type Yucatan pigs. Figure 42B shows the expression of the 15S1 transgene in pig neonatal kidneys relative to expression in wild-type Yucatan pigs. Figure 42C shows expression of the 15S1 transgene in pig adult kidneys relative to expression in wild-type Yucatan pigs. Figure 43A shows the expression of 15S1 transgenes TFPI, EPCR and THBD in pig fetal, neonatal and adult kidney tissues. Figure 43B shows the expression of 15S1 transgenic B2M HLA-E fusion protein and CD47 in pig fetal, neonatal and adult kidney tissues. Figure 43C shows the expression of 15S1 transgenes CD46 and CD55 in pig fetal, neonatal and adult kidney tissues. Figure 43D shows the expression of each of the 15S1 transgenes in two different fetal porcine kidney tissues. Figure 44 shows expression of CD47 by islet cells comprising nucleic acid containing the polycistronic cassette of Figure 2H ("15S4") or Figure 4A ("17M1"). Figure 45 shows that dissociated islet cells comprising the polycistronic cassette of Figure 2H ("15S4") are better protected from phagocytosis than islet cells comprising the polycistronic cassette of Figure 4A ("17M1") . Figure 46 provides an example of how to generate transgenic pigs comprising nucleic acids described herein. Figure 47A is a schematic representation of a standard promoter. Figure 47B is a schematic representation of a promoter comprising CpG islands. Figures 48A-48C show CD47 cistron expression in fibroblasts (Figure 48A), beta islet cells (Figure 48B) and pAEC-SV40 cells (Figure 48C) using promoters containing or lacking CpG islands. Figures 48D-48F show expression of CD46 in fibroblasts (Figure 48D), beta islet cells (Figure 48E) and pAEC-SV40 cells (Figure 48F) using promoters containing or lacking CpG islands. The x-axis of each of Figures 48A-48F represents the SEQ ID NO: of the promoter used to express the cistron. Figure 49 shows immunohistochemical images of islet cells comprising nucleic acid containing the polycistronic cassette of Figure 4A transplanted in diabetic mice. Images show staining for insulin, glucagon and nuclei in individual mice. Figure 50 shows the experimental design for the experiment of Example 16. Figure 51 shows blood glucose levels in NSG mice receiving neonatal porcine islet transplants comprising nucleic acids comprising the polycistronic cassette of Figure 4A. Figures 52A-52B show fasting blood glucose concentrations, total insulin, and porcine C-peptide in two cynomolgus monkeys that received neonatal porcine islet grafts containing the polycistronic protein of Figure 4A. Cassette nucleic acid. Figure 53A shows that cells expressing 17M1 are protected from NK cell-mediated lysis. Figure 53B shows that cells expressing 14P and 14S nucleic acids are protected from NK cell-mediated lysis compared to wild-type (WT) cells or K562 cancer cells that do not express 14P and 14S nucleic acids. Figure 54 shows that various cells expressing nucleic acids containing the polycistronic cassette "15S" of Figure 2D have functional THBD and PROCR, as indicated by elevated concentrations of activated protein C compared to cells not expressing 15S. The following cell types were evaluated: alveolar epithelial cells (AECs), kidney epithelial cells (KECs), human umbilical vein endothelial cells ("Hu UVECs"), porcine umbilical vein endothelial cells ("pig UVECs"), and epidermal-derived cells (EPDCs). Implementation

[0055] [Cross Reference to Related Application]

[0056] This application claims U.S. Provisional Application No. 63 / 113,650, filed November 13, 2020, U.S. Provisional Application No. 63 / 218,080, filed July 2, 2021; and U.S. Provisional Application No. 63 / 247,544 priority. Each of the foregoing applications is hereby incorporated by reference in its entirety. [Instructions for electronically submitting text files]

[0057] The contents of the text file submitted electronically herewith in its entirety are hereby incorporated by reference in its entirety: Copy of Sequence Listing in Computer Readable Format (File Name: EGEN_023_03WO_SeqList_ST25.txt, Date of Record: November 11, 2021, File Size : about 2.35 megabytes). [I.] [definition]

[0058] The terms "pig", "swine" and "porcine" are used interchangeably herein to refer to any animal related to the various breeds of the domestic pig species Sus scrofa.

[0059] When used in reference to a fragment or derivative of a protein or polypeptide, the term "biologically active" means that said fragment or derivative retains at least one measurable and / or detectable biological activity of the reference full-length protein or polypeptide . For example, a biologically active fragment or derivative of a CRISPR / Cas9 protein may be capable of binding a gRNA, sometimes referred to herein as a single guide RNA (sgRNA), binding a target DNA sequence when complexed with a guide RNA, and / or cleaving one or more strands of DNA.

[0060] When used in the context of a disease, injury or disorder, the terms "treatment", "treating", "alleviation" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect , and can also be used to refer to improving, alleviating and / or reducing the severity of one or more symptoms of the condition being treated. Said effect may be preventive in terms of fully or partially delaying the onset or recurrence of a disease, disorder or symptoms thereof, and / or in terms of partial or complete cure of a disease or disorder and / or attributable to said disease or disorder In terms of adverse effects, the effects may be therapeutic. "Treatment" as used herein encompasses any treatment of a disease or condition in a mammal, especially a human, and includes: (a) treatment in patients who may be susceptible to, but have not yet been diagnosed with, the disease or condition preventing the disease or condition from occurring in a subject; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) ameliorating the disease or condition (e.g., causing the disease or condition to regress, thereby improving one or more symptoms).

[0061] As used herein, the term "simultaneously" refers to an event that occurs simultaneously with another event, such as within seconds, milliseconds, microseconds, or less of the occurrence of the other event.

[0062] The term "knockout" ("KO") or "knocking out" as used herein refers to the deletion of a gene or a defective gene in a pig or other animal or any cell in a pig or other animal, inactivation or ablation. As used herein, KO can also refer to methods that perform or have performed deletion, inactivation or ablation of a gene or a portion thereof.

[0063] The term "knockin" ("KI") or "knocking in" as used herein refers to one or more nuclei of a gene in a pig or other animal or any cell in a pig or other animal. Nucleotide addition, substitution or mutation. As used herein, KI can also refer to a method by which an addition, substitution or mutation of one or more nucleotides of a gene or a portion thereof is performed or has been performed.

[0064] The term "PERV-free" with reference to porcine tissues, organs or cells refers to tissues, organs or cells that have at least 75% inactive porcine endogenous retrovirus (PERV) elements. The PERV element can be a gag, pol or env gene. In embodiments, the porcine tissue, organ or cell is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% PERV-free .

[0065] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to the specific number of nucleotide or amino acid residues that two or more sequences or subsequences have in common when compared. percentage. Percent identity can be calculated using the online available tool CLUSTALW2 or the Basic Local Alignment Search Tool (BLAST). The following default parameters can be used for CLUSTALW2 pairwise alignments: protein weight matrix = Gonnet; gap opening = 10; gap extension = 0.1.

[0066] As used herein, the term "polycistronic cassette" refers to a nucleic acid encoding two or more "cistronic elements" or genes under the control of a single promoter and polyA sequence. The term "polycistronic cassette" is used interchangeably herein with "transcription unit". In an embodiment, the polycistronic cassette comprises cDNA. In an embodiment, the polycistronic cassette comprises DNA. In embodiments, the polycistronic cassette comprises one or more introns. An exemplary polycistronic cassette is depicted in Figure 12A. Exemplary nucleic acids comprising multiple polycistronic cassettes are depicted in Figure 12B. II. Nucleic acids comprising one or more polycistronic cassettes

[0067] Porcine xenografts are broadly compatible with human organ size and physiology and are ethically accepted by the U.S. general population. However, xenografted porcine tissues trigger a complex series of events leading to graft rejection, including: hyperacute rejection due to the presence of preformed antibodies against porcine antigens, complement priming and hypercoagulability, and molecular Enhanced innate and adaptive immune responses due to incompatibility. The present disclosure uses genetic engineering methods to address current shortcomings of xenotransplantation.

[0068] The present disclosure provides nucleic acids comprising one or more polycistronic cassettes for genetic engineering of cells, organs, tissues and animals.

[0069] In embodiments, provided herein are nucleic acids comprising between about 1 and about 10 polycistronic cassettes. For example, the nucleic acid can comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 polycistronic cassettes. Each polycistronic cassette contains two or more cistrons selected from the group consisting of: CD46, CD55, CD59, THBD, TFPI, PROCR, CD39, B2M, HLA-E, CD47, A20, PD-L1, HO1, CTLA-4 (eg LEA29Y), XIAP and combinations thereof. In embodiments, the polycistronic cassette is flanked by insulator sequences, loxP sites, or a combination thereof.

[0070] In an embodiment, each cistron of the polycistronic cassette is separated by a 2A polypeptide or a sequence comprising an internal ribosome entry site (IRES). Each polycistronic cassette is under the control of a promoter. Exemplary promoters are described in Table B. Each polycistronic cassette also comprises one or more polyA (also referred to as "polyA") and / or terminator sequences. Exemplary poly A sequences and / or terminator sequences are provided in Table C.

[0071] When the name of a cistron, promoter, or polyA sequence contains a lowercase letter before the name of the cistron, promoter, or polyA sequence, the lowercase letter refers to the genus and species from which the cistron was isolated. For example, ssA20 refers to A20 from wild boar (Sus scrofa), hsCD46 refers to CD46 from Homo sapiens, mmA20 refers to A20 from Mus musculus, and btA20 refers to A20 from domestic cow (Bos musculus). taurus) A20. When a lowercase "s" is used before a cistron, promoter, or polyA sequence name, the s refers to wild boar. When the lowercase letter "h" is used before the name of a cistron, promoter, or polyA sequence, the h refers to Homo sapiens. When the lowercase "m" is used before a cistron, promoter, or polyA sequence name, the m refers to Mus musculus.

[0072] In an embodiment, the nucleic acid sequence of the cistron is provided in Table A. The NCBI Reference Sequences, Gene IDs and Protein IDs of Table A are incorporated herein by reference. In an embodiment, the cistron is a porcine cistron. In an embodiment, the cistron is a human cistron. In an embodiment, the cistron is a mouse cistron.

[0073] Table A cistron cistron-encoded protein Synonyms for protein names (if applicable) NCBI Gene ID One or more NCBI reference sequences: NCBI protein ID SEQ ID NO. B2M beta-2 microglobulin 567 NM_004048 NP_004039 HLA-E Major histocompatibility complex, class 1, E 3133 NM_005516 NP_005507 HO1 heme oxygenase-1 3162, 445512 NM_002133.3, NM_002133, NM_001004027 NP 002124, NP 001004027 63, 109, 165, 183, 189 A20 TNFα-induced protein 3 7128, 100622156 NM_001270508, NM_001270507.2, NM 001267890 NP 001257437, NP 001254819 64-65, 104, 182, 188 CD39 cluster of differentiation 39 953 NM_001776 NP_001767 67-70, 106 CD46 cluster of differentiation 46 Membrane cofactor protein (MCP) 4179 NM_002389.4, NM_153826.4, NM_172351.3, NM_153826.4 NP_758861, NP_722548 71-76, 185, 200 CD47 cluster of differentiation 47 961 NM_198793.3, NM_001777.4, NM_198793 NP_942088 77-83, 180 CD55 cluster of differentiation 55 Decay acceleration factor (DAF) 1604 NM_000574.5, NM_000574.4 NP_000565 84, 85, 107, 184 CD59 cluster of differentiation 59 MAC inhibitor protein 966 NM_203330, NM_000611.6, NM_203330.2 NP_976075 86, 108 CTLA-4 (eg, LEA29Y) CTLA-4 1493 NM_005214 NP_005205 87-88, 186 PD-L1 programmed cell death 1 ligand 1 NM_014143 NP_054862 89-91 PROCR endothelin c receptor 29126 NM_006404 NP_006395 92-93, 181 TFPI (also known as *TFPI) tissue factor pathway inhibitor 7035 NM_006287, NM 006287 NP_006278 94-96, 103, 187 THBD thrombomodulin 7056 NM_000361 NP_000352 97-102 and 166 XIAP X-linked inhibitor of apoptosis 331 XM_011531329, NM_001167 NP_001158 110 GFP green fluorescent protein 111 BFP blue fluorescent protein 242, 246

[0074] In an embodiment, the polycistronic cassette comprises nucleic acid encoding beta-2 microglobulin (B2M). The B2M gene encodes a serum protein that is found associated with major histocompatibility complex (MHC) class I heavy chains on the surface of nearly all nucleated cells. In an embodiment, the polycistronic cassette comprises nucleic acid encoding major histocompatibility complex class 1 E (HLA-E). HLA-E belongs to the HLA class I heavy chain paralogs. This class I molecule is a heterodimer comprising heavy and light chains (beta-2 microglobulin). In an embodiment, the polycistronic cassette comprises nucleic acid encoding both B2M and HLA-E (which is referred to as a "B2M HLA-E fusion protein"). In an embodiment, the B2M HLA-E fusion protein is encoded by a nucleic acid sequence selected from SEQ ID NO: 62, 66 or 105. In embodiments, the B2M HLA-E fusion protein comprises a peptide epitope. B2M HLA-E fusion proteins comprising peptide epitopes are referred to herein as "single-chain trimers". In an embodiment, the amino acid sequence of the epitope is VMAPRTLIL (SEQ ID NO: 197) or VMAPRTLFL (SEQ ID NO: 198). In an embodiment, the amino acid sequence of the epitope is MAPRTLIL (SEQ ID NO: 251) or MAPRTLFL (SEQ ID NO: 252). In an embodiment, the epitope is located at the N-terminus of the B2M-HLA-E fusion protein. There is the nucleic acid encoding of the sequence represented by SEQ ID NO: 23, 24, 26, 201, 202 and 204 comprising the B2M HLA-E fusion protein of peptide epitope, and described peptide epitope has the amine represented by SEQ ID NO: 251 amino acid sequence. having SEQ ID NO: 31, 32, 33, 34, 39, 30, 41, 42, 43, 44, 45, 46, 172, 173, 176, 177, 209-212, 216, 217, 219-226, The nucleic acids of the sequences represented by 229 and 230 encode a B2M HLA-E fusion protein comprising a peptide epitope having the amino acid sequence represented by SEQ ID NO: 198. There is the nucleic acid encoding of the sequence represented by SEQ ID NO: 25, 27-29, 35-37, 40, 203, 205-207, 213-215 and 218 comprising a B2M HLA-E fusion protein of a peptide epitope, said peptide The epitope has the amino acid sequence represented by SEQ ID NO: 197.

[0075] In embodiments, the polycistronic cassette comprises nucleic acid encoding heme oxygenase-1 (HO1). Heme oxygenase, an essential enzyme in the catabolism of heme, cleaves heme to form biliverdin and carbon monoxide (a putative neurotransmitter), which is then converted to biliverdin by biliverdin reductase. red pigment. Heme oxygenase activity is induced by its substrate heme and various non-heme substances. In an embodiment, the nucleic acid encoding HO1 is isolated from wild boar. In an embodiment, HO1 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NO: 63, 109, 165, 183 or 189.

[0076] In an embodiment, the polycistronic cassette comprises a nucleic acid encoding A20. The protein encoded by this gene is a zinc finger protein and ubiquitin editing enzyme, and has been shown to inhibit NF-κB initiation and TNF-mediated apoptosis. The encoded protein has both ubiquitin ligase and deubiquitinase activities, and participates in immune and inflammatory responses mediated by cytokines. In an embodiment, the nucleic acid encoding A20 is isolated from wild boar. In an embodiment, A20 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NO: 64-65, 104, 182 or 188.

[0077] In an embodiment, the polycistronic cassette comprises nucleic acid encoding cluster of differentiation 39 (CD39). The protein encoded by this gene is a plasma membrane protein, which can hydrolyze extracellular ATP and ADP into AMP. In an embodiment, CD39 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NOs: 67-70 and 106.

[0078] In an embodiment, the polycistronic cassette comprises nucleic acid encoding a cluster of differentiation 46 (CD46) protein. The protein encoded by this gene is a type I membrane protein and is a regulatory part of the complement system. The encoded protein has cofactor activity, which can inactivate complement components C3b and C4b through serum factor I, thereby protecting host cells from complement damage. In an embodiment, CD46 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NOs: 71-76 and 185. In an embodiment, CD46 comprises a splice site mutation in which the acceptor and donor sites are removed. CD46_LL (SEQ ID NO: 71), CD46da1 (SEQ ID NO: 72), CD46da2 (SEQ ID NO: 73), CD46da (SEQ ID NO: 72, 73, 185 or 200), and CD46i (SEQ ID NO: 74 -75) refers to a CD46 protein with a splice site mutation. In embodiments, the polycistronic cassette comprising CD46 comprises a CD46 isoform selected from the group consisting of CD46 C1, CD46 C2, CD46 BC1, CD46 BC2, and CD46 ABC1. These isoforms are described in the following reference, which is incorporated herein in its entirety: Human Genetics vol. 136, pp. 421-435 (2017).

[0079] In an embodiment, the polycistronic cassette comprises nucleic acid encoding a cluster of differentiation 47 (CD47) protein. This gene encodes a membrane protein that is involved in the increase in intracellular calcium concentration that occurs when cells adhere to the extracellular matrix. The encoded protein is also a receptor for the C-terminal cell-binding domain of thrombospondin and may function in membrane trafficking and signal transduction. CD47 contains an IgV domain, a transmembrane domain and a C-terminal domain. The CD47 C-terminal domain has four isoforms: isoform 1, isoform 2, isoform 3, and isoform 4. Isoform 1 is 4 amino acids in length and is expressed in epithelial and endothelial cells. Isoform 2 is 16 amino acids in length and is the most abundantly expressed isoform of CD47. Isoform 2 is expressed in hematopoietic, endothelial and epithelial cells. Isoform 3 is 23 amino acids in length, and isoform 4 is 36 amino acids in length. Isoforms 3 and 4 are expressed in neurons, gut and testis. In an embodiment, CD47 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NOs: 77-83 and 180. In embodiments, CD47 is isoform 1, 2, 3 or 4. In an embodiment, CD47 is CD47 isoform 2 (CD47-2). In an embodiment, CD47-2 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NO: 78, 83 and 180. In an embodiment, the polycistronic cassette comprises a nucleic acid encoding a CD47 protein having a S64A mutation, an S79A mutation, or a combination thereof as compared to SEQ ID NO: 201. The following article describes the S64A and S79A mutations and is hereby incorporated by reference in its entirety: J Biol Chem. 2011 Apr 29;286(17):14991-5002.

[0080] In an embodiment, the polycistronic cassette comprises nucleic acid encoding a cluster of differentiation 55 (CD55) protein. This gene encodes a glycoprotein involved in the regulation of the complement cascade. Binding of the encoded proteins to complement proteins accelerates their decay, thereby disrupting the cascade and preventing damage to the host cell. In an embodiment, CD55 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NO: 84, 85, 107 and 184.

[0081] In an embodiment, the polycistronic cassette comprises nucleic acid encoding a cluster of differentiation 59 (CD59) protein. This gene encodes a cell surface glycoprotein that regulates complement-mediated cell lysis and is involved in lymphocyte signal transduction. The CD59 protein is a potent inhibitor of the complement membrane attack complex, wherein during assembly of this complex it binds complement C8 and / or C9, thereby inhibiting the incorporation of multiple copies of C9 into the complex, which Intake is necessary for the formation of permeable pores. In an embodiment, CD59 is encoded by a nucleic acid having a sequence selected from SEQ ID NO: 86 or SEQ ID NO: 108.

[0082] In an embodiment, the polycistronic cassette comprises a nucleic acid encoding CTLA-4. In embodiments, the nucleic acid encodes the extracellular domain of CTLA-4. In embodiments, the extracellular domain of CTLA-4 comprises a L104E mutation, an A29Y mutation, or both relative to SEQ ID NO: 199. In embodiments, nucleic acids encoding CTLA-4 include nucleic acids encoding immunoglobulins or fragments thereof. In embodiments, the fragment of an immunoglobulin is a kappa light chain or a lambda light chain. In an embodiment, the fragment thereof is an immunoglobulin heavy chain. In embodiments, the fragment thereof is the CH1, CH2, CH3 or CH4 domain of an immunoglobulin heavy chain. In embodiments, the fragment thereof is a variable heavy domain or variable light domain of an immunoglobulin. In embodiments, the nucleic acid encoding CLTA-4 and immunoglobulin or fragments thereof is LEA29Y. In an embodiment, the nucleic acid encoding CTLA-4 and an immunoglobulin or fragment thereof comprises the extracellular domain of CTLA-4 (SEQ ID NO: 199) (which has a L104E mutation and an A29Y mutation relative to SEQ ID NO: 199) , and the hinge, CH2 and CH3 domains of human immunoglobulin 1. The nucleic acid sequence of LEA29Y encodes a protein with the following amino acid sequence: MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 196). LEA29Y binds human B7.1 / CD80 and B7.2 / CD86 with high affinity and is therefore a potent inhibitor of T cell co-stimulation via this pathway. The following references describe LEA29Y and are hereby incorporated by reference in their entirety: International Publication No. 2001 / 092337 and Wolf-van Buerck et al. Scientific Reports. 7: 3572 (2017). In an embodiment, CTLA-4 is encoded by a nucleic acid having a sequence selected from SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 186.

[0083] In an embodiment, the polycistronic cassette comprises nucleic acid encoding programmed cell death 1 ligand 1 (PD-L1). This gene encodes an immunosuppressive receptor ligand expressed by hematopoietic and non-hematopoietic cells such as T cells and B cells, as well as various types of tumor cells. The encoded protein is a type I transmembrane protein with immunoglobulin V-like and C-like domains. Interaction of this ligand with its receptor inhibits T cell priming and cytokine production. In an embodiment, PD-L1 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NO: 89-91.

[0084] In an embodiment, the polycistronic cassette comprises nucleic acid encoding the endothelin c receptor (PROCR) (used interchangeably with "EPCR"). EPCR binds activated protein C. In an embodiment, the EPCR is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NO: 92, 93 and 181.

[0085] In an embodiment, the polycistronic cassette comprises nucleic acid encoding a tissue factor pathway inhibitor (TFPI) protein. The protein encoded by TFPI inhibits factor X and inhibits VIIa / tissue factor activity. In an embodiment, TFPI is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NOs: 94-96, 103 and 187. In an embodiment, TFPI is a fusion between TFPI, the CD4 transmembrane domain (NM_000616) or a fragment thereof, and a P-selectin cytoplasmic tail (NM_003005) or a fragment thereof. In an embodiment, TFPI lacks a P-selectin tail and is referred to herein as TFPI (ΔP-Sel). In an embodiment, the CD4 transmembrane domain comprises amino acids 166 to 435 of CD4 having the NCBI RefSeq ID of NM_000616. In an embodiment, the P-selectin cytoplasmic tail comprises amino acids 797 to 830 of P-selectin having the NCBI RefSeq ID of NM_003005.

[0086] In an embodiment, the polycistronic cassette comprises nucleic acid encoding thrombomodulin (THBD). The protein encoded by this intronless gene is an endothelial-specific type I membrane receptor that binds thrombin. This binding results in the activation of protein C, which degrades coagulation factors Va and VIIIa and reduces thrombin production. In an embodiment, THBD is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NOs: 97-102 and 166. In an embodiment, THBD is "THBDda", which is a gene comprising a splice site mutation, having a sequence selected from any one of SEQ ID NOs. 99-102 or 166.

[0087] In an embodiment, the polycistronic cassette comprises nucleic acid encoding an X chromosome-linked inhibitor of apoptosis (XIAP) protein. In an embodiment, XIAP is encoded by a nucleic acid having the sequence of SEQ ID NO: 110. This gene encodes a protein belonging to the apoptosis inhibitor protein family.

[0088] In embodiments, any cistron described herein comprises a start codon or a stop codon. The polycistronic cassette is organized such that transcription proceeds from the 5' priming end of the cassette to the 3' end to facilitate the expression of each cistron. Individual cistrons can add or remove start or stop codons to facilitate polycistronic expression.

[0089] In embodiments, a polycistronic cassette comprises two or more cistrons that share a function, eg, genes involved in complement regulation, coagulation, innate immunity, inflammation and apoptosis, and cellular immunity. In embodiments, a polycistronic cassette comprises two or more cistrons with different functions.

[0090] In embodiments, the nucleic acids described herein comprise one or more polycistronic cassettes selected from the group consisting of coagulation cassettes, complement regulatory cassettes, complement regulatory and inflammation and apoptosis cassettes, innate immunity cassettes, innate immunity and inflammation and apoptosis cassette, inflammation and apoptosis cassette, complement regulation and innate immunity cassette, cellular immunity and coagulation cassette, apoptosis and coagulation cassette, and apoptosis and cellular immunity cassette.

[0091] In embodiments, the coagulation cassette comprises one or more cistrons selected from THBD, TFPI, CD39 and PROCR. In embodiments, the coagulation cassette comprises one or more cistrons selected from THBD and TFPI. In embodiments, the coagulation cassette comprises one or more cistrons selected from THBD, TFPI and CD39. In embodiments, the coagulation cassette comprises one or more cistrons selected from PROCR and THBD. In embodiments, the coagulation cassette comprises one or more cistrons selected from TFPI, PROCR and THBD.

[0092] In embodiments, the complement regulatory cassette comprises one or more cistrons selected from CD46, C59 and CD55. In embodiments, the complement regulatory cassette comprises one or more cistrons selected from CD46 and CD59. In embodiments, the complement regulatory cassette comprises one or more cistrons selected from CD46 and CD55. In embodiments, the complement regulatory cassette comprises one or more cistrons selected from CD46, CD55 and CD59. In embodiments, the complement regulatory cassette comprises one or more cistrons selected from CD55 and CD59. In embodiments, the nucleic acid comprising a complement regulatory cassette prevents complement deposition. For example, Figure 18D shows that expression of nucleic acids comprising cistrons CD46, CD55, CD59, or combinations thereof, reduces deposition of complement protein C3b compared to expression of nucleic acids lacking cistrons CD46, CD55, and CD59.

[0093] In embodiments, the complement regulation and inflammation and apoptosis cassette comprises one or more cistrons selected from the group consisting of CD46, CD55, CD59, A20, HO1 and PD-L1. In embodiments, the complement regulation and inflammation and apoptosis cassette comprises one or more cistrons selected from CD46, CD55, CD59, A20, HO1 and PD-L1. In embodiments, the complement regulation and inflammation and apoptosis cassette comprises one or more cistrons selected from CD46, CD55, A20 and HO1.

[0094] In embodiments, the innate immunity cassette comprises one or more cistrons selected from the group consisting of B2M, HLA-E, B2M HLA-E fusion protein, CD47, and combinations thereof. In an embodiment, the innate immunity cassette comprises a cistron encoding a B2M HLA-E fusion protein and a CD47 cistron.

[0095] In embodiments, the innate immunity and inflammation and apoptosis cassette comprises one or more cistrons selected from B2M, HLA-E, B2M HLA-E fusion protein, CD47, A20, HO1, PD-L1 and combinations thereof . In an embodiment, the innate immunity and inflammation and apoptosis cassette comprises a B2M cistron, an HLA-E cistron, a cistron encoding a B2M HLA-E fusion protein, a CD47 cistron, an A20 cistron, a HO1 One or more of cistrons and PD-L1 cistrons. In embodiments, the innate immunity and inflammation and apoptosis cassette comprises one or more cistrons selected from B2M, HLA-E, B2M HLA-E fusion protein, CD47, A20, and HO1. In embodiments, the innate immunity and inflammation and apoptosis cassette comprises one or more cistrons selected from the group consisting of cistrons encoding B2M HLA-E fusion proteins, CD47, A20, and HO1.

[0096] In embodiments, the inflammation and apoptosis cassette comprises one or more cistrons selected from A20, HO1 and PD-L1. In embodiments, the inflammation and apoptosis cassette comprises one or more cistrons selected from A20 and HO1. In embodiments, the inflammation and apoptosis cassette comprises one or more cistrons selected from A20 and PD-L1.

[0097] In embodiments, the complement regulation and innate immunity cassette comprises one or more cistrons selected from the group consisting of CD46, CD55, CD47, B2M, HLA-E, B2M HLA-E fusion proteins, and combinations thereof. In embodiments, the complement regulation and innate immunity cassette comprises one or more cistrons selected from CD46, CD55 and CD47.

[0098] In embodiments, the cellular immunity and coagulation cassette comprises one or more cistrons selected from LEA29Y, CD39, TFPI, THBD and PROCR. In an embodiment, the cellular immunity and coagulation cassette comprises a CTLA-4 (eg, LEA29Y) cistron and a CD39 cistron.

[0099] In embodiments, the apoptosis and coagulation cassette comprises one or more cistrons selected from THBD, TFPI, CD39, PROCR, and XIAP. In an embodiment, the apoptosis and coagulation cassette comprises a XIAP cistron and a CD39 cistron.

[0100] In embodiments, the apoptosis and cellular immunity cassette comprises one or more cistrons selected from A20, PD-L1, HO1 and CTLA-4 (eg, LEA29Y). In embodiments, the apoptosis and cellular immunity cassette comprises A20 and CTLA-4 (eg, LEA29Y) cistrons. In an embodiment, the apoptosis and cellular immunity cassette comprises A20 and HO1 cistrons. In embodiments, the A20 cistron, the HO1 cistron, or both are from wild boar.

[0101] In an embodiment, the coagulation and innate immunity cassette comprises one or more cistrons selected from the group consisting of: THBD cistron, TFPI cistron, CD39 cistron, PROCR cistron, B2M cistron, HLA- E cistron, cistron encoding B2M HLA-E fusion protein and CD47 cistron. In an embodiment, the coagulation and innate immunity cassette comprises a THBD cistron, a CD47 cistron, and a PROCR cistron.

[0102] In an embodiment, the nucleic acid comprises a first polycistronic cassette, a second polycistronic cassette, and a third polycistronic cassette, wherein said first polycistronic cassette is located in said second polycistronic cassette 5' side of the cassette, and the second polycistron cassette is located on the 5' side of the third polycistron cassette. In embodiments, the coagulation cassette is a first, second or third polycistronic cassette. In embodiments, the complement regulatory cassette is the first, second or third polycistronic cassette. In embodiments, the complement regulation and inflammation and apoptosis cassette is the first, second or third polycistronic cassette. In embodiments, the innate immunity cassette is the first, second or third polycistronic cassette. In embodiments, the innate immunity and inflammation and apoptosis cassette is the first, second or third polycistronic cassette. In embodiments, the inflammation and apoptosis cassette is the first, second or third polycistronic cassette. In embodiments, the complement regulation and innate immunity cassette is the first, second or third polycistronic cassette. In embodiments, the cellular immunity and coagulation cassette is the first, second or third polycistronic cassette. In embodiments, the apoptosis and coagulation cassette is the first, second or third polycistronic cassette. In embodiments, the apoptosis and cellular immunity cassette is the first, second or third polycistronic cassette. In embodiments, the coagulation and innate immunity cassette is the first, second or third polycistronic cassette.

[0103] In an embodiment, the nucleic acid comprises a first polycistronic cassette, a second polycistronic cassette, a third polycistronic cassette, and a fourth polycistronic cassette, wherein the first polycistronic cassette is located at The 5' side of the second polycistronic box, the second polycistronic box is located on the 5' side of the third polycistronic box, and the third polycistronic box is located in the The 5' side of the fourth polycistronic cassette. In embodiments, the coagulation cassette is a first, second, third or fourth polycistronic cassette. In embodiments, the complement regulatory cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the complement regulation and inflammation and apoptosis cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the innate immunity cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the innate immunity and inflammation and apoptosis cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the inflammation and apoptosis cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the complement regulation and innate immunity cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the cellular immunity and coagulation cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the apoptosis and coagulation cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the apoptosis and cellular immunity cassette is the first, second, third or fourth polycistronic cassette. In embodiments, the coagulation and innate immunity cassette is the first, second, third or fourth polycistronic cassette.

[0104] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a coagulation cassette, a complement regulatory cassette, and an innate immunity cassette. In embodiments, the coagulation cassette is located on the 5' side of the complement regulatory cassette. In embodiments, the complement regulatory cassette is located on the 5' side of the innate immunity cassette. In embodiments, the coagulation cassette is on the 5' side of the complement regulatory cassette, and the complement regulatory cassette is on the 5' side of the innate immunity cassette. In an embodiment, the coagulation cassette is located on the 5' side of the innate immunity cassette, and the innate immunity cassette is located on the 5' side of the complement regulation cassette. In an embodiment, the innate immunity cassette is located on the 5' side of the coagulation cassette, and the coagulation cassette is located on the 5' side of the complement regulatory cassette.

[0105] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises coagulation cassettes, complement regulatory cassettes, and innate immunity and inflammation and apoptosis cassettes. In embodiments, the coagulation cassette is located 5' to the complement regulatory cassette, and the complement regulatory cassette is located 5' to the innate immunity and apoptosis cassette. In embodiments, the complement regulatory cassette is located 5' to the innate immunity and apoptosis cassette. In embodiments, the coagulation cassette is located on the 5' side of the complement regulatory cassette.

[0106] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises coagulation cassettes, inflammation and apoptosis cassettes, and complement regulation and innate immunity cassettes. In an embodiment, the coagulation cassette is located 5' to the inflammation and apoptosis cassette, and the inflammation and apoptosis cassette is located 5' to the complement regulation and innate immunity cassette. In embodiments, the coagulation cassette is located 5' to the inflammation and apoptosis cassettes. In an embodiment, the inflammation and apoptosis cassettes are located 5' to the complement regulation and innate immunity cassettes.

[0107] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises coagulation cassettes, complement regulation and inflammation and apoptosis cassettes, and innate immunity cassettes. In an embodiment, innate immunity is located on the 5' side of the coagulation cassette, and the coagulation cassette is located on the 5' side of the complement regulation and inflammation and apoptosis cassettes.

[0108] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a coagulation cassette, a complement regulation cassette, an innate immunity cassette, and a cellular immunity and coagulation cassette. In an embodiment, the coagulation cassette is located on the 5' side of the innate immunity cassette. In an embodiment, the innate immunity cassette is located on the 5' side of the cellular immunity and coagulation cassette. In an embodiment, the cellular immunity and coagulation cassette is located on the 5' side of the complement regulatory cassette. In an embodiment, the coagulation cassette is located on the 5' side of the innate immunity cassette, the innate immunity cassette is located on the 5' side of the cellular immunity and coagulation cassette, and the cellular immunity and coagulation cassette is located on the 5' side of the complement regulation cassette.

[0109] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a coagulation cassette, a complement regulation cassette, an innate immunity cassette, and an apoptosis and inflammation cassette. In an embodiment, the coagulation cassette is on the 5' side of the innate immunity cassette, the innate immunity cassette is on the 5' side of the apoptosis and inflammation cassette, and the apoptosis and inflammation cassette is on the 5' side of the complement regulation cassette. In an embodiment, the coagulation cassette is on the 5' side of the innate immunity cassette, the innate immunity cassette is on the 5' side of the complement regulatory cassette, and the complement regulatory cassette is on the 5' side of the apoptosis and inflammation cassette.

[0110] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a coagulation cassette, a complement regulation cassette, an innate immunity cassette, and an apoptosis and coagulation cassette. In an embodiment, the coagulation cassette is on the 5' side of the innate immunity cassette, the innate immunity cassette is on the 5' side of the apoptosis and coagulation cassette, and the cellular immunity and coagulation cassette is on the 5' side of the complement regulation cassette.

[0111] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a coagulation cassette, a complement regulation cassette, an innate immunity cassette, and an apoptosis and cellular immunity cassette. In an embodiment, the coagulation cassette is on the 5' side of the innate immunity cassette, the innate immunity cassette is on the 5' side of the apoptosis and cellular immunity cassette, and the apoptosis and cellular immunity cassette is on the 5' side of the complement regulation cassette.

[0112] In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises coagulation and innate immunity cassettes, apoptosis and cellular immunity cassettes, and complement regulatory cassettes. In embodiments, the coagulation and innate immunity cassettes are located 5' to the apoptosis and cellular immunity cassettes, and the apoptosis and cellular immunity cassettes are located to the 5' side of the complement regulation cassettes.

[0113] In embodiments, the nucleic acid comprising at least one polycistronic cassette comprises one or more promoters, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 promoters. In embodiments, the nucleic acid comprising at least one polycistronic cassette contains a different promoter for each polycistronic cassette. A promoter is a DNA sequence that defines where transcription of the polycistronic cassette begins by RNA polymerase. In an embodiment, the promoter is derived from a porcine gene. In an embodiment, the promoter is derived from a mouse gene. In an embodiment, the promoter is derived from a human gene. In an embodiment, the promoter is the eukaryotic translation elongation factor 1α1 (abbreviated as “EF1α1” or “EEF1α1”) promoter. The EF1α1 promoter is a constitutive promoter. In an embodiment, the promoter is a CAG promoter. The CAG promoter contains the cytomegalovirus enhancer fused to the chicken β-actin promoter. In an embodiment, the promoter is the actin gamma 1 (ACTG1) promoter. In an embodiment, the promoter is the heat shock protein family A member 8 (HSPA8) promoter. In an embodiment, the promoter is the ubiquitin C (UBC) promoter. In an embodiment, the promoter is the intracellular adhesion molecule 2 (ICAM2) promoter. In embodiments, the promoter comprises the EF1α1, CAG, ACTG1, HSPA8, UBC or ICAM2 promoters. In embodiments, the promoter comprises an extension at the 5' or 3' end. In embodiments, the extension is an intron or a fragment thereof. Introns may be obtained from endogenous promoter genes (eg EF1α1, CAG, ACTG1, HSPA8, UBC or ICAM2) or provided from other sources.

[0114] In embodiments, the promoter comprises CpG islands. CpG islands are regions with elevated GC content and high density of CpG dinucleotides. In an embodiment, the CpG island is located 5' to the exon sequence of the promoter. In embodiments, the CpG island comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% GC content . In embodiments, the CpG island comprises about 500 base pairs (bp) to about 3 kilobase pairs (kb). In embodiments, the CpG island comprises about 500 bp to about 5 kb. In embodiments, the CpG island comprises about 1 kb to about 3 kb. For example, a CpG island can comprise at least about 500 bp, at least about 1 kb, at least about 1.5 kb, at least about 2 kb, at least about 2.5 kb, at least about 3 kb, at least about 3.5 kb, at least about 4 kb, at least about 4.5 kb Or at least about 5 kb. In contrast, standard promoters are about 100-200 base pairs in length. In embodiments, the CpG island contains a ratio of the observed number to the expected number of CpG dinucleotides greater than 0.60. In embodiments, the CpG island comprises unmethylated CpG dinucleotides. In embodiments, the CpG island comprises methylated CpG dinucleotides. The following references describe CpG islands: J Mol Bio. 1987 Jul 20;196(2):261-82; Nat Rev Mol Cell Biol. 2018 Oct;19(10):621-637; Biochem Biophys Res Commun. 2009 May 15; 382(4): 643-645. Each of these references is incorporated herein in its entirety.

[0115] Non-limiting examples of promoters contained in the nucleic acids or polycistronic cassettes described herein are shown in Table B.

[0116] Form B [name] [SEQ ID NO.] CAG 126-128, 247, 250 hsACTG1 129 hsEF1α1, also known as "hEF1α1" 130-132, 231-232 hsHSPA8 133, 236-238 mmEF1α1, also known as "mEF1a1" 134-135 ssEEF1α1, also known as "ssEEF1A1(ATACseq)" 136-138, 179, 233-235 ssHSPA8, also known as "ssHSPA8 (ATACseq)" 139-140, 142-145, 236-238 sUBC 141, 168, 178 ssICAM2 167

[0117] In embodiments, each promoter within the nucleic acid is distinct. In an embodiment, the first promoter and the second promoter within the nucleic acid are the same. In embodiments, each polycistronic cassette of the nucleic acid comprises a different promoter. In embodiments, the promoter is a forward promoter on the sense strand. In an embodiment, the promoter is an inverted promoter on the antisense strand.

[0118] In Fig. 1A-Fig. 1D, Fig. 2A-Fig. 2J, Fig. 3A-Fig. 3C, Fig. 4A-Fig. 4H, Fig. 5A-Fig. indicated by the arrow.

[0119] In embodiments, any cassette described herein may comprise any promoter from Table B. Exemplary arrangements of promoters and polycistronic cassettes are found in FIGS. 1A-1D , 2A-2J , 3A-3C , 4A-4H , 5A-5F and 6 .

[0120] In an embodiment, provided herein is a nucleic acid comprising a first polycistronic cassette, a second polycistronic cassette, and a third polycistronic cassette from 5' to 3'. In an embodiment, the first polycistronic cassette and the second polycistronic cassette are under the control of promoters that initiate transcription of cassettes on different strands. For example, a first polycistronic cassette can comprise a forward promoter, and a second polycistronic cassette can comprise a reverse promoter. Alternatively, the first polycistronic cassette can contain a reverse promoter, and the second polycistronic cassette can contain a forward promoter. In an embodiment, the second polycistronic cassette and the third polycistronic cassette are under the control of promoters that initiate transcription of cassettes on different strands. For example, a second polycistronic cassette can comprise a forward promoter, and a third polycistronic cassette can comprise a reverse promoter. Alternatively, the second polycistronic cassette can contain a reverse promoter, and the third polycistronic cassette can contain a forward promoter. In an embodiment, the first polycistronic cassette is under the control of the forward promoter, the second polycistronic cassette is under the control of the reverse promoter, and the third polycistronic cassette is under the control of the forward promoter under control. In an embodiment, the first polycistronic cassette is under the control of the reverse promoter, the second polycistronic cassette is under the control of the forward promoter, and the third polycistronic cassette is under the control of the reverse promoter under control.

[0121] In an embodiment, provided herein is a nucleic acid comprising a first polycistronic cassette, a second polycistronic cassette, a third polycistronic cassette, and a fourth polycistronic cassette from 5' to 3'. In an embodiment, the first polycistronic cassette and the second polycistronic cassette are under the control of promoters that initiate transcription of cassettes on different strands. For example, a first polycistronic cassette can comprise a forward promoter, and a second polycistronic cassette can comprise a reverse promoter. Alternatively, the first polycistronic cassette can contain a reverse promoter, and the second polycistronic cassette can contain a forward promoter. In an embodiment, the second polycistronic cassette and the third polycistronic cassette are under the control of promoters that initiate transcription of cassettes on different strands. For example, a second polycistronic cassette can comprise a forward promoter, and a third polycistronic cassette can comprise a reverse promoter. Alternatively, the second polycistronic cassette can contain a reverse promoter, and the third polycistronic cassette can contain a forward promoter. In an embodiment, the third polycistronic cassette and the fourth polycistronic cassette are under the control of promoters that initiate transcription of cassettes on different strands. For example, a third polycistronic cassette can comprise a forward promoter, and a fourth polycistronic cassette can comprise a reverse promoter. Alternatively, the third polycistronic cassette can contain a reverse promoter, and the fourth polycistronic cassette can contain a forward promoter. In an embodiment, the first polycistronic cassette is under the control of the reverse promoter, the second polycistronic cassette is under the control of the forward promoter, and the third polycistronic cassette is under the control of the reverse promoter. and the fourth polycistronic cassette is under the control of the forward promoter. In an embodiment, the first polycistronic cassette is under the control of the forward promoter, the second polycistronic cassette is under the control of the reverse promoter, and the third polycistronic cassette is under the control of the forward promoter. under the control of , and the fourth polycistronic cassette is under the control of the reverse promoter.

[0122] In embodiments, the nucleic acid comprising at least one polycistronic cassette comprises one or more polyA sequences. In an embodiment, the polyA sequence is from the human growth hormone (GH) gene. In an embodiment, the poly A sequence is from the beta globin (HBB) gene. In an embodiment, the poly A is from the SV40 virus. In embodiments, the poly A sequence is synthetic poly A (pA). See Table C for exemplary poly A sequences. In embodiments, any cassette described herein may comprise any poly A sequence from Table C. Exemplary arrangements of poly A sequences within a polycistronic cassette are found in FIGS. 1A-1D , 2A-2J , 3A-3C , 4A-4H , 5A-5F and 6 . In embodiments, the nucleic acids described herein comprise one or more polyA sequences from Table C located on the 5' side of the polycistronic cassette. In embodiments, the polyA sequence comprises a terminator. In embodiments, the nucleic acids described herein comprise one or more polyA sequences from Table C located 3' to the polycistronic cassette.

[0123] Form C [name] [SEQ ID NO.] btGH pA also known as "bGH pA" 112, 120-122, 159, 190 wxya 113, 161 hsHBB pA also known as "HBB pA" 114-116, 191 wxya 160 synthetic pA 117, 125 SPA hsACTB pA is also known as "SPAActBpA" and "SPA ACTB", "SPA / ACTB" and "SPA / hsActB" 118 SV40pA is also known as "SV40 late pA" 119, 162 SPAACT1 123 SPA / PTCH2pA 124, 240 SPA / CCNB 239 wxya 192

[0124] In an embodiment, the polyA sequence comprises synthetic pA (SEQ ID NO: 117 or SEQ ID NO: 125) and the transcription terminator ACTB (SEQ ID NO: 158). In embodiments, the polyA sequence comprises synthetic pA and a transcription terminator. In an embodiment, the transcription terminator is ACTB. In an embodiment, ACTB has the nucleic acid sequence of SEQ ID NO: 118. In an embodiment, the presence of a polyA sequence and a terminator sequence terminates transcription of the polycistronic cassette. In an embodiment, transcription of the polycistronic cassette is more efficiently terminated when the polycistronic cassette comprises both a poly-A sequence and a terminator than when the polycistronic cassette comprises only a poly-A sequence. In an embodiment, the terminator is selected from any terminator sequence described in the following document, which is hereby incorporated by reference in its entirety: Nojima et al. Cell Reports 3, 1080-1092 (2013).

[0125] In embodiments, the nucleic acid comprises a polyA sequence and a transcription terminator (eg, ACTB) on the 5' or 3' side of the first or last polycistronic cassette within the nucleic acid. Inclusion of a transcription terminator on the 5' or 3' side of the first or last polycistronic cassette terminates transcription mediated by the upstream genomic promoter. Therefore, only polycistronic cassettes are transcribed.

[0126] In an embodiment, the cistrons within a polycistronic cassette are separated by a nucleic acid sequence encoding a 2A polypeptide (also referred to herein as a "2A peptide"). The term 2A polypeptide refers to a self-cleaving peptide that induces ribosome jumping during protein translation in cells. In embodiments, the polycistronic cassette comprises one or more 2A polypeptides selected from the group consisting of E2A, F2A, P2A and T2A polypeptides. In an embodiment, the polycistronic cassette encodes an E2A polypeptide from a nucleic acid sequence selected from SEQ ID NO.1 and 9. In an embodiment, the polycistronic cassette comprises a F2A polypeptide encoded by a nucleic acid having a sequence selected from SEQ ID NOs. 2 and 10. In an embodiment, the polycistronic cassette comprises a P2A polypeptide encoded by a nucleic acid having a sequence selected from SEQ ID NOs. 3-6, 12-14 and 169. In an embodiment, the polycistronic cassette comprises a T2A polypeptide encoded by a nucleic acid having the sequence SEQ ID NO: 11. Additional 2A polypeptides are described by Liu et al. and are incorporated herein by reference in their entirety: Liu et al. Scientific Reports, Volume 7, Article 2193 (2017). In embodiments, each nucleic acid encoding a 2A polypeptide within the nucleic acids described herein encodes a different amino acid sequence.

[0127] In an embodiment, when a polycistronic cassette containing cistrons separated by 2A peptides is expressed, the expressed cistrons may comprise N-terminal prolines, C-terminal fusions, C-terminal fusions of 2A peptides or fragments thereof. or a combination thereof. In the Examples, the represented cistron comprises a C-terminal fusion of a 2A peptide, where the 2A peptide lacks a C-terminal proline.

[0128] In an example, when a cistron located at the 5' end of a 2A peptide is expressed, the expressed cistron comprises a C-terminal fusion of the 2A peptide, wherein the 2A peptide lacks a C-terminal proline. In an embodiment, when a cistron located at the 3' end of the 2A peptide is expressed, the expressed cistron contains an N-terminal proline. In an embodiment, when the cistron is located at the 5' end of the first 2A peptide and at the 3' end of the second 2A peptide, the expressed cistron comprises an N-terminal proline and a C-terminal fusion of the 2A peptide, wherein The C-terminal fusion of the 2A peptide lacks the C-terminal proline of the 2A peptide.

[0129] In an embodiment, the cistrons within a polycistronic cassette are separated by an internal ribosome entry site (IRES). In an embodiment, the nucleic acid described herein comprises one or more IRESs having the nucleic acid sequence of any one of SEQ ID NO: 11, 194 and 195. In the Examples, when a polycistronic cassette containing cistrons separated by IRESs is expressed, the expressed cistrons are wild-type proteins.

[0130] In an embodiment, a nucleic acid provided herein comprises a first insulator sequence on the 5' side of the 5' polycistronic cassette and a second insulator sequence on the 3' side of the 3' polycistronic cassette. In an embodiment, the insulator is HS4. In an embodiment, HS4 has a sequence selected from any one of SEQ ID NOs. 49-58, 163 and 164. In an embodiment, HS4 prevents transcriptional interference from the promoter at the genomic integration site.

[0131] In an embodiment, the nucleic acid provided herein comprises: a first inverted terminal repeat (ITR) located on the 5' side of the 5' polycistronic box and having the sequence of SEQ ID NO: 17 or 18, and The second ITR having the sequence of SEQ ID NO: 15 or 16 on the 3' side of the polycistronic cassette. Inclusion of an ITR in a nucleic acid provided herein enables integration of the nucleic acid into a genome (eg, a porcine genome) via PiggyBac transposon-mediated gene transfer.

[0132] In embodiments, the nucleic acids provided herein comprise a first loxP site on the 5' side of the 5' polycistronic cassette and a second loxP site on the 3' side of the 3' polycistronic cassette. loxP sites are nucleic acid sequences that mediate mitotic recombination by recombinases. Inclusion of a loxP site in a nucleic acid provided herein enables integration of the nucleic acid into a genome (eg, a porcine genome) via recombinase. Maizels et al. describe genome engineering using loxP and recombinases and are hereby incorporated by reference in their entirety: Maizels et al. J Immunol. 2013; 191(1). In an embodiment, the first or the second loxP site has a nucleic acid sequence selected from SEQ ID NO.146-150 and 244. In embodiments, the region between the first loxP site and the second loxP site of a nucleic acid described herein is inserted into the genome by recombination.

[0133] In embodiments, nucleic acids provided herein comprise 5' and 3' guide RNA (gRNA) target sequences (eg, FOS_sg1 or AAVS_sg3 of Figures 5A-5F). gRNA target sequences are recognized by guide RNAs and nucleases such as Cas9. The region between the 5' guide RNA target sequence and the 3' gRNA sequence is cleaved by the guide RNA and nucleases. In embodiments, a nucleic acid moiety comprising a plurality of polycistronic cassettes described herein between the 5' and 3' gRNA target sequences is integrated into a cell, tissue, organ or animal. In an embodiment, a nucleic acid sequence located between a 5' gRNA target sequence and a 3' gRNA target sequence is cloned into a cell, tissue, organ or animal using homology-independent targeted integration (HITI). Suzuki et al. describe in vivo genome editing using HITI and are hereby incorporated by reference in their entirety: Suzuki et al. Nature. 2016 Dec 1;540(7631):144-149. In an embodiment, a nucleic acid sequence located between the 5' and 3' gRNA target sequences is cloned into a cell, tissue, organ or animal using CRISPR-mediated homology-directed repair (HDR). Hsu et al. describe in vivo genome editing using CRISPR-mediated HDR and are hereby incorporated by reference in their entirety: Hsu et al. Cell 157, 1262–1278 (2014). In an embodiment, the 5' gRNA target sequence is FOS (SEQ ID NO: 47) or AAVS (SEQ ID NO: 48). In an embodiment, the 3' gRNA target sequence is FOS (SEQ ID NO: 47) or AAVS (SEQ ID NO: 48).

[0134] Exemplary arrangements of loxP sites, insulator sequences, guide RNA target sequences, and polycistronic cassettes are presented in Table D.

[0135] Form D [arrange() [5'] [arrive] [3'] [)] First ITR first loxP site first insulator sequence polycistronic cassette second insulator sequence second loxP site Second ITR first guide RNA target sequence Second guide RNA target sequence first lox P site first insulator sequence polycistronic cassette second insulator sequence second loxP site Second guide RNA target sequence first guide RNA target sequence first guide RNA target sequence Second guide RNA target sequence First ITR first insulator sequence first loxP site polycistronic cassette second loxP site second insulator sequence Second guide RNA target sequence first guide RNA target sequence Second ITR first loxP site first insulator sequence polycistronic cassette second insulator sequence second loxP site first insulator sequence first loxP site polycistronic cassette second loxP site second insulator sequence first guide RNA target sequence Second guide RNA target sequence First ITR first loxP site first insulator sequence polycistronic cassette second insulator sequence second loxP site Second guide RNA target sequence first guide RNA target sequence Second ITR first guide RNA target sequence Second guide RNA target sequence first insulator sequence first lox P site polycistronic cassette second loxP site second insulator sequence Second guide RNA target sequence first guide RNA target sequence

[0136] In embodiments, the nucleic acids described herein further comprise an initiation codon. In embodiments, the nucleic acids described herein further comprise a stop codon. In the examples, a stop codon is found at the 3' end of each polycistronic cassette. In embodiments, the nucleic acids described herein comprise a Kozak sequence. In an embodiment, the Kozak sequence is the nucleic acid of SEQ ID NO: 59 or 61.

[0137] In embodiments, the nucleic acids described herein comprise one or more ubiquitous chromatin opening elements (UCOEs). Neville et al. describe the UCOE and are incorporated herein by reference in their entirety: Neville et al. Biotechnology Advances 35 (2017) 557-564. In embodiments, the polycistronic cassettes of the nucleic acids described herein are separated by UCOE. In an embodiment, the UCOE is selected from Can 6-3, A2CBX3, CBX3, CBX3* and SRF1. In an embodiment, the UCOE comprises any one of the sequences defined by SEQ ID NO: 19-22, 157 and 193.

[0138] In embodiments, nucleic acids provided herein comprise fluorescent proteins. In an embodiment, the fluorescent protein is located on the 5' side of the 5' polycistronic cassette. In an embodiment, the fluorescent protein is located on the 3' side of the 3' polycistronic cassette. In embodiments, the fluorescent protein is located 5' to the ITR, gRNA target sequence, loxP site, or a combination thereof. In an example, fluorescent proteins are used for negative selection. In an embodiment, the fluorescent protein is green fluorescent protein. In an embodiment, the fluorescent protein is GFP having the nucleic acid sequence of SEQ ID NO: 111. In an embodiment, the fluorescent protein is BFP having the nucleic acid sequence of SEQ ID NO: 242 or SEQ ID NO: 246.

[0139] In embodiments, any of the nucleic acids described herein, or fragments thereof, are integrated into a chromosome. In embodiments, fragments thereof are contiguous portions of nucleic acids described herein, for example, the fragments may be about 20%, about 25%, about 30%, about 35%, about 30% of the nucleotides of any nucleic acid described herein. 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% , about 99% or about 100%. In embodiments, the fragment comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 100% of the nucleotides of any nucleic acid described herein.

[0140] In embodiments, fragments comprising individual components of the nucleic acids described herein are amplified or synthesized from genomic DNA by polymerase chain reaction. Adjacent fragments overlap by at least 50 base pairs at their 5' or 3' ends. The fragments are assembled into full-length nucleic acids using yeast homologous recombination. The plasmid containing the nucleic acid is transformed into E. coli and amplified therein. exemplary nucleic acid

[0141] Exemplary nucleic acids comprising multiple polycistronic cassettes are shown in FIGS. 1A-1D , 2A-2J , 3A-3C , 4A-4H , 5A-5F and 6 . In an embodiment, the nucleic acid comprising a plurality of polycistronic cassettes has a sequence selected from any one of SEQ ID NOs: 23-46, 172-177.

[0142] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The mouse EF1α1 promoter of NO: 134-135, and the hsHBB poly A (“hsHBB pA” or “HBB pA”) sequence selected from SEQ ID NO. 114-116 and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO. ID NOs: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NOs: 94-96, 103 and 187, (b) a complement regulation box, the complement regulation box comprising: the first cistron encoding CD46 , the second cistron encoding CD59, the human EF1α1 promoter selected from SEQ ID NO: 130-132, the btGH pA sequence selected from any one of SEQ ID NO. 112, 120-122, 159 and 190, and combinations thereof , wherein the nucleotide sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86, 108; and an innate immunity box, said innate immunity box comprising: encoding B2M HLA - the first cistron of the E fusion protein, the second cistron encoding CD47, the CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB selected from SEQ ID NO. 123-125 and 154-156 Poly A sequences and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180. In an embodiment, CD46 is CD46_LL comprising the nucleic acid sequence of SEQ ID NO: 71. In embodiments, the UCOE separates the complement regulatory cassette from the innate immunity cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20.

[0143] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The mouse EF1α1 promoter of NO: 134-135, and the hsHBB poly A (“hsHBB pA” or “HBB pA”) sequence selected from SEQ ID NO. 114-116 and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO. ID NOs: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NOs: 94-96, 103 and 187, (b) a complement regulation box, the complement regulation box comprising: the first cistron encoding CD46 , the second cistron encoding CD59, the human EF1α1 promoter selected from SEQ ID NO: 130-132, the btGH pA sequence selected from any one of SEQ ID NO. 112, 120-122, 159 and 190, and combinations thereof , wherein the nucleotide sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86, 108; and an innate immunity box, said innate immunity box comprising: encoding B2M HLA - the first cistron of the E fusion protein, the second cistron encoding CD47, the CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB selected from SEQ ID NO. 123-125 and 154-156 Poly A sequences and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180. In embodiments, the UCOE separates the complement regulatory cassette from the innate immunity cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20.

[0144] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The mouse EF1α1 promoter of NO: 134-135, and the hsHBB poly A (“hsHBB pA” or “HBB pA”) sequence selected from SEQ ID NO. 114-116 and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO. ID NOs: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NOs: 94-96, 103 and 187, (b) a complement regulation box, the complement regulation box comprising: the first cistron encoding CD46 , the second cistron encoding CD59, the human EF1α1 promoter selected from SEQ ID NO: 130-132, the btGH pA sequence selected from any one of SEQ ID NO: 112, 120-122, 159 and 190, and combinations thereof , wherein the nucleotide sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86, 108; and innate immunity and inflammation and apoptosis box, the innate immunity and inflammation and apoptosis cassettes comprising: the first cistron encoding the B2M HLA-E fusion protein, the second cistron encoding CD47, the third cistron encoding A20, the fourth cistron encoding HO1, The CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB polyA sequence selected from SEQ ID NO. 123-125 and 154-156 and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein Selected from SEQ ID NO:62,66,105, wherein the nucleotide sequence of CD47 is selected from SEQ ID NO:77-83 and 180, wherein the nucleotide sequence of A20 is selected from SEQ ID NO:64-65,104,182 and 188, Wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189. In an embodiment, an IRES (SEQ ID NO: 11) separates the second and third cistrons of the innate immunity and inflammation and apoptosis cassettes. In an embodiment, the UCOE separates the complement regulatory cassette from the innate immunity and inflammation and apoptosis cassettes. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20.

[0145] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39 Tricistronic, mouse EF1α1 promoter selected from SEQ ID NOs: 134-135, and hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from SEQ ID NOs. 114-116, and combinations thereof , wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, and wherein the nucleotide sequence of CD39 is selected from SEQ ID NO: 67- 70, (b) a complement regulation cassette, the complement regulation cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a third cistron encoding CD55, selected from SEQ ID NO: 130 -132 human EF1α1 promoter, btGH pA sequence selected from any one of SEQ ID NO: 112, 120-122, 159 and 190 and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86 and 108, wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; and innate immunity and inflammation and apoptosis box, said The innate immunity and inflammation and apoptosis cassette contains: the first cistron encoding the B2M HLA-E fusion protein, the second cistron encoding CD47, the third cistron encoding A20, the fourth cistron encoding HO1 A promoter, a fifth cistron encoding PD-L1, a CAG promoter selected from SEQ ID NO: 126-128, and a SPAActB polyA sequence selected from SEQ ID NO. 123-125 and 154-156, and combinations thereof, Wherein the nucleotide sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleotide sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 77-83 and 180, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: ID NO: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89-91 . In an embodiment, an IRES (SEQ ID NO: 11) separates the second and third cistrons of the innate immunity and inflammation and apoptosis cassettes. In an embodiment, the UCOE separates the complement regulatory cassette from the innate immunity and inflammation and apoptosis cassettes. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20.

[0146] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The mouse EF1α1 promoter of NO: 134-135, and the hsHBB poly A (“hsHBB pA” or “HBB pA”) sequence selected from SEQ ID NO. 114-116 and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO. ID NOs: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NOs: 94-96, 103 and 187, (b) a complement regulation box, the complement regulation box comprising: the first cistron encoding CD46 , the second cistron encoding CD59, the human EF1α1 promoter selected from SEQ ID NO: 130-132, the btGH pA sequence selected from any one of SEQ ID NO. 112, 120-122, 159 and 190, and combinations thereof , wherein the nucleotide sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86, 108; and an innate immunity box, said innate immunity box comprising: encoding B2M HLA - the first cistron of the E fusion protein, the second cistron encoding CD47, the CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB selected from SEQ ID NO. 123-125 and 154-156 Poly A sequences and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180. In an embodiment, CD46 is CD46_LL comprising the nucleic acid sequence of SEQ ID NO: 71. In embodiments, the UCOE separates the complement regulatory cassette from the innate immunity cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the UCOE located on the 5' or 3' side of the coagulation cassette is an SRF having the sequence of SEQ ID NO: 22.

[0147] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39 Tricistronic, mouse EF1α1 promoter selected from SEQ ID NOs: 134-135, and hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from SEQ ID NOs. 114-116, and combinations thereof , wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, and wherein the nucleotide sequence of CD39 is selected from SEQ ID NO: 67- 70, (b) a complement regulation cassette, the complement regulation cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a third cistron encoding CD55, selected from SEQ ID NO: 136 The ssEF1α1 promoter of -138, the btGH pA sequence selected from any one of SEQ ID NOs. 112, 120-122, 159 and 190, and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOs: 71-76, 185 and 200, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86, 108, wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; and innate immunity and inflammation and apoptosis box, the innate The immune and inflammation and apoptosis cassette contains: the first cistron encoding B2M HLA-E fusion protein, the second cistron encoding CD47, the third cistron encoding A20, the fourth cistron encoding HO1 , the fifth cistron encoding PD-L1, the CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB polyA sequence selected from SEQ ID NO. 123-125 and 154-156 and combinations thereof, wherein The nucleic acid sequence of coding described B2M HLA-E fusion protein is selected from SEQ ID NO:62,66,105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO:77-83 and 180, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89-91. In an embodiment, an IRES (SEQ ID NO: 11) separates the second and third cistrons of the innate immunity and inflammation and apoptosis cassettes. In an embodiment, the UCOE separates the complement regulatory cassette from the innate immunity and inflammation and apoptosis cassettes. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3 UCOE having the sequence of SEQ ID NO: 21. In embodiments, the UCOE is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the UCOE located on the 5' or 3' side of the coagulation cassette is a Can 6-3 UCOE having the sequence of SEQ ID NO: 19. In an embodiment, CD46 is CD46i having a nucleic acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75.

[0148] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39 a tricistronic, ssUBC promoter having the sequence of SEQ ID NO: 141, and an hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191, And combination thereof, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, wherein the nucleotide sequence of CD39 is selected from SEQ ID NO : 67-70, (b) complement regulation box, described complement regulation box comprises: the first cistron of coding CD46, the second cistron of coding CD59, the 3rd cistron of coding CD55, selected from SEQ ID The ssEF1α1 promoter of NO: 136-138, the btGH pA sequence selected from SEQ ID NO. 112, 120-122, 159, 190 and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86, 108, wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; and innate immunity and inflammation and apoptosis box, the innate The immune and inflammation and apoptosis cassette contains: the first cistron encoding B2M HLA-E fusion protein, the second cistron encoding CD47, the third cistron encoding A20, the fourth cistron encoding HO1 , the fifth cistron encoding PD-L1, the CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB polyA sequence selected from SEQ ID NO. 123-125 and 154-156 and combinations thereof, wherein The nucleic acid sequence of coding described B2M HLA-E fusion protein is selected from SEQ ID NO:62,66,105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO:77-83 and 180, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89-91. In an embodiment, an IRES (SEQ ID NO: 11) separates the second and third cistrons of the innate immunity and inflammation and apoptosis cassettes. In an embodiment, the UCOE separates the complement regulatory cassette from the innate immunity and inflammation and apoptosis cassettes. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3 UCOE having the sequence of SEQ ID NO: 21. In an embodiment, CD46 is CD46i having a nucleic acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75.

[0149] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding PROCR, having a SEQ ID NO : the ssUBC promoter of the sequence 19-22, 157 or 193, and the hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, Wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of PROCR is selected from SEQ ID NO: 92, 93 and 181, (b) inflammation and apoptosis box, said inflammation and apoptosis The dead box comprises: a first cistron encoding A20, a second cistron encoding HO1, a ssEF1α1 promoter selected from SEQ ID NOs: 136-138 and 179, a promoter selected from SEQ ID NOs: 112, 120-122, The btGH pA sequences of 159, 190 and combinations thereof, wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; and a complement regulation and innate immunity cassette comprising: a first cistron encoding CD46, a second cistron encoding CD47, a third cistron encoding CD55, selected from The CAG promoter of SEQ ID NO:126-128, and be selected from the SPAActB polyA sequence of SEQ ID NO.123-125 and 154-156 and combination thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO:71-76, 185 and 200, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184. In an embodiment, the UCOE separates the inflammation and apoptosis cassette from the complement regulation and innate immunity cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3 UCOE having the sequence of SEQ ID NO: 21. In an embodiment, CD46 is CD46i having a nucleic acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75.

[0150] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding PROCR, a third cistron encoding TFPI a tricistronic, an ssUBC promoter having the sequence of SEQ ID NO: 141, and an hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and Its combination, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, and wherein the nucleotide sequence of PROCR is selected from SEQ ID NO: 92,93 and 181, and wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94- 96, 103 and 187, (b) an innate immunity cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, selected from the group consisting of SEQ ID NO: 139 -140 of the ssHSPA8 promoter, and btGH pA sequences selected from SEQ ID NO.112, 120-122, 159, 190 and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; and (c) a complement regulation box, the complement regulation box comprising: the first cistron encoding CD46, the first cistron encoding CD55 The second cistron, the CAG promoter selected from SEQ ID NO: 126-128, and the hsGH poly A sequence having the sequence of SEQ ID NO: 113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184. In embodiments, the UCOE separates the innate immunity cassette from the complement regulatory cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3 UCOE having the sequence of SEQ ID NO: 21. In embodiments, poly A is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the polyA is SV40 pA having the sequence of SEQ ID NO: 119. In an embodiment, CD46 is CD46da having the sequence of SEQ ID NO: 73 or SEQ ID NO: 72. In an embodiment, THBD is THBDda having a sequence selected from SEQ ID NO: 99-102. In embodiments, the B2M HLA-E fusion protein comprises an HLA-G epitope. In an embodiment, CD47 is CD47-2 having the sequence of SEQ ID NO: 80, 81 or 83 (eg NM_198793). In embodiments, poly A is located on the 5' or 3' side of the complement regulatory cassette. In an embodiment, the poly A located on the 5' or 3' side of the complement regulatory cassette is SPAActB pA having the sequence of SEQ ID NO: 118.

[0151] In some embodiments, provided herein are nucleic acids comprising: (a) an innate immunity cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding a CD47 An antitron, an ssUBC promoter having the sequence of SEQ ID NO: 141, and an hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191, and combinations thereof , wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, (b) coagulation box, the The coagulation box comprises: a first cistron encoding THBD, a second cistron encoding PROCR, a third cistron encoding TFPI, a hsHSPA8 promoter having the sequence of SEQ ID NO: 133, and a cistron selected from SEQ ID NO: 133. The btGH pA sequence of NO.112,120-122,159,190 and combination thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO:97-102 and 166, wherein the nucleic acid sequence of PROCR is selected from SEQ ID NO:92,93 and 181, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; and (c) a complement regulation box, the complement regulation box comprising: the first cistron encoding CD46, the second cistron encoding CD55 Dicistronic, CAG promoter selected from SEQ ID NO: 126-128, and hsGH poly A sequence having the sequence of SEQ ID NO: 113 and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71- 76, 185 and 200, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184. In embodiments, the UCOE separates the innate immunity cassette from the complement regulatory cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In embodiments, poly A is located on the 5' or 3' side of the innate immunity cassette. In an embodiment, the polyA is SV40 pA having the sequence of SEQ ID NO: 119. In an embodiment, CD46 is CD46da having the sequence of SEQ ID NO: 73 or SEQ ID NO: 72. In an embodiment, THBD is THBDda having a sequence selected from SEQ ID NO: 99-102. In embodiments, the B2M HLA-E fusion protein comprises an HLA-G epitope. In an embodiment, CD47 is CD47-2 having the sequence of SEQ ID NO: 80, 81 or 83 (eg NM_198793). In embodiments, poly A is located on the 5' or 3' side of the complement regulatory cassette. In an embodiment, the poly A located on the 5' or 3' side of the complement regulatory cassette is SPAActB pA having the sequence of SEQ ID NO: 118. In an embodiment, the nucleic acid comprises a gRNA target sequence selected from SEQ ID NO: 47-48.

[0152] In some embodiments, provided herein are nucleic acids comprising: (a) an innate immunity cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding a CD47 An antitron, an ssUBC promoter having the sequence of SEQ ID NO: 141, and an hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191, and combinations thereof , wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, (b) coagulation box, the The coagulation box comprises: a first cistron encoding THBD, a second cistron encoding PROCR, a third cistron encoding TFPI, a hsHSPA8 promoter having the sequence of SEQ ID NO: 133, and a cistron selected from SEQ ID NO: 133. The btGH pA sequence of NO.112,120-122,159,190 and combination thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO:97-102 and 166, wherein the nucleic acid sequence of PROCR is selected from SEQ ID NO:92,93 and 181, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; and (c) a complement regulation box, the complement regulation box comprising: the first cistron encoding CD46, the second cistron encoding CD55 Dicistronic, CAG promoter selected from SEQ ID NO: 126-128, and hsGH poly A sequence having the sequence of SEQ ID NO: 113 and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71- 76, 185 and 200, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184. In embodiments, the UCOE separates the innate immunity cassette from the complement regulatory cassette. In an embodiment, UCOE has a sequence selected from SEQ ID NO: 19-22 and 157. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In embodiments, poly A is located on the 5' or 3' side of the innate immunity cassette. In an embodiment, the polyA is SV40 pA having the sequence of SEQ ID NO: 119. In an embodiment, CD46 is CD46da having the sequence of SEQ ID NO: 73 or SEQ ID NO: 72. In an embodiment, THBD is THBDda having a sequence selected from SEQ ID NO: 99-102. In embodiments, the B2M HLA-E fusion protein comprises an HLA-G epitope. In an embodiment, CD47 is CD47-2 having the sequence of SEQ ID NO: 80, 81 or 83 (eg NM_198793). In embodiments, poly A is located on the 5' or 3' side of the complement regulatory cassette. In an embodiment, the poly A located on the 5' or 3' side of the complement regulatory cassette is SPAActB pA having the sequence of SEQ ID NO: 118. In an embodiment, the nucleic acid comprises a gRNA target sequence selected from SEQ ID NO: 47-48.

[0153] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding PROCR, a third cistron encoding TFPI a tricistronic, an ssUBC promoter having the sequence of SEQ ID NO: 141, and an hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and Its combination, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, and wherein the nucleotide sequence of PROCR is selected from SEQ ID NO: 92,93 and 181, and wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94- 96, 103 and 187, (b) an innate immunity cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, selected from the group consisting of SEQ ID NO: 136 -138 and 179 ssEF1α1 promoters, and btGH pA sequences selected from SEQ ID NO.112, 120-122, 159, 190 and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO. NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; and (c) a complement regulation box, which comprises: the first cistron encoding CD46, the first cistron encoding The second cistron of CD55, the CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB polyA sequence selected from SEQ ID NO. 123-125 and 154-156 and combinations thereof, wherein the nucleic acid sequence of CD46 Selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184. In embodiments, the UCOE separates the innate immunity cassette from the complement regulatory cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3 UCOE having the sequence of SEQ ID NO: 21. In embodiments, poly A is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the polyA is SV40 pA having the sequence of SEQ ID NO: 119. In an embodiment, CD46 is CD46da having the sequence of SEQ ID NO: 73 or SEQ ID NO: 72. In an embodiment, THBD is THBDda having a sequence selected from SEQ ID NO: 99-102. In embodiments, the B2M HLA-E fusion protein comprises an HLA-G epitope. In an embodiment, CD47 is CD47-2 having the sequence of SEQ ID NO: 80, 81 or 83 (eg NM_198793). In embodiments, poly A is located on the 5' or 3' side of the complement regulatory cassette. In an embodiment, the poly A located on the 5' or 3' side of the complement regulatory cassette is SPAActB pA having the sequence of SEQ ID NO: 118. In an embodiment, the nucleic acid comprises a gRNA target sequence selected from SEQ ID NO: 47-48.

[0154] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The mouse EF1α1 promoter of NO: 134-135, and the hsHBB poly A (“hsHBB pA” or “HBB pA”) sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, wherein THBD The nucleic acid sequence is selected from SEQ ID NO: 97-102 and 166, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, (b) a complement regulation box, the complement regulation box comprising: coding CD46 The first cistron, the second cistron encoding CD55, the human EF1α1 promoter selected from SEQ ID NO: 130-132, the btGH pA sequence selected from SEQ ID NO. 112, 120-122, 159, 190, and Its combination, wherein the nucleotide sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; And innate immunity box, described innate immunity The cassette comprises: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a CAG promoter selected from SEQ ID NO: 126-128, and a cistron selected from SEQ ID NO. 123-125 and the SPAActB polyA sequences of 154-156 and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77 -83 and 180. In embodiments, the UCOE separates the complement regulatory cassette from the innate immunity cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the UCOE located on the 5' or 3' side of the coagulation cassette is an SRF having the sequence of SEQ ID NO: 22. In an embodiment, CD46 is CD46i having a nucleic acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75.

[0155] In some embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising a first cistron encoding THBD, a second cistron encoding PROCR, selected from the group consisting of SEQ ID The mouse EF1α1 promoter of NO: 134-135, and the hsHBB poly A (“hsHBB pA” or “HBB pA”) sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, wherein THBD The nucleic acid sequence is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of PROCR is selected from SEQ ID NO: 92, 93 and 181, (b) a complement regulation box, the complement regulation box comprising: the first coding CD46 A cistron, a second cistron encoding CD55, a human EF1α1 promoter selected from SEQ ID NO: 130-132, a btGH pA sequence selected from SEQ ID NO. 112, 120-122, 159, 190, and combinations thereof , wherein the nucleotide sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; and (c) innate immunity and inflammation and apoptosis The innate immunity, inflammation and apoptosis cassettes include: the first cistron encoding B2M HLA-E fusion protein, the second cistron encoding CD47, the third cistron encoding A20, the encoding HO1 The fourth cistron of , the CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB polyA sequence selected from SEQ ID NO. 123-125 and 154-156 and combinations thereof, wherein the B2M HLA is encoded -The nucleotide sequence of the E fusion protein is selected from SEQ ID NO: 62,66,105, wherein the nucleotide sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, and wherein the nucleotide sequence of A20 is selected from SEQ ID NO: 64-65 , 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 1895. In an embodiment, an IRES having the sequence of SEQ ID NO: 11 separates the B2M HLA-E fusion protein and CD47 from A20 and HO1. In some embodiments, the UCOE separates the complement regulatory cassette from the innate immunity cassette. In an embodiment, UCOE has a sequence selected from SEQ ID NO: 19-22 and 157. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22 and 157 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the UCOE located on the 5' or 3' side of the coagulation cassette is an SRF having the sequence of SEQ ID NO: 22. In an embodiment, CD46 is CD46i having a nucleic acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75.

[0156] In some embodiments, provided herein are nucleic acids comprising: (a) an innate immunity cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding a CD47 An antitron, a mouse EF1α1 promoter selected from SEQ ID NOs: 134-135, and a hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, (b) blood coagulation A box comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39, a human EF1α1 promoter selected from SEQ ID NO: 130-132, btGH pA sequences selected from SEQ ID NO: 112, 120-122, 159, 190 and combinations thereof, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO : 94-96, 103 and 187, wherein the nucleotide sequence of CD39 is selected from SEQ ID NO: 67-70; and (c) complement regulation and inflammation and apoptosis box, said complement regulation and inflammation and apoptosis box comprises : the first cistron encoding CD46, the second cistron encoding CD59, the third cistron encoding CD55, the fourth cistron encoding A20, the fifth cistron encoding HO1, and the encoding PD-L1 The sixth cistron, the CAG promoter selected from SEQ ID NO: 126-128, and the SPAActB polyA sequence selected from SEQ ID NO. 123-125 and 154-156 and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from From SEQ ID NO: 71-76, 185 and 200, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO: 86, 108, wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184, wherein A20 The nucleic acid sequence is selected from SEQ ID NO: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89-91. In an embodiment, an IRES having the sequence of SEQ ID NO: 11 separates CD46, CD55 and CD59 from A20, HO1 and PD-L1. In some embodiments, the UCOE separates the complement regulatory cassette from the innate immunity cassette. In an embodiment, UCOE has a sequence selected from SEQ ID NO: 19-22 and 157. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22 and 157 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the UCOE located on the 5' or 3' side of the coagulation cassette is an SRF having the sequence of SEQ ID NO: 22. In an embodiment, CD46 is CD46i having a nucleic acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75.

[0157] In some embodiments, provided herein are nucleic acids comprising: (a) an innate immunity cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding a CD47 An antitron, an ssUBC promoter having the sequence of SEQ ID NO: 141, and an hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191, and combinations thereof , wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, (b) coagulation box, the The coagulation box comprises: a first cistron encoding THBD, a second cistron encoding TFPI, an hsHSPA8 promoter selected from SEQ ID NO: 133, an hsHSPA8 promoter selected from SEQ ID NO. 112, 120-122, 159, 190 The btGH pA sequence and combination thereof, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; and (c) complement regulation 123-123- The SPAActB polyA sequences of 125 and 154-156 and combinations thereof, wherein the nucleotide sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, and wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184. In embodiments, the UCOE separates the complement regulation cassette from the coagulation cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the innate immunity cassette. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the innate immunity cassette. In an embodiment, the UCOE located on the 5' or 3' side of the innate immunity cassette is a SRF having the sequence of SEQ ID NO: 22. In an embodiment, CD46 is CD46i having a nucleic acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75.

[0158] In some embodiments, provided herein are nucleic acids comprising: (a) an innate immunity cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding a CD47 An antitron, an ssUBC promoter having the sequence of SEQ ID NO: 141, and an hsHBB poly A ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191, and combinations thereof , wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, (b) coagulation box, the The coagulation box comprises: a first cistron encoding THBD, a second cistron encoding TFPI, an hsHSPA8 promoter selected from SEQ ID NO: 133, an hsHSPA8 promoter selected from SEQ ID NO. 112, 120-122, 159, 190 The btGH pA sequence and combination thereof, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187; and (c) complement regulation and inflammation and apoptosis box, said complement regulation and inflammation and apoptosis box comprises: the first cistron encoding CD46, the second cistron encoding CD55, the third cistron encoding A20, the encoding HO1 The fourth cistron of , the CAG promoter selected from SEQ ID NO: 126-128, and the hsGH poly A sequence having the sequence of SEQ ID NO: 113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76 , 185 and 200, wherein the nucleotide sequence of CD55 is selected from SEQ ID NO:84,85,107 and 184, wherein the nucleotide sequence of A20 is selected from SEQ ID NO:64-65,104,182 and 188, wherein the nucleotide sequence of HO1 selected from SEQ ID NO: 63, 109, 165, 183 and 189. In an embodiment, an IRES having the sequence of SEQ ID NO: 11 separates CD46 and CD55 from A20 and HO1. In embodiments, the UCOE separates the complement regulation cassette from the coagulation cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22, 157 and 193 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the UCOE located on the 5' or 3' side of the coagulation cassette is an SRF having the sequence of SEQ ID NO: 22. In an embodiment, CD46 is CD46da having a nucleic acid sequence selected from SEQ ID NO: 72 or SEQ ID NO: 73.

[0159] In an embodiment, provided herein are nucleic acids comprising: (a) an innate immunity cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding a CD47 promoter, the ssUBC promoter having the sequence of SEQ ID NO: 141, and the hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, Wherein the nucleotide sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleotide sequence of CD47 is selected from SEQ ID NO: 77-83 and 180, (b) coagulation box, the The coagulation box comprises: the first cistron encoding THBD, the second cistron encoding TFPI, the third cistron encoding CD39, the hsHSPA8 promoter selected from SEQ ID NO: 133, the hsHSPA8 promoter selected from SEQ ID NO: 112 , btGH pA sequences of 120-122, 159, 190 and combinations thereof, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, wherein the nucleotide sequence of CD39 is selected from SEQ ID NO: 67-70; and (c) complement regulation and inflammation and apoptosis box, said complement regulation and inflammation and apoptosis box comprises: the first sequence encoding CD46 The second cistron encoding CD55, the third cistron encoding CD59, the fourth cistron encoding A20, the fifth cistron encoding HO1, the sixth cistron encoding PD-L1, A CAG promoter selected from SEQ ID NO: 126-128, and a hsGH polyA sequence having the sequence of SEQ ID NO: 113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein CD55 The nucleotide sequence of A20 is selected from SEQ ID NO:84,85,107 and 184, wherein the nucleotide sequence of CD59 is selected from SEQ ID NO:86 and SEQ ID NO:108, wherein the nucleotide sequence of A20 is selected from SEQ ID NO:64-65 , 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89-91. In an embodiment, an IRES having the sequence of SEQ ID NO: 11 separates CD46, CD55 and CD59 from A20, HO1 and PD-L1. In embodiments, the UCOE separates the complement regulation cassette from the coagulation cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO: 20. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22 and 157 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, a UCOE having a sequence selected from SEQ ID NO: 19-22 and 157 is located on the 5' or 3' side of the coagulation cassette. In an embodiment, the UCOE located on the 5' or 3' side of the coagulation cassette is an SRF having the sequence of SEQ ID NO: 22. In an embodiment, CD46 is CD46da having a nucleic acid sequence selected from SEQ ID NO: 72 or SEQ ID NO: 73.

[0160] In an embodiment, provided herein are nucleic acids comprising: (a) an innate immunity cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding a CD47 promoter, the ssUBC promoter having the sequence of SEQ ID NO: 141, and the hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, Wherein the nucleotide sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleotide sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (b) coagulation box, the The coagulation box comprises: a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39, a ssHSPA8 promoter selected from SEQ ID NO: 142-145, a promoter selected from SEQ ID NO : btGH pA sequences of 112, 120-122, 159, 190 and combinations thereof, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, wherein the nucleotide sequence of CD39 is selected from SEQ ID NO: 67-70; and (c) complement regulation and inflammation and apoptosis box, said complement regulation and inflammation and apoptosis box comprises: the first CD46 encoding One cistron, second cistron encoding CD55, third cistron encoding CD59, fourth cistron encoding A20, fifth cistron encoding HO1, sixth cistron encoding PD-L1 A promoter, a CAG promoter selected from SEQ ID NO: 126-128, and a hsGH poly A sequence with the sequence of SEQ ID NO: 113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, Wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NO: 86 and SEQ ID NO: 108, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO: 64 -65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89-91. In an embodiment, an IRES having the sequence of SEQ ID NO: 11 separates CD46, CD55 and CD59 from A20, HO1 and PD-L1. In embodiments, the UCOE separates the complement regulation cassette from the coagulation cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3*UCOE having the sequence of SEQ ID NO: 21. In embodiments, the nucleic acid comprises a polyA sequence on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence located at the 5' or 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 119 or SEQ ID NO: 162.

[0161] In an embodiment, the following nucleic acid is provided herein, the nucleic acid comprising: (a) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The ssHSPA8 promoter of NO: 142-145, the SPA PTCH2 pA sequence with the sequence of SEQ ID NO: 124, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, (b) innate immune box, said innate immune box comprises the first cistron encoding B2M HLA-E fusion protein, the second cistron encoding CD47, having SEQ ID NO The ssUBC promoter of the sequence : 141, and the hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, wherein the B2M HLA is encoded -The nucleic acid sequence of the fusion protein is selected from SEQ ID NO: 62,66,105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (c) cellular immunity and coagulation box, said cellular immunity and The coagulation cassette comprises a first cistron encoding CTLA-4 (eg, LEA29Y), a second cistron encoding CD39, a ssEF1α1 promoter selected from SEQ ID NOs: 136-138 and 179, and a cistron having SEQ ID NOS: 112 , 120-122, 159 and 190 of the btGH poly A sequence, wherein the nucleic acid sequence of CTLA-4 is selected from SEQ ID NO: 87-88 and 186, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NO: 67-70; And (d) a complement regulation box, said complement regulation box comprising a first cistron encoding CD46, a second cistron encoding CD55, a CAG promoter selected from SEQ ID NO: 126-128, and a cistron having SEQ ID NO: 126-128 The hsGH polyA sequence of NO: 113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, and the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184. In embodiments, the nucleic acid comprises a polyA sequence on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence located at the 5' or 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 119. In embodiments, the nucleic acid comprises a fluorescent protein located on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the fluorescent protein located on the 5' or 3' side of all polycistronic cassettes is green fluorescent protein having the sequence of SEQ ID NO: 111. In an embodiment, CD46 is CD46da having the nucleic acid sequence of SEQ ID NO: 72. In an embodiment, THBD is THBDda having a sequence selected from SEQ ID NO. 99-102. In an embodiment, TFPI comprises TFPI and a CD4 transmembrane domain. In an embodiment, TFPI has the sequence of SEQ ID NO: 94. In an embodiment, the UCOE separates the complement regulation cassette from the cellular immunity and coagulation cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3*UCOE having the sequence of SEQ ID NO: 21.

[0162] In an embodiment, the following nucleic acid is provided herein, the nucleic acid comprising: (a) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The ssHSPA8 promoter of NO: 142-145, the SPA PTCH2 pA sequence with the sequence of SEQ ID NO: 124, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, (b) innate immune box, said innate immune box comprises the first cistron encoding B2M HLA-E fusion protein, the second cistron encoding CD47, having SEQ ID NO The ssUBC promoter of the sequence : 141, and the hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, wherein the B2M HLA is encoded The nucleic acid sequence of -E fusion protein is selected from SEQ ID NO: 62,66,105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (c) inflammation and apoptosis box, described inflammation and The apoptosis cassette comprises a first cistron encoding A20, a second cistron encoding PD-L1, a ssEF1α1 promoter selected from SEQ ID NOs: 136-138 and 179, and a cistron having SEQ ID NOs: 112, 120 - the btGH poly A sequence of the sequence of 122, 159, 190, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NO: 89- 91; and (d) a complement regulatory cassette comprising a first cistron encoding CD46, a second cistron encoding CD55, a CAG promoter selected from SEQ ID NOs: 126-128, and having The hsGH poly A sequence of the sequence of SEQ ID NO: 113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184 . In embodiments, the nucleic acid comprises a polyA sequence on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence located at the 5' or 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 119. In embodiments, the nucleic acid comprises a fluorescent protein located on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the fluorescent protein located on the 5' or 3' side of all polycistronic cassettes is green fluorescent protein having the sequence of SEQ ID NO: 111. In an embodiment, CD46 is CD46da having the nucleic acid sequence of SEQ ID NO: 72. In an embodiment, THBD is THBDda having a sequence selected from SEQ ID NO. 99-102. In an embodiment, TFPI comprises TFPI and a CD4 transmembrane domain. In an embodiment, TFPI has the sequence of SEQ ID NO: 94. In an embodiment, the UCOE separates the complement regulatory cassette from the inflammation and apoptosis cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3*UCOE having the sequence of SEQ ID NO: 21.

[0163] In an embodiment, the following nucleic acid is provided herein, the nucleic acid comprising: (a) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The ssHSPA8 promoter of NO: 142-145, the SPA PTCH2 pA sequence with the sequence of SEQ ID NO: 124, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, (b) innate immune box, said innate immune box comprises the first cistron encoding B2M HLA-E fusion protein, the second cistron encoding CD47, having SEQ ID NO The ssUBC promoter of the sequence : 141, and the hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, wherein the B2M HLA is encoded The nucleic acid sequence of -E fusion protein is selected from SEQ ID NO: 62,66,105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (c) apoptosis and coagulation box, said apoptosis The death and coagulation cassette comprises a first cistron encoding XIAP, (ii) a second cistron encoding CD39, a ssEF1α1 promoter selected from SEQ ID NOs: 136-138 and 179, and a cistron selected from SEQ ID NO: 136-138 and 179. 112, 120-122, 159, 190 in the btGH poly A sequence of any one sequence, wherein the nucleic acid sequence of XIAP is selected from SEQ ID NO: 110, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NO: 106, and (d) A complement regulatory cassette comprising a first cistron encoding CD46, a second cistron encoding CD55, a CAG promoter selected from SEQ ID NOs: 126-128, and a cistron with SEQ ID NO: 113 The hsGH polyA sequence of sequence, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; And in the embodiment , the nucleic acid comprises a polyA sequence positioned at the 5' or 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence located at the 5' or 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 119. In embodiments, the nucleic acid comprises a fluorescent protein located on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the fluorescent protein located on the 5' or 3' side of all polycistronic cassettes is green fluorescent protein having the sequence of SEQ ID NO: 111. In an embodiment, CD46 is CD46da having the nucleic acid sequence of SEQ ID NO: 72. In an embodiment, THBD is THBDda having a sequence selected from SEQ ID NO. 99-102. In an embodiment, TFPI comprises TFPI and a CD4 transmembrane domain. In an embodiment, TFPI has the sequence of SEQ ID NO: 94. In an embodiment, the UCOE separates the complement regulatory cassette from the apoptosis and coagulation cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3*UCOE having the sequence of SEQ ID NO: 21.

[0164] In an embodiment, the following nucleic acid is provided herein, the nucleic acid comprising: (a) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, selected from the group consisting of SEQ ID The ssHSPA8 promoter of NO: 142-145, the SPA PTCH2 pA sequence with the sequence of SEQ ID NO: 124, wherein the nucleotide sequence of THBD is selected from SEQ ID NO: 97-102 and 166, wherein the nucleotide sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187, (b) innate immune box, said innate immune box comprises the first cistron encoding B2M HLA-E fusion protein, the second cistron encoding CD47, having SEQ ID NO The ssUBC promoter of the sequence : 141, and the hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, wherein the B2M HLA is encoded -The nucleic acid sequence of the fusion protein is selected from SEQ ID NO: 62,66,105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (c) cell apoptosis and cellular immunity box, the cell The apoptosis and cellular immunity cassette comprises a first cistron encoding A20, a second cistron encoding CTLA-4 (eg, LEA29Y), a ssEF1α1 promoter selected from SEQ ID NOs: 136-138 and 179, and a cistron with SEQ ID NO: 136-138 and 179. The btGH poly A sequence of any one of ID NO:112,120-122,159 and 190, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO:64-65,104,182 and 188, wherein the nucleic acid sequence of CTLA-4 The sequence is selected from SEQ ID NO: 87-88 and 186; and (d) a complement regulatory cassette comprising a first cistron encoding CD46, a second cistron encoding CD55, selected from SEQ ID NO : 126-128 CAG promoter, and hsGH poly A sequence with the sequence of SEQ ID NO: 113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184. In embodiments, the nucleic acid comprises a polyA sequence on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence located at the 5' or 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 119. In embodiments, the nucleic acid comprises a fluorescent protein located on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the fluorescent protein located on the 5' or 3' side of all polycistronic cassettes is green fluorescent protein having the sequence of SEQ ID NO: 111. In an embodiment, CD46 is CD46da having the nucleic acid sequence of SEQ ID NO: 72. In an embodiment, THBD is THBDda having a sequence selected from SEQ ID NO. 99-102. In an embodiment, TFPI comprises TFPI and a CD4 transmembrane domain. In an embodiment, TFPI has the sequence of SEQ ID NO: 94. In an embodiment, the UCOE separates the complement regulatory cassette from the apoptosis and cellular immunity cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3*UCOE having the sequence of SEQ ID NO: 21.

[0165] In an embodiment, provided herein are nucleic acids comprising: (a) an innate immunity cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding a CD47 promoter, the ssUBC promoter having the sequence of SEQ ID NO: 141, and the hsHBB polyA ("hsHBB pA" or "HBB pA") sequence selected from any one of SEQ ID NOs. 114-116 and 191 and combinations thereof, Wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (b) inflammation and apoptosis A cassette, the inflammation and apoptosis cassette comprising: a first cistron encoding A20, a second cistron encoding HO1, a third cistron encoding PD-L1, selected from SEQ ID NO: 139-140 , the ssHSPA8 promoter of 142-145, and the btGH poly A sequence having a sequence selected from any one of SEQ ID NO: 112, 120-122, 159, 190, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO: 64- 65, 104, 182 and 188, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189, wherein the nucleotide sequence of PD-L1 is selected from SEQ ID NO: 89-91; (c) complement A regulatory cassette comprising a first cistron encoding CD46, a second cistron encoding CD55, a third cistron encoding CD59, a CAG promoter selected from SEQ ID NO: 126-128, And the btHBB polyA sequence with the sequence of SEQ ID NO: 160, wherein the nucleotide sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200, and wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NO: 86 and SEQ ID NO: 108; and (d) a coagulation box, which comprises: the first cistron encoding THBD, the second cistron encoding TFPI Antitron, the ssICAM2 promoter with the sequence of SEQ ID NO: 167, and the hsGH poly A sequence with the sequence of SEQ ID NO: 113 or SEQ ID NO: 161, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO: 97- 102 and 166, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO: 94-96, 103 and 187. In an embodiment, TFPI has the sequence of SEQ ID NO: 96. In an embodiment, the UCOE separates the complement regulatory cassette from the inflammation and apoptosis cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In an embodiment, the UCOE is a CBX3*UCOE having the sequence of SEQ ID NO: 21. In embodiments, the nucleic acid comprises a polyA sequence on the 5' or 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence located at the 5' or 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 119 or SEQ ID NO: 162.

[0166] In embodiments, provided herein are nucleic acids comprising: (a) a coagulation and innate immunity cassette comprising: (i) a first cistron encoding a THBD protein, wherein THBD's Nucleic acid sequence is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding CD47 protein, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (ii) coding EPCR protein The third cistron, wherein the nucleotide sequence of EPCR is selected from SEQ ID NO: 94-96, 103 and 187; (iii) selected from the ssUBC promoter of SEQ ID NO: 141, 168 and 178; and (iv) selected From the hsGH poly A sequence of SEQ ID NO.113 and 161; (b) apoptosis and cellular immunity box, described apoptosis and cellular immunity box comprise (i) the first cistron of coding A20 albumen, wherein A20 The nucleic acid sequence of HO1 is selected from SEQ ID NO: 64-65, 104, 182 and 188; (ii) the second cistron of encoding HO1 protein, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; (iii) selected from the ssHSPA8 promoter of SEQ ID NO: 139-140 and 142-145; and (iv) selected from the btGH poly A sequence of SEQ ID NO. 112, 120-122, 159, 190; and (c) a complement regulation box, the complement regulation box comprising: (i) the first cistron encoding CD46 protein, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) encoding The second cistron of the CD55 protein, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NO: 84, 85, 107 and 184; (iii) selected from the CAG promoter of SEQ ID NO: 126-128; and (iv) selected hsHBB poly A sequence from SEQ ID NO. 114-116,191. In an embodiment, the UCOE separates the apoptosis and cellular immunity cassettes from the complement regulatory cassettes. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In embodiments, the nucleic acid comprises a polyA sequence on the 5' side of all polycistronic cassettes. In an embodiment, the polyA sequence at the 5' side of all polycistronic cassettes is btHBB pA having the nucleic acid sequence of SEQ ID NO: 160. In embodiments, the nucleic acid comprises a polyA sequence on the 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence at the 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 162 or 119.

[0167] In embodiments, provided herein are nucleic acids comprising: (a) a coagulation and innate immunity cassette comprising: (i) a first cistron encoding a THBD protein, wherein THBD's Nucleic acid sequence is selected from SEQ ID NO: 97-102 and 166; (ii) the second cistron of coding CD47 protein, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NO: 77-83 and 180; (ii) coding EPCR protein The third cistron, wherein the nucleotide sequence of EPCR is selected from SEQ ID NO: 94-96, 103 and 187; (iii) selected from the ssUBC promoter of SEQ ID NO: 141, 168 and 178; and (iv) selected From the hsGH poly A sequence of SEQ ID NO.113 and 161; (b) apoptosis and cellular immunity box, described apoptosis and cellular immunity box comprise (i) the first cistron of coding A20 albumen, wherein A20 The nucleotide sequence of is selected from SEQ ID NO: 64-65, 104, 182 and 188; (ii) the second cistron of coding LEA29Y protein, wherein the nucleotide sequence of LEA29Y is selected from SEQ ID NO: 186; (iii) is selected from The ssHSPA8 promoter of SEQ ID NO: 139-140 and 142-145; and (iv) the btGH poly A sequence selected from SEQ ID NO. 112, 120-122, 159, 190; The complement regulation box comprises: (i) a first cistron encoding CD46 protein, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NO: 71-76, 185 and 200; (ii) a second cistron encoding CD55 protein , wherein the nucleotide sequence of CD55 is selected from SEQ ID NO: 84,85, 107 and 184; (iii) selected from the CAG promoter of SEQ ID NO: 126-128; and (iv) selected from SEQ ID NO. 114-116 , 191 hsHBB poly A sequence. In an embodiment, the UCOE separates the apoptosis and cellular immunity cassettes from the complement regulatory cassettes. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In embodiments, the nucleic acid comprises a polyA sequence on the 5' side of all polycistronic cassettes. In an embodiment, the polyA sequence at the 5' side of all polycistronic cassettes is btHBB pA having the nucleic acid sequence of SEQ ID NO: 160. In embodiments, the nucleic acid comprises a polyA sequence on the 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence at the 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 162 or 119.

[0168] In embodiments, provided herein are nucleic acids comprising one or more polycistronic cassettes selected from: (a) a coagulation cassette comprising: (i) a first polycistronic cassette encoding a THBD protein A cistron, wherein the nucleotide sequence encoding the THBD protein is selected from SEQ ID NO: 97-102 and 166; (ii) a second cistron encoding the TFPI protein, wherein the nucleotide sequence encoding the TFPI protein is selected from SEQ ID NO: 97-102 and 166; ID NO: 94-96, 103 and 187; (iii) selected from the ssUBC promoter of SEQ ID NO: 141, 168, 178; (iv) selected from the hsGH poly A sequence of SEQ ID NO. 113 and 161; (b ) an innate immunity box, the innate immunity box comprising: (i) a first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47 protein, wherein the nucleic acid sequence encoding the CD47 protein is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 139-140 , 142-145 of the ssHSPA8 promoter; and (iv) selected from the btGH polyA sequence of SEQ ID NO.112, 120-122, 159, 190; and (c) a complement regulatory box, the complement regulatory box comprising: ( i) the first cistron encoding CD46 protein, wherein the nucleic acid sequence encoding the CD46 protein is selected from SEQ ID NO: 71-76, 185 and 200; (ii) the second cistron encoding CD55 protein, wherein encoding The nucleic acid sequence of the CD55 protein is selected from SEQ ID NO: 84,85, 107 and 184; (iii) selected from the CAG promoter of SEQ ID NO: 126-128; hsHBB poly A sequences of 116 and 191. In embodiments, the UCOE separates the innate immunity cassette from the complement regulatory cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In embodiments, the nucleic acid comprises a polyA sequence on the 5' side of all polycistronic cassettes. In an embodiment, the polyA sequence at the 5' side of all polycistronic cassettes is btHBB pA having the nucleic acid sequence of SEQ ID NO: 160. In embodiments, the nucleic acid comprises a polyA sequence on the 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence at the 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 162 or 119.

[0169] In embodiments, provided herein are nucleic acids comprising one or more polycistronic cassettes selected from: (a) a coagulation cassette comprising: (i) a first polycistronic cassette encoding a THBD protein A cistron, wherein the nucleotide sequence encoding the THBD protein is selected from SEQ ID NO: 97-102 and 166; (ii) a second cistron encoding the TFPI protein, wherein the nucleotide sequence encoding the TFPI protein is selected from SEQ ID NO: 97-102 and 166; ID NO: 94-96, 103 and 187; (iii) selected from the ssUBC promoter of SEQ ID NO: 141, 168, 178; (iv) selected from the hsGH poly A sequence of SEQ ID NO. 113 and 161; (b ) an innate immunity box, the innate immunity box comprising: (i) a first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NO: 62, 66, 105; (ii) the second cistron encoding CD47 protein, wherein the nucleic acid sequence encoding the CD47 protein is selected from SEQ ID NO: 77-83 and 180; (iii) selected from SEQ ID NO: 139-140 , 142-145 of the ssHSPA8 promoter; and (iv) selected from the btGH polyA sequence of SEQ ID NO.112, 120-122, 159, 190; (c) a complement regulatory box, said complement regulatory box comprising: (i ) a first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding the CD46 protein is selected from SEQ ID NO: 71-76, 185 and 200; (ii) a second cistron encoding a CD55 protein, wherein encoding the The nucleic acid sequence of the CD55 protein is selected from SEQ ID NO: 84, 85, 107 and 184; (iii) is selected from the CAG promoter of SEQ ID NO: 126-128; and (iv) has the nucleic acid sequence of SEQ ID NO: 192 ocHBB poly A sequence; and (d) inflammation and apoptosis box, said inflammation and apoptosis box comprising: (i) the first cistron of coding A20 protein, wherein the nucleic acid sequence of A20 is selected from SEQ ID NO : 64-65, 104, 182 and 188; (ii) the second cistron of encoding HO1 protein, wherein the nucleotide sequence of HO1 is selected from SEQ ID NO: 63, 109, 165, 183 and 189; (iii) selected from ssEEF1α1 promoter of SEQ ID NO: 136-138, 179; and (iv) hsHBB poly A sequence selected from SEQ ID NO. 114-116 and 191. In embodiments, the UCOE separates the innate immunity cassette from the complement regulatory cassette. In an embodiment, the UCOE has a sequence selected from SEQ ID NO: 19-22, 157 and 193. In embodiments, the nucleic acid comprises a polyA sequence on the 5' side of all polycistronic cassettes. In an embodiment, the polyA sequence at the 5' side of all polycistronic cassettes is btHBB pA having the nucleic acid sequence of SEQ ID NO: 160. In embodiments, the nucleic acid comprises a polyA sequence on the 3' side of all polycistronic cassettes. In an embodiment, the polyA sequence at the 3' side of all polycistronic cassettes is SV40 pA having the nucleic acid sequence of SEQ ID NO: 162 or 119. III. Cells, Tissues, Organs and Animals

[0170] Porcine xenografts are broadly compatible with human organ size and physiology and are ethically accepted by the U.S. general population. However, xenografted porcine tissues trigger a complex series of events leading to graft rejection, including: hyperacute rejection due to the presence of preformed antibodies against porcine antigens, complement priming and hypercoagulability, and molecular Enhanced innate and adaptive immune responses due to incompatibility. The present disclosure uses genetic engineering methods to address current shortcomings of xenotransplantation.

[0171] Specifically, there are many immune and functional challenges involving innate and adaptive immune function. Complement and coagulation-mediated dysfunction arise from molecular incompatibility between donor porcine tissue and human physiology and contribute to acute xenograft failure. Preformed antibodies against the alpha-1,3-galactosyl-galactose (αGal) epitope initiate hyperacute graft rejection by initiating complement. Genetic inactivation of the glycoprotein GGTA1 reduces this rapid graft destruction. Protection was further improved by overexpression of the human complement regulatory protein (hCRP) CD46 (membrane cofactor protein), CD55 (complement decay accelerating factor) and CD59 (MAC inhibitory protein) genes.

[0172] Most non-Gal xenoantibodies recognize sialyl N-glycolylneuraminic acid (Neu5Gc) synthesized by the CMAH protein. This gene is inactive in humans, therefore, porcine Neu5Gc is immunogenic in humans. Therefore, porcine CMAH may have to be inactivated for clinical success in xenotransplantation. Although complement regulator expression and GGTA1 knockdown reduced hyperacute rejection, these genetic modifications did not affect acute vascular rejection (AVR).

[0173] Coagulopathy, including thrombotic microangiopathy and systemic consumptive coagulopathy, persists even in the presence of GGTA1 knockout and hCRP overexpression, mainly due to differences between pigs and nonhuman primates (NHPs). Caused by molecular incompatibility of the coagulation system.

[0174] Although others have attempted to generate transgenic pigs for safe xenotransplantation, these transgenic pigs carry only a limited number of transgenes due to limitations in construction capacity and transcriptional interference between transgenes. These approaches proved insufficient to overcome xenograft incompatibility. For example, US Patent Publication No. 2018 / 0249688 utilizes polycistronic expression vectors with different combinations of transgenes. Importantly, these polycistronic vectors contained only 4 transgenes and were used to generate pigs with 6 genetic modifications including KO of αGal (GTKO). In this disclosure, a combination of KO, KI and genomic replacement strategies was utilized. For the first time PERV-free pigs expressing more than 6 transgenes from a single locus were generated.

[0175] The examples described and disclosed herein provide a platform to achieve a greater number of genetic modifications within the same pig. Through this work, pig cells were genetically modified with more than six transgenes to generate immunocompatible cells, tissues, organs, pigs and offspring. Using CRISPR-Cas9, multiple genes were functionally knocked out, including GGTA1, CMAH, and β4GALNT2, to eliminate glycans recognized by preformed human anti-pig antibodies. PiggyBAC-mediated integration or CRISPR-mediated integration is used to integrate one or more of the polycistronic cassettes described herein (e.g., coagulation cassettes, complement regulatory cassettes, innate immunity cassettes, innate immunity, and inflammation and apoptosis cassettes cassette, cellular immunity and coagulation cassette, apoptosis and coagulation cassette, apoptosis and cellular immunity cassette, inflammation and apoptosis cassette, complement regulation and innate immunity cassette, complement regulation and inflammation and apoptosis cassette). In addition, porcine endogenous retrovirus (PERV) genes were knocked out in pig cells by CRISPR-Cas9.

[0176] In embodiments, provided herein are cells comprising a polycistronic cassette described herein. In embodiments, provided herein are cells comprising the nucleic acids described herein. In embodiments, the cells are porcine cells. In embodiments, the cells are primary porcine cells. In embodiments, the cells are cloned. In an embodiment, clonal cells are selected and genotyped. Cells with the correct genotype were expanded to support porcine cloning. Cells comprising one or more polycistronic cassettes described herein at genomic safe harbor loci were cloned into pigs by somatic cell nuclear transfer. In embodiments, provided herein are genetically modified animals comprising cells comprising one or more polycistronic cassettes described herein. In embodiments, the genetically modified animal is bred with a non-genetically modified animal. In embodiments, the genetically modified animal is bred with another genetically modified animal. Provided herein are the progeny resulting from breeding a genetically modified animal with another genetically modified animal or a non-genetically modified animal. In an embodiment, provided herein is a cell, tissue or organ from a progeny.

[0177] In an embodiment, the pig donor cells used to generate the transgenic pigs are substantially free of PERV, exhibit knockout of GGTA1, CMAH, and β4GALNT2, exhibit knock-in of one or more polycistronic cassettes described herein , or exhibit any combination of the preceding characteristics.

[0178] The present disclosure provides cells, tissues, organs and animals having a plurality of modified genes, and methods of producing them. In an embodiment, the cells, tissues, organs are obtained from animals or are animals. In embodiments, the animal is a mammal. In embodiments, the mammal is a non-human mammal, such as an equine, primate, porcine, bovine, ovine, caprine, canine or feline. In embodiments, the mammal is a porcine mammal.

[0179] Modification of genes according to the present disclosure is used to improve molecular compatibility between donor and recipient and reduce adverse events, including hyperacute rejection, acute humoral rejection, thrombotic microangiopathy, and chronic vascular disease. For example, hyperacute rejection occurs over a very short time span, usually within minutes to hours after transplantation, and is caused by preformed antibodies that initiate complement and transplanted endothelial cells, leading to procoagulant changes , which leads to hemostasis and eventual destruction of the transplanted organ. In certain embodiments, the cells, tissues, organs and animals produce reduced hyperacute rejection.

[0180] In an embodiment, the present disclosure provides one or more cells, tissues, organs or animals having a plurality of modified genes. In an embodiment, the cell, tissue, organ or animal has been genetically modified such that multiple genes have been added, deleted, inactivated, destroyed, part thereof has been excised, or the gene sequence has been changed. In an embodiment, the cell, tissue, organ or animal has from about 5 to about 20 modified genes, for example about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, About 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 genes have been modified. In embodiments, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 genes that have been modified are expressed from a single locus. In embodiments, 5, 10 or 12 genes that have been modified are expressed from a single locus. In the example, the 12 genes that have been modified are expressed from a single locus. In embodiments, the cell, tissue, organ or animal has more than 20, more than 15, more than 10, more than 5, more than 3, or more than 2 genes that have been modified. In embodiments, the cell, tissue, organ or animal has more than 10, more than 5, more than 3, more than 2 or more than 1 genes that have been modified. In embodiments, said cell, tissue, organ or animal has one copy of said modified gene, and in other embodiments said cell, tissue, organ or animal has more than one copy of said one or more modified genes, such as more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20. More than 25, more than 30, more than 35, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90 or more than 100 copies of said modified gene. In embodiments, the cell has between 1 copy and about 100 copies, between 1 copy and about 90 copies, between 1 copy and about 80 copies, between 1 copy and about 70 copies Between, between 1 copy and about 60 copies, between 1 copy and about 50 copies, between 1 copy and about 40 copies, between 1 copy and about 30 copies, about 5 One or more of between copies and about 20 copies, between about 10 copies and about 15 copies, between about 10 copies and about 15 copies, or between 1 copy and about 5 copies modified gene.

[0181] In an embodiment, the present disclosure provides one or more cells, tissues, organs or animals having multiple copies of one or more modified genes. For example, the cell, tissue, organ or animal can have 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 15, about 20, about 25, about 30 or more copies of a One or more modified genes.

[0182] In embodiments, the one or more cells are primary cells. In embodiments, the one or more cells are somatic cells. In embodiments, the one or more cells are postnatal cells. In embodiments, the one or more cells are adult cells (eg, adult otic fibroblasts). In embodiments, the one or more cells are fetal / embryonic cells (eg, embryonic blastomeres). In embodiments, the one or more cells are germline cells. In embodiments, the one or more cells are oocytes. In embodiments, the one or more cells are stem cells. In embodiments, the one or more cells are cells from a primary cell line. In an embodiment, the one or more cells are selected from: epithelial cells, liver cells, granulosa cells, adipocytes. In specific embodiments, the one or more cells are fibroblasts. In an embodiment, the fibroblasts are female fetal fibroblasts. In embodiments, the one or more cells are in vitro. In embodiments, the one or more cells are in vivo. In embodiments, the one or more cells are single cells. In embodiments, the one or more cells are members of a colony of cells. In an embodiment, the cells are islet cells.

[0183] In embodiments, the one or more cells are porcine cells. Non-limiting examples of breeds of porcine cell origin or derivation include any of the following breeds of pigs: American Landrace, American Yorkshire, Aksai Black Pied, Angeln Saddleback saddleback), Appalachian English, Arapawa Island, Auckland Island, Australian Yorkshire, Babi Kampung, Bachuan ( Ba Xuyen), Bantu, Basque, Bazna, Beijing Black, Belarus Black Pied, Belgian Landrace Landrace), Bengal Brown Shannaj, Bentheim Black Pied, Berkshire, Bisaro, Bangur, Black Slavonian, Black Canarian, Breitovo, British Landrace, British Lop, British Saddleback, Bulgarian White Bulgarian White, Cambrough, Cantonese, Celtic, Chato Murciano, Chester White, Chiangmai Blackpig ), Choctaw Hog, Creole, Czech Improved White, Danish Landrace, Danish Protest, Dermantsi Pied), Li Yan, Duroc, Dutch Landrace, East Landrace, East Balkan, Essex, Estonian Bacon (Estonian Bacon), Fengjing, Finnish Landrace, Forest Mountain, French Landrace, Gascon, German Landrace ), Gloucestershire Old Spots, Gottingen minipig, Grice, Guinea Hog, Hampshire, Hunter (Hante), Hereford, Hezuo, Hogan Hog, Huntington Black Hog, Iberian, Italian Landrace, Japanese Landrace, Jeju Black, Jinhua, Kakhetian, Kele, Kemerovo, Korean native pig (Korean Native), Krskopolje, Kunekune, Lamcombe, Large Black, Large Black-White, Large Large White, Latvian White, Leicoma, Lithuanian Native, Lithuanian White, Lincolnshire Curly-Coated, Livny, Malhado de Alcobaca, Mangalitsa, Meishan, Middle White, Minzhu, Minokawa Buta, Mong Cai, Mora Romagnola, Mora Pigs (Moura), Mukota, Mulefoot, Murom, Myrhorod, Nero dei Nebrodi, Neijiang, New Zealand New Zealand), Ningxiang, North Caucasian, North Siberian, Norwegian Landrace, Norwegian Yorkshire, Ossabaw Island ), Oxford Sandy and Black, Pakchong 5, Philippine Native, Pietrain, Poland China, Red Wattle, Saddleback Pigs, Semirechensk, Siberian Black Pied, Small Black, Small White, Spots, Surabaya Babi, Swabian- Hall), Swedish Landrace, Swallow Belied Mangalitza, Taihu pig, Tamworth, Thuoc Nhieu, Tibetan , Tokyo-X, Tsivilsk, Turopolje, Ukrainian Spotted Steppe, Ukrainian White Steppe, Urzhum, Vietnamese Potbelly, Welsh, Wessex Saddleback, West French White, Windsnyer, Wuzhishanm, Yanan , Yorkshire and Yorkshire blue and white pigs (Yorkshire Blue and White). In an embodiment, the porcine cells are large Yorkshire and Yucatan porcine cells.

[0184] In an embodiment, the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes have been modified by adding, deleting, inactivating, destroying, excising parts thereof, or the genes Part of the sequence has been changed.

[0185] In an embodiment, a cell, tissue, organ or animal of the disclosure comprises one or more mutations that inactivate one or more genes. In embodiments, the cell, tissue, organ or animal comprises one or more mutations or epigenetic changes that result in reduced or eliminated expression of one or more genes with the one or more mutations. In an embodiment, said one or more genes are inactivated by genetically modifying one or more nucleic acids present in said cell, tissue, organ or animal. In embodiments, inactivation of one or more genes is confirmed by assays. In embodiments, the assay is an infectivity assay, reverse transcriptase PCR assay, RNA-seq, real-time PCR, or ligated PCR localization assay. specific genotype

[0186] In order to provide cells, tissues, organs, and animals that are safe and effective for human clinical use, the cells, tissues, organs, and animals of the present disclosure (e.g., donor pigs) are genetically engineered to have enhanced complement (i.e. complement toxicity), coagulation, inflammation (i.e. apoptosis / inflammation), immunity (i.e. cytotoxicity) and / or immune regulatory systems, making them compatible in humans. Novel combinations of knockout (KO), knockin (KI) (also referred to herein as transgenic (TG)) and / or genome replacement strategies provide enhanced complement, coagulation, inflammation, immune and / or immunomodulatory systems. Expression of genes that make pig cells and organs compatible with humans was assessed using various methods. Non-limiting examples of such methods include flow cytometry, immunohistochemistry, and RNA sequencing.

[0187] For example, cells, tissues, organs, and animals lacking expression of major xenogenic carbohydrate antigens due to genetic KO have reduced or eliminated humoral rejection during xenotransplantation. The three major heterologous carbohydrate antigens include those generated by the glycosyltransferases / glycosylhydrolases GGTA1, CMAH, and B4GALNT2. The purpose of loss-of-function of these genes is to reduce and / or eliminate the binding of pre-formed anti-pig antibodies to the endothelium of the porcine graft.

[0188] Insertion of key complement, coagulation, inflammation, immunity and / or immunomodulatory factors into one or more genomic loci, such as safe harbor genomic loci such as AAVS1, will help regulate the human complement system as well as natural killer (NK) , macrophage and T cell function. Non-limiting examples include overexpression of hCD46, hCD55 and hCD59 by KI to inhibit the human complement cascade; humanization of vWF to prevent unregulated platelet sequestration and thrombotic microangiopathy, e.g. by humanizing A1 of the porcine vWF sequence domain and / or flanking regions; KI of B2M-HLA-E SCT to confer protection against human NK cell cytotoxicity and humanization of porcine cells; and KI of CD47, CD39, THBD, TFPI, A20 to act to the effects of immunosuppressants, immunomodulators and / or anticoagulants.

[0189] In an embodiment, the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes or parts thereof have been modified by addition, deletion, inactivation, destruction, removal of parts thereof. In an embodiment, the present disclosure provides isolated cells, tissues, organs or animals having a plurality of modified genes. In an embodiment, the modified genes include one or more of the following: αGGTA1, β4GalNT2, CMAH, THBD, TFPI, CD39, HO1, CD46, CD55, CD59, major histocompatibility complex class I E Single chain trimer (HLA-E SCT), A20, PD-L1, CD47, porcine leukocyte antigen 1 (SLA-1), SLA-2, SLA-3, vWF, B2M, DQA, DRA, PROCR, B2M, HLA-E, CTLA-4 (eg LEA29Y) and XIAP.

[0190] In embodiments, the modified gene is GGTA1, B4GalNT2, CMAH, or any combination thereof. In embodiments, GGTA1, B4GalNT2 and / or CMAH are genetically KO. In an embodiment, the modified genes are MHC-I genes SLA-1, SLA-2 and SLA-3, MHC-II genes DQA and DRA, endogenous vWF, CD9, asialoglycoprotein receptor, At least one complement inhibitor gene (eg, C3, CD46, CD55, and CD59) and any combination thereof. In embodiments, CD46, CD55 and / or CD59 are genetically KI.

[0191] In one embodiment, the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising CD46, CD55, CD59, THBD, TFPI, EPCR, CD39, B2M, HLA- E, CD47, A20, PD-L1, HO1, CTLA-4 (eg, LEA29Y), XIAP, or any combination thereof. In one embodiment, a cell, tissue, organ or animal of the present disclosure has been further genetically modified to have reduced or no expression of GGTA1, B4GalNT2, CMAH or any combination thereof, for example by genetic KO.

[0192] The cells, tissues, organs or animals of the present disclosure may be genetically modified by any method. In embodiments, a cell, tissue, organ or animal of the disclosure is modified with a nucleic acid comprising a plurality of polycistronic cassettes. Non-limiting examples of suitable methods for the knockout (KO), knockin (KI) and / or genome replacement strategies disclosed and described herein include the use of Cas9, Cas12a (Cpf1 ) or other CRISPR endonucleases, Argonaute nucleic acid CRISPR mediation of endonucleases, transcriptional initiator-like (TAL) effector and nucleases (TALENs), zinc finger nucleases (ZFNs), expression vectors, transposon systems (e.g. PiggyBac transposase), or any combination thereof genetic modification.

[0193] A cell, tissue, organ or animal of the present disclosure may be PERV-free. For example, cells, tissues, organs or animals of the present disclosure may have a copy of PERV functionally deleted from their genome. A cell, tissue, organ or animal of the present disclosure may have a functionally inactive copy of PERV in its genome. PERV represents a risk factor if porcine cells, tissues or organs are transplanted into human recipients. PERV was released from normal pig cells and became infectious. PERV-A and PERV-B are polytropic viruses that infect cells of several species, including humans (for example, they are heterotropic); whereas PERV-C is an affinity virus that infects only porcine cells . Non-limiting methods of functionally deleting copies of PERV are disclosed and described in Niu 2017 and WIPO Publication No. WO2018 / 195402, both of which are incorporated herein by reference in their entireties. In embodiments, pigs are genetically engineered to be free of PERV-A, PERV-B, or PERV-C (or any combination thereof).

[0194] In an embodiment, an additional gene of a cell, tissue, organ or animal of the present disclosure is modified by adding, deleting, inactivating, disrupting, excising parts thereof or parts of the gene sequence. In an embodiment, the modification comprises deletion of one or more of the following genes: MHC-I genes SLA-1, SLA-2 and SLA-3, MHC-II genes DQA and DRA, endogenous vWF, CD9 , asialoglycoprotein receptor, and C3, and express one or more of the following transgenes: PD-L1, exogenous vWF, HLA-E, HLA-G, B2M, and CIITA-DN. In an embodiment, the modification includes deletion of one or more of the following genes: α-galactosyltransferase 1, GGTA1, β4GALN2, and CMAH, and expression of one or more of the following transgenes: CD46, CD55, CD59, CD47, HO1, A20, TNFR1-Ig, CD39, THBD, TFPI, EPCR, PD-1, CTLA-4 (eg LEA29Y), CD73, SOD3, CXCL12, FasL, CXCR3, CD39L1, GLP-1R, M3R, IL35, IL12A and EB13. In an embodiment, the modified gene is CD46, CD55, CD59, CD47, HO1, A20, TNFR1-Ig, CD39, THBD, TFPI, EPCR, PD-1, CTLA-4 (such as LEA29Y), CD73, SOD3 , CXCL12, FasL, CXCR3, CD39L1, GLP-1R, M3R, IL35, IL12A, and EB13.

[0195] In an embodiment, a cell, tissue, organ or animal of the present disclosure comprises one or more genetically modified genes. A genetically modified gene may contain an addition of one or more nucleotides, a deletion of one or more nucleotides, or a mutation of one or more nucleotides. In embodiments, the genetically modified gene is disrupted. In embodiments, the transcription and / or translation of the genetically modified gene is disrupted. In embodiments, the genetically modified gene is inactivated. In embodiments, the cell, tissue, organ or animal comprises a transgene or portion thereof that is not present in a wild-type cell, tissue, organ or animal. In embodiments, the transgene is a foreign gene. In embodiments, the transgene is an endogenous gene. In an embodiment, a cell, tissue, organ or animal of the present disclosure comprises one or more nucleic acids comprising one or more polycistronic cassettes as described herein.

[0196] In embodiments, the modified gene is an MHC class I gene. In an embodiment, the modified MHC class I gene includes one or more of the following SLA-1, SLA-2, SLA-3 and B2M. In an embodiment, the modified gene is SLA-1, SLA-2 and / or SLA-3. In embodiments, the modified gene is B2M. In an embodiment, the modified MHC class I gene includes one or more of the following SLA-1, SLA-2, SLA-3 and B2M. In an embodiment, the modified B2M, SLA-1, SLA-2 and / or SLA-3 genes and / or parts thereof are used with human HLA-E gene, human HLA-G gene, human B2M gene and / or Human (dominant negative mutant class II trans promoter) CIITA-DN gene and / or partial replacement thereof. In embodiments, the modified gene is conditionally and / or inducibly modified. In embodiments, conditional and / or inducible promoters are used to conditionally and / or inducibly modify the one or more modified genes. In an embodiment, provided herein is an isolated cell, tissue, organ, or animal comprising a B2M, SLA-1, SLA-2, or SLA-3 gene, or any combination of genes, conditionally altered and expressed by human HLA - At least a part of replacement of E gene, human HLA-G gene, human B2M gene and / or human CIITA-DN gene.

[0197] In an embodiment, provided herein is a cell, tissue, organ or animal comprising a modified MHC class II gene. In embodiments, the modified MHC class II gene is DRQ, DRA, or any combination thereof. A genetically modified MHC class II gene may contain an addition of one or more nucleotides, a deletion of one or more nucleotides, or a mutation of one or more nucleotides. In embodiments, the genetically modified MHC class II gene is disrupted. In embodiments, the transcription and / or translation of the genetically modified MHC class II genes is disrupted. In embodiments, the genetically modified MHC class II genes are inactivated. In embodiments, the cell, tissue, organ or animal comprises an MHC class II transgene or portion thereof that is not present in a wild-type cell, tissue, organ or animal. In embodiments, the modified gene is conditionally and / or inducibly modified. In embodiments, conditional and / or inducible promoters are used to conditionally and / or inducibly modify the one or more modified genes. In embodiments, said isolated cell, tissue, organ or animal comprises conditionally altered DRQ and / or DRA genes or any combination thereof.

[0198] In an embodiment, provided herein is a cell, tissue, organ or animal comprising a modified vWF gene and / or a vWF-related gene. Genetically modified vWF genes and / or vWF-related genes may contain additions of one or more nucleotides, deletions of one or more nucleotides, or mutations of one or more nucleotides. In embodiments, the genetically modified vWF gene and / or vWF-related genes are disrupted. In embodiments, the transcription and / or translation of the genetically modified vWF gene and / or vWF-related genes is disrupted. In embodiments, the genetically modified vWF gene and / or vWF-related genes are inactivated. In an embodiment, the cell, tissue, organ or animal comprises a vWF gene and / or a vWF-related gene transgene or part thereof that is absent in a wild-type cell, tissue, organ or animal. In an embodiment, the modified vWF gene and / or part thereof is replaced with a human vWF gene and / or part thereof. In an embodiment, the modified vWF gene, the modified vWF-related gene and / or one or more parts thereof are replaced with a human vWF gene, one or more human vWF-related genes and / or parts thereof. In embodiments, the modified vWF gene and / or vWF-related gene is conditionally and / or inducibly modified. In embodiments, conditional and / or inducible promoters are used to conditionally and / or inducibly modify the one or more modified genes. In an embodiment, the isolated cell, tissue, organ or animal comprises conditionally altered vWF, vWF-related genes, one or more parts thereof, or any combination thereof, and human vWF gene, at least part of human vWF gene, The conditionally altered gene is replaced by one or more other human vWF-related genes, at least part of one or more human vWF-related genes, or any combination thereof. In an embodiment, the vWF gene is modified using a gRNA designed to initiate HDR replacement in the endogenous porcine genome and cut near the region to be replaced by the human sequence.

[0199] In embodiments, provided herein are cells, tissues, organs or animals comprising one or more modified complement genes. In an embodiment, the complement gene is C3. A genetically modified complement gene may contain an addition of one or more nucleotides, a deletion of one or more nucleotides, or a mutation of one or more nucleotides. In embodiments, the genetically modified complement gene is disrupted. In embodiments, the transcription and / or translation of the genetically modified complement genes is disrupted. In embodiments, the genetically modified complement genes are inactivated. In embodiments, the cell, tissue, organ or animal comprises a complement gene or portion thereof that is not present in a wild-type cell, tissue, organ or animal. In embodiments, said modified C3 gene and / or complement gene is conditionally and / or inducibly modified. In embodiments, conditional and / or inducible promoters are used to conditionally and / or inducibly modify the one or more modified genes. In embodiments, the isolated cell, tissue, organ or animal comprises conditionally altered C3, complement genes, one or more portions thereof, or any combination thereof. In an embodiment, the C3 gene is modified using gRNA.

[0200] In embodiments, the modified gene is a knockout of C3. In embodiments, the modified gene is a knock-in of PD-L1. In an embodiment, the modified gene is humanized vWF of porcine vWF. In an embodiment, the modified gene is a conditional knock-in of the MHC-I genes SLA-1, SLA-2 and SLA-3.

[0201] In embodiments, the host elicits no or substantially no immune response to the genetically modified cell, tissue or organ.

[0202] In an embodiment, the present disclosure provides a nucleic acid obtained from any of the cells disclosed herein. In embodiments, one or more nucleic acids in the cell are genetically modified such that one or more genes in the cell are altered, or otherwise the genome of the cell is modified. In embodiments, the gene or portion thereof is genetically modified using any genetic modification system known in the art and / or disclosed herein. In an embodiment, the genetic modification system is a TALEN, a zinc finger nuclease and / or a CRISPR-based system. In an embodiment, the genetic modification system is a CRISPR-Cas9 system. In an embodiment, the genetic modification system is a Class II Type II CRISPR system. In an embodiment, the genetic modification system is a Class II Type V CRISPR system. In an embodiment, the cell is genetically modified such that one or more genes or parts thereof in the cell are inactivated, and the cell is further genetically modified such that one or more of the cell A gene or part thereof that induces an immune response if said cell (or a tissue or organ cloned / derived from said cell) is transplanted into a human body is reduced. In embodiments, the cell is genetically modified to increase expression of one or more human genes or portions thereof. In embodiments, the cell is genetically modified to increase the expression of one or more humanized genes or portions thereof. In an embodiment, the cell is genetically modified such that one or more genes or parts thereof in the cell are inactivated, and the cell is further genetically modified such that one or more of the cell Increased expression of a gene that suppresses an immune response if said cell (or a tissue or organ cloned / derived from said cell) is transplanted into a human. In an embodiment, the cell is genetically modified such that one or more genes or parts thereof in the cell are inactivated, and the cell is further genetically modified such that one or more of the cell gene that induces an immune response if the cell (or a tissue or organ cloned / derived from the cell) is transplanted into a human body is reduced, and the cell is further genetically modified such that the An increase in the expression of one or more genes in said cells that would suppress an immune response if said cells (or tissues or organs cloned / derived from said cells) were transplanted into a human.

[0203] In an embodiment, the present disclosure provides embryos cloned from genetically modified cells. The following reference describes the generation of embryos from genetically modified cells and is incorporated herein by reference in its entirety: Kim et al. (2016). PLoS ONE: 11(7): e0160289. In embodiments, one or more genetically modified nucleic acids are extracted from a genetically modified cell and cloned into a different cell. For example, in somatic cell nuclear transfer, a genetically modified nucleic acid from a genetically modified cell is introduced into an enucleated oocyte. In examples, oocytes can be enucleated by partial zona pellucida dissection near the polar body, followed by extrusion of the cytoplasm at the dissected region. In the Examples, the genetically modified cells were injected into enucleated oocytes arrested in meiosis 2 using an injection pipette with a sharp beveled tip. Oocytes arrested in meiosis 2 are often referred to as "eggs". In an embodiment, embryos are produced by fusion and priming of oocytes. In embodiments, the oocyte is fused by delivering electrical pulses to the oocyte. Such embryos may be referred to herein as "genetically modified embryos." In embodiments, genetically modified embryos are primed by treatment with 6-dimethylaminopurine (6-DMAP), ionomycin, or a combination thereof. In an embodiment, a genetically modified embryo is treated with a histone deacetylase inhibitor (HDACi). In an embodiment, the genetically modified embryos are transferred to the oviduct of a recipient female pig. In an embodiment, the genetically modified embryos are transferred to the oviduct of a recipient female pig 12 hours to 4 days after priming. For example, at about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 1.25 days, about 1.5 days, about 1.75 days, about 2 days, about 2.25 days, about 2.5 days, about 2.75 days, about 3 days, about 3.25 days, about 3.5 days, about 3.75 days , or about 4 days, the genetically modified embryos are transferred to the oviducts of recipient female pigs. In an embodiment, the genetically modified embryos are transferred to the oviduct of a recipient female pig 20 to 24 hours after priming. See, eg, Cibelli 1998 and US Patent No. 6,548,741. In an embodiment, the recipient female is checked for pregnancy approximately 20-21 days after the transfer of the genetically modified embryo.

[0204] In an embodiment, a genetically modified embryo is grown into a postnatal genetically modified animal. In embodiments, the postnatal genetically modified animal is a neonatal genetically modified animal. In embodiments, the genetically modified pig is a juvenile genetically modified animal. In embodiments, the genetically modified animal is an adult genetically modified animal (eg, older than 5-6 months). In embodiments, the genetically modified animal is a female genetically modified animal. In an embodiment, the animal is a male genetically modified animal. In embodiments, the genetically modified animal is bred with a non-genetically modified animal. In embodiments, the genetically modified animal is bred with another genetically modified animal. In embodiments, the genetically modified pig is bred to another genetically modified animal with reduced or no active virus. In an embodiment, the genetically modified animal is bred with a second genetically modified animal that has been genetically modified such that cells from the second genetically modified animal , tissues or organs are unlikely to induce an immune response if transplanted into humans.

[0205] In embodiments, the genetically modified animal is an animal with one or more modified genes maintained for at least one month, at least 6 months, at least 1 year, at least 5 years, at least 10 years after pregnancy The same or similar level of expression or inactivation of one or more modified genes. In embodiments, the genetically modified animal remains genetically modified even after delivery from a non-virally inactivated surrogate or after being in a facility / space with other non-virally inactivated animals, as the genetically modified A modified pig has one or more modified genes.

[0206] In an embodiment, the present disclosure provides cells, tissues or organs obtained from any of the postnatal genetically modified pigs described herein. In an embodiment, the cell, tissue or organ is selected from liver, kidney, lung, heart, pancreas, muscle, blood and bone. In specific embodiments, the organ is liver, kidney, lung or heart. In an embodiment, the cells from said postnatal genetically modified pig are selected from the group consisting of pancreatic islets, lung epithelial cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, liver cells, non-parenchymal liver cells, gallbladder epithelial cells, gallbladder endothelial cells , bile duct epithelial cells, bile duct endothelial cells, liver vascular epithelial cells, liver vascular endothelial cells, sinusoidal cells, choroid plexus cells, fibroblasts, supporting cells, neuronal cells, stem cells and adrenal chromaffin cells. In embodiments, the genetically modified organs, tissues or cells have been isolated from their natural environment (ie, from the pig in which they are growing). In an embodiment, separation from the natural environment means general physical separation from the natural environment, for example, removal from the genetically modified donor animal, and changing the proximity of the genetically modified organ, tissue, or cell to their direct contact. Relationship of cells (eg, by dissociation). IV. Methods of Producing Cells, Tissues, Organs or Animals

[0207] The present disclosure provides methods of producing a cell, tissue, organ or animal having one or more nucleic acids comprising one or more polycistronic cassettes described herein. In an embodiment, the present disclosure provides methods of inactivating, deleting or otherwise disrupting one or more genes, or portions thereof, in any of the cells disclosed herein, the methods comprising administering to the cell a pair of genes A specific gene editing agent, wherein the agent destroys the transcription and / or translation of the gene. In embodiments, the agent targets the start codon of the gene and inhibits transcription of the gene. In embodiments, the agent targets an exon in the gene, and the agent induces a frameshift mutation in the gene. In embodiments, the reagent introduces an inactivating mutation into the gene. In embodiments, the agent inhibits the transcription of the gene.

[0208] In an embodiment, the present disclosure provides a method of altering one or more genes or portions thereof in vivo, the method comprising administering to the cell a gene editing agent specific for the gene, wherein the agent alters The sequence of the gene, such as by humanizing the gene or otherwise altering the native (eg, wild-type) sequence of the gene.

[0209] In an embodiment, the present disclosure provides methods of expressing one or more nucleic acids comprising one or more polycistronic cassettes or portions thereof comprising administering to a cell comprising one or more polycistronic A gene editing agent for a polycistronic cassette or a portion thereof, wherein the reagent introduces a nucleic acid sequence comprising one or more polycistronic cassettes. In an embodiment, the reagent is a nucleic acid sequence, such as a plasmid, a vector, and the like. In embodiments, the nucleic acid sequence includes one or more nucleic acid sequences, such as promoters, transgenes and / or additional genes. In embodiments, said nucleic acid sequence or portion thereof is derived from one or more species and / or one or more sources. In embodiments, the species is the species that will receive the genetically modified cells, tissues or organs. In an embodiment, the species is human. In other embodiments, the species is non-human, such as mammals, animals, bacteria and / or viruses.

[0210] In embodiments, any agent disclosed herein is a polynucleotide. In embodiments, said polynucleotide encodes one or more of the nucleases and / or nickases and / or RNA or DNA molecules described herein. In embodiments, the polynucleotide agent is introduced into one or more cells. In embodiments, the polynucleotide is introduced into the one or more cells in such a manner that the one or more cells transiently express the polynucleotide. In embodiments, the polynucleotide is introduced into the one or more cells in such a manner that the one or more cells stably express the polynucleotide. In embodiments, the polynucleotide is introduced in such a way that the polynucleotide is stably incorporated into the genome of the cell. In embodiments, the polynucleotide is introduced together with one or more transposable elements. In an embodiment, the transposable element is a polynucleotide sequence encoding a transposase. In an embodiment, the transposable element is a polynucleotide sequence encoding PiggyBac transposase. In embodiments, said transposable element is inducible. In embodiments, the transposable element is doxycycline inducible. In embodiments, said polynucleotide further comprises a selectable marker. In embodiments, the selectable marker is a puromycin resistance marker. In embodiments, the selectable marker is a fluorescent protein (eg, GFP).

[0211] In embodiments, the reagent is a nuclease or nickase for targeting DNA in cells. In embodiments, the agent specifically targets a gene and inhibits its expression. In embodiments, the agent comprises a transcriptional repressor domain. In embodiments, the transcriptional repressor domain is a Krüppel-associated box (KRAB).

[0212] In embodiments, the reagent is any programmable nuclease. In embodiments, the agent is a natural homing meganuclease. In embodiments, the reagent is a TALEN-based reagent, a ZFN-based reagent, or a CRISPR-based reagent, or any biologically active fragment, fusion, derivative or combination thereof. CRISPR-based reagents include, for example, Class II, Type II and Type V systems, including various species variants of, for example, Cas9 and Cpf1. In embodiments, the agent is a deaminase or a nucleic acid encoding a deaminase. In embodiments, cells are genetically engineered to stably and / or transiently express TALEN-based reagents, ZFN-based reagents, and / or CRISPR-based reagents.

[0213] In embodiments, any nucleic acid described herein, or fragment thereof, is integrated into the genome of a host cell. Nucleic acids can be integrated into cells by recombinase-mediated cassette exchange, PiggyBac transposon-mediated gene transfer, CRISPR-mediated homology-dependent recombination, or CRISPR-mediated non-homology-dependent targeted integration.

[0214] In embodiments, any nucleic acid described herein, or fragment thereof, is integrated into a host cell at a genomic safe harbor locus comprising a landing pad. Figures 28A and 28C provide exemplary landing pads. Non-limiting examples of genomic safe harbor loci include AAVS1, CCR5, and Fos5. In an embodiment, the landing pad comprises Ank2 gene, Cpsf4 gene, C-Mos gene, Fos1 gene or adeno-associated virus integration site (AAVS). US Patent No. 8,980,579 describes chromosome landing platforms and is incorporated herein by reference in its entirety. In embodiments, the landing pad comprises an adeno-associated virus integration site (AAVS) or a Fos1 gene. In an embodiment, the landing pad comprises an insulator sequence (eg HS4). In embodiments, the landing pad comprises one or more loxP sites. In embodiments, the landing pad comprises two loxP sites. In embodiments, the landing pad comprises a polyA sequence. In embodiments, said polyA sequence comprises a terminator. In embodiments, the landing pad comprises a marker, such as blue fluorescent protein (BFP). In embodiments, the landing pad comprises a promoter. In an embodiment, the promoter comprises any one of the sequences in Table B. In an embodiment, the polyA sequence comprises any one of the sequences in Table C. In an embodiment, from 5' to 3', the landing pad comprises a first insulator sequence, a loxP site, a promoter, a fluorescent protein, a poly A sequence, a second loxP site, a second insulator site and a poly A sequence. In an embodiment, from 5' to 3', the landing rack comprises a first loxP site, a promoter, a fluorescent protein, a poly A sequence and a second loxP site. In an embodiment, the landing pad comprises the nucleic acid sequence of SEQ ID NO: 248 or 249.

[0215] In embodiments, the landing pad is integrated into a genomic safe harbor locus by recombinase-mediated cassette exchange. In embodiments, the landing gear is integrated into a genomic safe harbor locus by CRISPR-Cas9 Homology Directed Repair (HDR).

[0216] In an embodiment, provided herein is a cell, organ or tissue comprising a landing pad. In an embodiment, provided herein is a cell, organ or tissue comprising a landing pad comprising the nucleic acid sequence of SEQ ID NO: 248 or 249.

[0217] In embodiments, after introducing the landing pad into the cell, any of Figures 1A-1D, 2A-2J, 3A-3C, 4A-4H, 5A-5F, or 6 The polycistronic cassette is introduced into the cell. In an embodiment, the polycistronic cassette of any one of Figures 1A-1D, 2A-2J, 3A-3C, 4A-4H, 5A-5F, or 6 replaces the landing pad. Poly A sequence and marker gene. Integration of polycistronic cassettes can be performed using recombinase-mediated cassette exchange or CRISPR-Cas9 HDR. Figure 28B provides an illustration of the integration of the 15S5 payload (see Figure 2I) into the landing gear of Figure 28A. In embodiments, a nucleic acid that integrates into a genomic safe harbor locus comprises sequences homologous to the genomic integration sites at the 5' and 3' ends of the nucleic acid. In an embodiment, the nucleic acid comprising the polycistronic cassette of any one of Figures 1A-1D, Figure 2A-2J, Figure 3A-3C, Figure 4A-4H, Figure 5A-5F or Figure 6 comprises Sequences homologous to the genomic integration sites at the 5' and 3' ends of a nucleic acid.

[0218] In an embodiment, provided herein is a method of producing a genetically modified cell comprising (i) knocking out GGTA1, CMAH, and β4GALNT2; (ii) knocking out one or more PERV elements (e.g., PERV pol, env, gag genes or combinations thereof); (iii) knocking in any one or more of the polycistronic cassettes described herein. The aforementioned methods can be carried out in any order of the following: (i), (ii), (iii); (i), (iii), (ii); (ii), (i), (iii); (ii), (iii), (i); (iii), (i), (ii); or (iii), (ii), (i). In an embodiment, the tissue, organ or animal comprises cells produced according to any of the foregoing methods. In embodiments, the cells are porcine cells. In embodiments, the cells are primary porcine cells. In embodiments, the cells are human cells. V. Treatment

[0219] In embodiments, any genetically modified cell, tissue or organ disclosed herein can be used to treat a subject of a different species than the genetically modified cell. In embodiments, the present disclosure provides methods of transplanting any of the genetically modified cells, tissues or organs described herein into a subject in need thereof. In embodiments, the subject is a human. In embodiments, the subject is a non-human primate. In embodiments, the subject is a pig, ape, human, dog, cat, monkey, lemur, chimpanzee, bonobo, orangutan, gibbon, cow, horse, bird, sheep or gorilla.

[0220] In an embodiment, the genetically modified organ used in any of the methods disclosed herein may be selected from heart, lung, liver, eye, pituitary, thyroid, parathyroid, esophagus, thymus, adrenal gland, appendix of a genetically modified pig , bladder, gallbladder, small intestine, large intestine, small intestine, kidney, pancreas, spleen, stomach, skin and / or prostate. In embodiments, the genetically modified tissue used in any of the methods disclosed herein may be selected from genetically modified porcine cartilage (e.g., esophageal cartilage, knee cartilage, ear cartilage, nasal cartilage), muscle (such as but not limited to Smooth muscle and heart (eg, heart valves), tendons, ligaments, bone (eg, bone marrow), cornea, middle ear, and veins. In embodiments, genetically modified cells for use in any of the methods disclosed herein include blood cells, skin hair follicles, hair follicles, and / or stem cells. Compositions of the present disclosure can also be administered to any part of an organ or tissue (eg, a part of the eye, such as the cornea).

[0221] In an embodiment, a heart, lung, liver, kidney, pancreas, or spleen is isolated from a pig that has been genetically modified to contain (a) deletion or disruption of GGTA1, CMAH, and B4GALNT2; (b) from a single pig genome CD46, CD55, CD59, THBD, TFPI, PROCR, CD39, B2M, HLA-E, CD47, A20, PD-L1, HO1, CTLA-4 (such as LEA29Y), XIAP, and combinations thereof (such as its human or humanized copies); and (c) functional deletion of all PERV copies. In the Examples, hearts, lungs, livers, kidneys, pancreas or spleens are isolated from pigs that have been genetically modified to contain (a) functional disruptions of GGTA1, CMAH and B4GALNT2; (b) from a single pig genome CD46, CD55, CD59, THBD, TFPI, PROCR, CD39, B2M, HLA-E, CD47, A20, PD-L1, HO1, CTLA-4 (e.g. LEA29Y), XIAP transgenes and combinations thereof expressed by multiple transgene cassettes (e.g. and (c) functional inactivation of all PERV copies. In certain embodiments, the pig has been further genetically modified to have a deletion of humanized vWF, ASGR1 and / or a deletion of the B2M gene.

[0222] In an embodiment, the organ is stored at a temperature between 2°C and about 37°C prior to transplantation. For example, in embodiments, the organ is kept at about 2ºC, about 3ºC, about 4ºC, about 5ºC, about 6ºC, about 7ºC, about 8ºC, about 9ºC, about 10ºC, about 11ºC, about 12ºC, about 13ºC, about 14ºC, about 15ºC, 16ºC, 17ºC, 18ºC, 19ºC, 20ºC, 21ºC, 22ºC, 23ºC, 24ºC, 25ºC, 26ºC, 27ºC, 28ºC, 29ºC, 30ºC, 31ºC, Store at about 32ºC, about 33ºC, about 34ºC, about 35ºC, about 36ºC, or about 37ºC. In embodiments, the organ is stored at a temperature of about 2°C to about 8°C, about 4°C to about 8°C, or about 20°C to about 25°C. In Examples, organs are stored on ice prior to transplantation.

[0223] In embodiments, the organ is stored in an organ preservation solution prior to transplantation. In an embodiment, the organ preservation solution contains histidine, tryptophan, ketoglutarate, sodium chloride, human serum albumin, dextran 40, glucose, mannitol, sucrose, citrate, hydroxyethyl - Piperazine-ethanesulfonic acid (HEPES), sodium ion, potassium ion, lactobionate, raffinose, glutathione, allopurinol, magnesium ion, trehalose, N-acetylcysteine , dibutyryl cAMP, nitroglycerin, or combinations thereof. In an embodiment, the organ preservation solution is oxygenated. In an embodiment, the organ preservation solution comprises 0.9% sodium chloride. Organ storage in histidine-tryptophan ketoglutarate.

[0224] In an embodiment, the organ is perfused with a perfusate prior to transplantation. When referring to an organ, the term "perfusing" means pumping fluid (eg, perfusate) through the organ. In embodiments, the perfusate is delivered to the organ through the circulatory or lymphatic system. In embodiments, the perfusate is delivered to the blood vessels of the organ. In an embodiment, the perfusate is an organ preservation solution. In embodiments, the perfusate includes plasma, whole blood, red blood cells, hemoglobin, human serum albumin, or combinations thereof. In an embodiment, the perfusate comprises heparinized human whole blood. In embodiments, the perfusate is oxygenated.

[0225] In embodiments, the perfusate comprises an antibody or antibody fragment thereof that binds glycoprotein 1B (GPIb). The antibody fragment thereof may be a variable heavy domain, variable light domain, constant light chain, constant heavy chain domain (CH1, CH2, CH3 or CH4) or any combination thereof. In an embodiment, the antibody fragment thereof is an antigen-binding fragment (Fab). In embodiments, the antibody or fragment thereof that binds GPIb is a murine or human antibody. In embodiments, the perfusate comprises eptifibatide. In embodiments, the perfusate comprises eptifibatide and an antibody or antibody fragment thereof that binds glycoprotein 1B (GPIb).

[0226] In embodiments, the organ is perfused at a pressure between 30 mmHg and about 100 mmHg. For example, placing an organ at about 30 mmHg, about 35 mmHg, about 40 mmHg, about 45 mmHg, about 50 mmHg, about 55 mmHg, about 60 mmHg, about 65 mmHg, about 70 mmHg, about 75 mmHg, about 80 mmHg, about Perfuse at a pressure of 85 mmHg, about 90 mmHg, about 95 mmHg, or about 100 mmHg. In embodiments, the organ is perfused at a pressure of about 40 mmHg to about 75 mmHg.

[0227] In embodiments, the organ is perfused at a temperature between about 2°C and about 37°C. For example, in embodiments, the organ is kept at about 2ºC, about 3ºC, about 4ºC, about 5ºC, about 6ºC, about 7ºC, about 8ºC, about 9ºC, about 10ºC, about 11ºC, about 12ºC, about 13ºC, about 14ºC, about 15ºC, 16ºC, 17ºC, 18ºC, 19ºC, 20ºC, 21ºC, 22ºC, 23ºC, 24ºC, 25ºC, 26ºC, 27ºC, 28ºC, 29ºC, 30ºC, 31ºC, Infusing at a temperature of about 32ºC, about 33ºC, about 34ºC, about 35ºC, about 36ºC or about 37ºC. In embodiments, the organ is perfused at a temperature of about 20°C to about 25°C.

[0228] In embodiments, the organ is perfused for about 5 minutes to about 2 days. For example, perfusing the organ for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours , about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 5.25 hours, about 5.5 hours, about 5.75 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours , about 1 day, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, or about 2 sky.

[0229] In embodiments, the organ is perfused and then stored at a temperature of about 4°C to about 8°C.

[0230] In an embodiment, a subject receiving a transplant of an organ that has been stored and / or perfused according to any of the methods described herein exhibits a The subjects had lower troponin I levels of between about 50% and about 100% compared to those tested. For example, subjects who received transplants of organs that had been stored and / or perfused according to any of the methods described herein exhibited higher troponin I levels than those who received organs that had not been stored and / or perfused according to one of the preceding methods. The transplanted subject's Troponin I level is ...

Claims

1. A nucleic acid molecule comprising: (a) a first polycistronic cassette comprising: (i) a first cistronic cassette encoding an EPCR protein; and (ii) a second cistronic cassette encoding a coagulation regulatory protein (THBD) protein; (b) a second polycistronic cassette comprising: (i) a third cistronic cassette encoding an A20 protein; and (ii) a fourth cistronic cassette encoding an HO1 protein; and (c) a third polycistronic cassette comprising: (i) a fifth cistronic cassette encoding a CD46 protein; (ii) a sixth cistronic cassette encoding a CD55 protein; and (iii) a seventh cistronic cassette encoding a CD47 protein.

2. The nucleic acid molecule of claim 1, wherein the cistron encoding the CD46 protein or the cistron encoding the THBD protein contains a splicing site mutation.

3. The nucleic acid molecule of claim 1, wherein the cistron encoding the CD46 protein contains a splice site mutation and encodes CD46_LL, CD46dal, CD46a2, CD46da, or CD46i.

4. The nucleic acid molecule of claim 1, wherein the CD47 protein is CD47 isotype 1 or CD47 isotype 2.

5. The nucleic acid molecule of claim 1, wherein the CD46 protein is a CD46 isotype selected from the group consisting of: CD46 C1, CD46 C2, CD46 BC1, CD46 BC2 and CD46 ABC1.

6. The nucleic acid molecule of claim 1 further comprises a ubiquitous chromatin opening element.

7. The nucleic acid molecule as requested in item 6, wherein the chromatin opening element is Can 6-3, A2CBX3, CBX3, CBX3* or SRF1.

8. The nucleic acid molecule of claim 1, wherein the first polycistronic cassette, the second polycistronic cassette and the third polycistronic cassette are separated by ubiquitous chromatin open elements.

9. As in request item 1, the nucleic acid molecule, wherein: (a) The first cistron encoding the EPCR protein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence selected from the group consisting of SEQ ID NO: 92, 93, and 181; (b) The second cistron encoding the THBD protein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence selected from the group consisting of SEQ ID NO: 97 to 102 and 166; (c) The third cistron encoding the A20 protein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence selected from the group consisting of SEQ ID NO: 92, 93, and 181; (d) The fourth cistron encoding the HO1 protein contains a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence selected from the group consisting of SEQ ID NO: 63, 109, 165, 183, and 189; (e) The fifth cistron encoding the CD46 protein contains a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence selected from the group consisting of SEQ ID NO: 71 to 76, 185, and 200; (f) The sixth cistron encoding the CD55 protein contains a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence selected from the group consisting of SEQ ID NO: 63, 109, 165, 183, and 189; (g) The seventh cistron encoding the CD47 protein contains a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of the group consisting of SEQ ID NOs: 77 to 83 and 180.

10. As in request item 9, the nucleic acid molecule, wherein: (a) The first cistron encoding the EPCR protein contains a nucleic acid sequence that is at least 95% identical to the sequence selected from the group consisting of SEQ ID NO: 92, 93, and 181; (b) The second cistron encoding the THBD protein contains a nucleic acid sequence that is at least 95% identical to the sequence selected from the group consisting of SEQ ID NO: 97 to 102 and 166; (c) The third cistron encoding the A20 protein contains a nucleic acid sequence that is at least 95% identical to the sequence selected from the group consisting of SEQ ID NO: 64, 65, 104, 182, and 188; (d) The fourth cistron encoding the HO1 protein contains a nucleic acid sequence that is at least 95% identical to the sequence selected from the group consisting of SEQ ID NO: 63, 109, 165, 183, and 189; (e) The fifth cistron encoding the CD46 protein contains a nucleic acid sequence that is at least 95% identical to the sequence selected from the group consisting of SEQ ID NO: 97 to 102 and 166. (f) The sixth cistron encoding the CD55 protein contains a nucleic acid sequence that is at least 95% identical to the sequence selected from the group consisting of SEQ ID NO: 84, 85, 107 and 184; and (g) The seventh cistron encoding the CD47 protein contains a nucleic acid sequence that is at least 95% identical to the sequence selected from the group consisting of SEQ ID NO: 77 to 83 and 180.

11. As in request item 9, the nucleic acid molecule, wherein: (a) The first cistron encoding the EPCR protein contains a nucleic acid sequence that is at least 99% identical to the sequence selected from the group consisting of SEQ ID NO: 92, 93, and 181; (b) The second cistron encoding the THBD protein contains a nucleic acid sequence that is at least 99% identical to the sequence selected from the group consisting of SEQ ID NO: 97 to 102 and 166; (c) The third cistron encoding the A20 protein contains a nucleic acid sequence that is at least 99% identical to the sequence selected from the group consisting of SEQ ID NO: 64, 65, 104, 182, and 188; (d) The fourth cistron encoding the HO1 protein contains a nucleic acid sequence that is at least 99% identical to the sequence selected from the group consisting of SEQ ID NO: 63, 109, 165, 183, and 189; (e) The fifth cistron encoding the CD46 protein contains a nucleic acid sequence that is at least 99% identical to the sequence selected from the group consisting of SEQ ID NO: 97 to 102 and 166. (f) The sixth cistron encoding the CD55 protein contains a nucleic acid sequence that is at least 99% identical to the sequence selected from the group consisting of SEQ ID NO: 84, 85, 107 and 184; and (g) The seventh cistron encoding the CD47 protein contains a nucleic acid sequence that is at least 99% identical to the sequence selected from the group consisting of SEQ ID NO: 77 to 83 and 180.

12. As in request item 9, the nucleic acid molecule, wherein: (a) The first cistron encoding the EPCR protein contains a nucleic acid sequence that is 100% identical to the sequence selected from the group consisting of SEQ ID NO: 92, 93, and 181; (b) The second cistron encoding the THBD protein contains a nucleic acid sequence that is 100% identical to the sequence selected from the group consisting of SEQ ID NO: 97 to 102 and 166; (c) The third cistron encoding the A20 protein contains a nucleic acid sequence that is 100% identical to the sequence selected from the group consisting of SEQ ID NO: 64, 65, 104, 182, and 188; (d) The fourth cistron encoding the HO1 protein contains a nucleic acid sequence that is 100% identical to the sequence selected from the group consisting of SEQ ID NO: 63, 109, 165, 183, and 189; (e) The fifth cistron encoding the CD46 protein contains a nucleic acid sequence that is 100% identical to the sequence selected from the group consisting of SEQ ID NO: 97 to 102 and 166. (f) The sixth cistron encoding the CD55 protein contains a nucleic acid sequence that is 100% identical to the sequence selected from the group consisting of SEQ ID NO: 84, 85, 107 and 184; and (g) The seventh cistron encoding the CD47 protein contains a nucleic acid sequence that is 100% identical to the sequence selected from the group consisting of SEQ ID NO: 77 to 83 and 180.

13. As in request item 1, the nucleic acid molecule, wherein: (a) The first polycistrans cassette includes a first promoter, wherein the first promoter contains at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 126 to 145, 167, 168, 178, 179, 231 to 238, and 250; (b) The second polycistrans cassette includes a second promoter, wherein the second promoter contains at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 126 to 145, 167, 168, 178, 179, 231 to 238, and 250; and (c) The third polycistrans cassette includes a third promoter, wherein the third promoter contains a sequence identical to a sequence selected from the group consisting of SEQ ID NO: 126 to 145, 167, 168, 178, 179, 231 to 238, and 250. NO: The sequences of groups consisting of 126 to 145, 167, 168, 178, 179, 231 to 238 and 250 are at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical.

14. As in request item 1, the nucleic acid molecule, wherein: (a) The first polycis-trans subbox contains at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a first polyA sequence selected from the group consisting of SEQ ID NO: 112 to 125, 154 to 156, 159 to 162, 190 to 192, 239, and 240; (b) The second polycis-trans subbox contains at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a second polyA sequence selected from the group consisting of SEQ ID NO: 112 to 125, 154 to 156, 159 to 162, 190 to 192, 239, and 240; and (c) The third polycis-trans subbox contains a first polyA sequence selected from SEQ ID NO: 112 to 125, 154 to 156, 159 to 162, 190 to 192, 239, and 240; NO: The sequences of groups consisting of 112 to 125, 154 to 156, 159 to 162, 190 to 192, 239 and 240 are at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical third cluster A sequences.

15. The nucleic acid molecule of claim 1, further comprising: (a) a nucleic acid sequence encoding a 2A polypeptide, wherein (i) one or more proteins in the first polycistronic cassette are separated by a sequence encoding a 2A polypeptide that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 10, 12 to 14, and 169; or (ii) one or more proteins in the second or third polycistronic cassette are separated by a 2A polypeptide encoding a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 10, 12 to 14, and 169; and / or (b) an internal ribosome entry site (IRES) sequence, wherein the IRES sequence is separated by ...c) an internal ribosome entry site (IRES) sequence, wherein the IRES sequence is separated by a sequence selected from the group consisting of SEQ ID NOs: 1 to 10, 12 to 1 The sequences of the groups consisting of NO: 11, 194 and 195 are at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical.

16. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a continuous nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

30.

17. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule contains a continuous nucleic acid sequence that is at least 95% identical to SEQ ID NO:

30.

18. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule contains a continuous nucleic acid sequence that is at least 98% identical to SEQ ID NO:

30.

19. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule comprises the sequential nucleic acid sequence of SEQ ID NO:

30.

20. The nucleic acid molecule of request item 1 further includes the ssUBC promoter.

21. The nucleic acid molecule of claim 20, wherein the ssUBC promoter comprises any one of SEQ ID NO: 141, 168 and 178.

22. A genetically modified pig cell comprising a nucleic acid molecule as claimed in any one of claims 1 to 21.

23. The genetically modified porcine cells of claim 22 further comprising the knockout of one, two, or three of the genes encoding glycoprotein α-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylaminohydrolase (CMAH), and β-1,4-N-acetylgalactosyltransferase 2 (β4GALNT2).

24. The genetically modified pig cell of claim 22, wherein the cistron of the first polycistronic cassette, the cistron of the second polycistronic cassette, and the cistron of the third polycistronic cassette are integrated into a single locus of the genetically modified pig cell.

25. The genetically modified pig cells of claim 22, wherein the nucleic acid molecule is integrated into a single genomic safe harbor locus selected from the group consisting of AAVS1, CCR5 and Fos5.

26. The genetically modified porcine cells of claim 22, wherein porcine endogenous retroviral (PERV) elements in the genetically modified porcine cells are knocked out.

27. A genetically modified organ comprising a nucleic acid molecule as claimed in any one of claims 1 to 21.

28. A genetically modified organ as claimed in claim 27, wherein the genetically modified organ is size compatible with human organs.

29. The genetically modified organ as claimed in claim 27, wherein the genetically modified organ is the liver.

30. The genetically modified organ as claimed in claim 27, wherein the genetically modified organ is a kidney.

31. The genetically modified organ as claimed in claim 27, wherein the genetically modified organ is the heart.

32. A method for genetically modifying porcine cells, comprising: (i) knocking out porcine endogenous retroviral (PERV) elements; (ii) knocking out the glycoprotein α-1,3-galactosyltransferase (GGTA1) gene, the cytidine monophosphate-N-acetylaminohydrolase (CMAH) gene, and the β-1,4-N-acetylgalactosyltransferase 2 (β4GALNT2) gene; and (iii) knocking in the first polycistronic cassette, the second polycistronic cassette, and the third polycistronic cassette of a nucleic acid molecule as claimed in any one of claims 1 to 21.

33. The method of claim 32, wherein the method comprises knocking out porcine endogenous retrovirus (PERV) pol elements.

34. An ex vivo procedure for liver perfusion, the procedure comprising: (a) providing an ex vivo liver from a genetically modified pig, wherein the genetically modified pig comprises a nucleic acid molecule as claimed in any one of claims 1 to 21 and a plurality of inactivated endogenous carbohydrate antigen genes, wherein the plurality of inactivated endogenous carbohydrate antigen genes comprise glycoprotein α-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydrolase (CMAH) and β-1,4-N-acetylgalactosyltransferase 2 (β4GALNT2); and (b) perfusing the liver ex vivo with a perfusion fluid comprising plasma, whole blood, erythrocytes, hemoglobin, human serum albumin or a combination thereof, thereby producing a perfused liver.

35. Use of any one of claims 1 to 21 in the preparation of a composition for producing a genetically modified pig organ for transplantation into a human individual.

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