CN117157406A - Cells, tissues, organs and animals having one or more modified genes for enhancing xenograft survival and tolerance - Google Patents

Cells, tissues, organs and animals having one or more modified genes for enhancing xenograft survival and tolerance Download PDF

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Publication number
CN117157406A
CN117157406A CN202180090204.7A CN202180090204A CN117157406A CN 117157406 A CN117157406 A CN 117157406A CN 202180090204 A CN202180090204 A CN 202180090204A CN 117157406 A CN117157406 A CN 117157406A
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sequence
nos
group
identity
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J·莱
覃文宁
阚一楠
J·克拉布特雷
M·尤德
D·赫加
D·安格勒斯·阿尔博雷斯
R·阿南德
P·佩拉特
R·厄恩斯特
V·帕拉加斯
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E Create Biotechnology Co ltd
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E Create Biotechnology Co ltd
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Priority claimed from PCT/US2021/059265 external-priority patent/WO2022104155A1/en
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Abstract

The present disclosure relates to cells, tissues, organs and/or animals having one or more modified genes for enhancing xenograft survival and/or tolerance. Furthermore, the present disclosure relates to methods of making and using the cells, tissues, organs and/or animals having one or more of the modified genes.

Description

Cells, tissues, organs and animals having one or more modified genes for enhancing xenograft survival and tolerance
Cross Reference to Related Applications
The application requires U.S. provisional application No. 63/113,650 filed 11/13/2020, U.S. provisional application No. 63/218,080 filed 7/2/2021; and U.S. provisional application No. 63/247,544 filed on 9 and 23 of 2021. Each of the foregoing applications is incorporated by reference herein in its entirety.
Description of electronically submitted text files
The contents of the text files submitted electronically herein together are incorporated herein by reference in their entirety: a computer readable format copy of the sequence listing (file name: egen_023_03wo_seqlist_st25.txt, recording date: 2021, 11/11, file size: about 2.35 megabytes).
Background
The shortage of human organs and tissues for transplantation has been increasing over the past few decades and is one of the most important unmet medical needs. Xenografts have the potential to supply transplanted organs for chronic organ failure patients almost limitlessly. The similarity of organ size and physiology, together with the elimination of molecular incompatibility by genetic engineering, makes pigs the choice donor for kidney xenografts. Preclinical studies have shown that porcine kidney xenografts can extend the life of non-human primate recipients for weeks to months (Higginbotham 2015, iwase 2015 b). However, due to the evolutionary distance between pigs and humans, porcine organs trigger rejection of the human immune system in a variety of forms, including (i) hyperacute rejection; (ii) Acute humoral rejection, consisting of thrombomodulin dysfunction and type II Endothelial Cell (EC) activation with leukocyte recruitment; (iii) Thrombotic microangiopathy consisting of intravascular thrombosis with platelet consumption and EC activation, fibrin deposition and thrombosis due to lack of thrombotic modulation, and (iv) chronic vascular disease. These adverse events are due, at least in part, to molecular incompatibilities between the donor and recipient, particularly with respect to genes involved in the complement, coagulation, inflammation, and immune response systems. Clinical use of xenogeneic organs (e.g., pigs) is hampered by these immunological incompatibilities, which have so far prevented the use of porcine cells, tissues and vascularized porcine organs in clinical xenografts.
Over the last two decades, several genetic modifications have been identified that reduce the inter-species incompatibility between pigs and humans. However, these previously identified genetic modifications have not achieved long term xenograft survival. Furthermore, the technical limitations of large-scale genome engineering prevent the integration of these modifications in a single animal.
Disclosure of Invention
There is a need to develop porcine cells, tissues, organs and/or porcine animals with novel combinations of genetic modifications for xenografts and to develop related methods.
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 the use of such cells, tissues, organs and animals in xenografts.
Genetic modification improves xenograft compatibility by introducing several modifications in pigs. These modifications eliminate the expression of antigens known to stimulate host versus graft responses. In addition, overexpression of proteins helps to modulate additional responses to the graft to optimize graft survival in the graft host. Disclosed herein are cassettes that express proteins that provide control of a host response. The cassettes may be used in combination in nucleic acids, also referred to herein as payloads, that are introduced into the genome of a pig.
The payload may contain a coagulation cassette that expresses a plurality of proteins, each of which may modulate one or more aspects of the host coagulation response. For example, expressed proteins that reduce or eliminate graft-related coagulation may include THBD and TFPI. THBD reduces blood clotting by converting thrombin from procoagulant to antithrombin. TFPI blocks activation of blood coagulation by inhibiting factor vila and prothrombinase.
The payload may contain a complement regulatory cassette that expresses a plurality of proteins, each of which may modulate one or more aspects of the host complement response, thereby reducing complement deposition and thereby reducing the anti-graft immune response. The expressed protein that reduces or eliminates complement deposition on the graft may include CD46, CD55, or CD59.CD46 is a cofactor that contributes to inactivation of complement components C3b and C4b by serum factor I. CD55 indirectly blocks the formation of membrane attack complexes by limiting the amplification of complement cascade convertases. Membrane attack complexes form on the cell membrane surface following complement activation, and the membrane attack complexes allow complement-mediated cell lysis to occur. CD59 prevents C9 from polymerizing and forming a membrane attack complex.
The payload may contain an innate immune cassette that reduces the innate immune response of the host against the graft. Expressed proteins that inhibit innate immune attack against the graft may include B2M, HLA-E and CD 47. 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 the innate immunity modulating component; such as a20 and HO 1. A20 reduced NF- κb activation in response to external stimulus. HO1 prevents vascular inflammation by cleaving heme groups resulting in the formation of release of biliverdin, carbon monoxide and ferrous iron.
Provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS 126-145 and 167-168; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding CD59, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOs 86, 108; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162; and
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162.
In embodiments, the nucleic acid comprises at least two of the polycistronic cassettes. In embodiments, the nucleic acid comprises three of the polycistronic cassettes. In embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs 24, 25, 29, 202, 203 and 207.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding CD59, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOs 86, 108; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 the group consisting of SEQ ID NOS 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, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A third cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (iv) A fourth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (v) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (vi) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (vi) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162.
In embodiments, the nucleic acid comprises at least two of the polycistronic cassettes. In embodiments, the nucleic acid comprises three of the polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO. 26 or SEQ ID NO. 204.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A third cistron encoding CD39, wherein the nucleic acid sequence of CD39 is selected from the group consisting of SEQ ID NOS 67-70 and SEQ ID NO 106; (iv) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; and (v) a poly A sequence selected from the group consisting of SEQ ID NOS.112-125, 154-156, 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 NOS 1-10, 12-14 and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding CD59, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOs 86, 108; (iii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iv) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (v) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 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 NOS 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, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A third cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (iv) A fourth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (v) A fifth cistron encoding PD-L1, wherein the nucleic acid sequence of PD-L1 is selected from the group consisting of SEQ ID NOS 89-91; (vi) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (vii) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (vii) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162.
In embodiments, the nucleic acid comprises at least two of the polycistronic cassettes. In embodiments, the nucleic acid comprises three of the polycistronic cassettes. In embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs 23, 27, 28, 201, 205 and 206.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding a PROCR, wherein the nucleic acid sequence of the PROCR is selected from the group consisting of SEQ ID NOs 92, 93 and 181; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(b) An inflammatory and apoptotic cassette comprising: (i) A first cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (ii) A second cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; and (iv) a poly A sequence selected from the group consisting of SEQ ID NOS.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 the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162; and (c) a complement-modulating and innate immune cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iv) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (v) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (vi) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162.
In embodiments, a nucleic acid disclosed herein comprises at least two of the polycistronic cassettes. In embodiments, the nucleic acid comprises three of the polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO. 30 or SEQ ID NO. 208.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding a PROCR, wherein the nucleic acid sequence of the PROCR is selected from the group consisting of SEQ ID NOs 92, 93 and 181; (iii) A third cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iv) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (v) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 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 NOS 1-10, 12-14 and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162; and
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOS.31-34 and 209-212.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding a PROCR, wherein the nucleic acid sequence of the PROCR is selected from the group consisting of SEQ ID NOs 92, 93 and 181; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 the group consisting of SEQ ID NOS 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, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A third cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (iv) A fourth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (v) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; and (vi) a poly A sequence selected from the group consisting of SEQ ID NOS.112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof, and (vii) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS.1-10, 12-14, and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO. 37 or SEQ ID NO. 215.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A third cistron encoding CD39, wherein the nucleic acid sequence of CD39 is selected from the group consisting of SEQ ID NOS 67-70 and SEQ ID NO 106; (iv) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (v) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 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 NOS 1-10, 12-14 and 159-162;
(b) A complement regulatory and inflammatory and apoptotic cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding CD59, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOs 86, 108; (iii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iv) A fourth cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (v) A fifth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (vi) A sixth cistron encoding PD-L1, wherein the nucleic acid sequence of PD-L1 is selected from the group consisting of SEQ ID NOS 89-91; (vii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (viii) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; (ix) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162, and
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO. 35 or SEQ ID NO. 213.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162, and
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from the group consisting of SEQ ID NOS.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 NOS.1-10, 12-14, and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs 36, 40, 214 and 218.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; and (iv) a poly A sequence selected from the group consisting of SEQ ID NOS.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(b) A complement regulatory and inflammatory and apoptotic cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A third cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (iv) A fourth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (v) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; and (vi) a poly A sequence selected from the group consisting of SEQ ID NOS.112-125, and 154-156, 159-162, 190-192, and combinations thereof; (vii) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162; and
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOS: 1-10, 12-14 and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs 41, 42, 219 and 220.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A third cistron encoding CD39, wherein the nucleic acid sequence of CD39 is selected from the group consisting of SEQ ID NOS 67-70 and SEQ ID NO 106; (iv) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (v) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; (vi) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162,
(b) A complement regulatory and inflammatory and apoptotic cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding CD59, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOs 86, 108; (iii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iv) A fourth cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (v) A fifth cistron encoding HO1, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (vi) A sixth cistron encoding PD-L1, wherein the nucleic acid sequence of PD-L1 is selected from the group consisting of SEQ ID NOS 89-91; (vii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (viii) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; and (ix) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162, and
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOS: 1-10, 12-14 and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises three of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs 38, 39, 173, 216, 217 and 226.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOs 1-10, 12-14 and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162,
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from the group consisting of SEQ ID NOS.112-125, and 154-156, 159-162, 190-192, and combinations thereof, (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS.1-10, 12-14, and 159-162; and
(d) A cellular immunity and coagulation cassette comprising: (i) A first cistron encoding LEA29Y, wherein the nucleic acid sequence of LEA29Y is selected from the group consisting of SEQ ID NOS 87-88; (ii) A second cistron encoding CD39, wherein the nucleic acid sequence of CD39 is selected from the group consisting of SEQ ID NOS 67-70 and SEQ ID NO 106; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; and (iv) a poly A sequence selected from the group consisting of SEQ ID NOS.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 NOS.1-10, 12-14, and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acids comprise at least three of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO. 43 or SEQ ID NO. 221.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from the group consisting of SEQ ID NOS.112-125, and 154-156, 159-162, 190-192, and combinations thereof, (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS.1-10, 12-14, and 159-162; and
(d) An inflammatory and apoptotic cassette comprising: (i) A first cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (ii) A second cistron encoding PD-L1, wherein the nucleic acid sequence of PD-L1 is selected from the group consisting of SEQ ID NOS 89-91; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from the group consisting of SEQ ID NOS.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 NOS.1-10, 12-14, and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acids comprise at least three of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs 44, 172, 222 and 225.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162,
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from the group consisting of SEQ ID NOS.112-125, and 154-156, 159-162, 190-192, and combinations thereof, (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS.1-10, 12-14, and 159-162, and
(d) An apoptosis and clotting cassette comprising: (i) A first cistron encoding XIAP, wherein the nucleic acid sequence of XIAP is selected from the group consisting of SEQ ID No. 110; (ii) A second cistron encoding CD39, wherein the nucleic acid sequence of CD39 is selected from the group consisting of SEQ ID NO. 106; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; and (iv) a poly A sequence selected from the group consisting of SEQ ID NOS.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 NOS.1-10, 12-14, and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acids comprise at least three of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO. 45 or SEQ ID NO. 223.
Provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding THBD, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding TFPI, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from the group consisting of SEQ ID NOS.112-125, and 154-156, 159-162, 190-192, and combinations thereof, (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS.1-10, 12-14, and 159-162;
(b) A complement regulatory cassette comprising: (i) A first cistron encoding CD46, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding CD55, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, and 154-156, 159-162, 190-192, and combinations thereof; (v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162;
(c) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding CD47, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from the group consisting of SEQ ID NOS.112-125, and 154-156, 159-162, 190-192, and combinations thereof, (v) at least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS.1-10, 12-14, and 159-162, and
(d) An apoptosis and cellular immune cassette comprising: (i) A first cistron encoding A20, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (ii) A second cistron encoding LEA29Y, wherein the nucleic acid sequence of LEA29Y is selected from the group consisting of SEQ ID NOS 87-88; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; and (iv) a poly A sequence selected from the group consisting of SEQ ID NOS.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 NOS.1-10, 12-14, and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acids comprise at least three of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO. 46 or SEQ ID NO. 224.
In embodiments, provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A clotting and innate immune cassette comprising: (i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOS 97-102, 166 and 265-266; (ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOS 77-83 and 180; (iii) A third cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iv) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (v) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (vi) at least one 2A polypeptide encoded by a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 159-162;
(b) An apoptosis and cellular immune cassette comprising: (i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (ii) A second cistron encoding HO1 protein, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) Poly A sequences selected from the group consisting of SEQ ID NOS.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 NOS.1-10, 12-14, and 159-162; and
(c) A complement regulatory cassette comprising: (i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOS: 1-10, 12-14 and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acids comprise at least three of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO:174 or SEQ ID NO: 227.
In embodiments, provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A clotting and innate immune cassette comprising: (i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOS 97-102, 166 and 265-266; (ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOS 77-83 and 180; (iii) A third cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iv) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (v) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; and (vi) at least one 2A polypeptide encoded by a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 159-162;
(b) An apoptosis and cellular immune cassette comprising: (i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (ii) A second cistron encoding a CTLA-4 protein, wherein the nucleic acid sequence of CTLA-4 is selected from the group consisting of SEQ ID NOs 87-88, 174 and 186; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOs 1-10, 12-14 and 159-162; and
(c) A complement regulatory cassette comprising: (i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, 253-258, 262-264; (ii) A third cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof; (v) At least one 2A polypeptide encoded by a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acids comprise at least three of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO 175 or SEQ ID NO 228.
In embodiments, provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding said THBD protein is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOs 1-10, 12-14 and 159-162;
(b) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence encoding said CD47 protein is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOs 1-10, 12-14 and 159-162;
(c) A complement regulatory cassette comprising: (i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding said CD46 protein is selected from the group consisting of SEQ ID NOs 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence encoding said CD55 protein is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOS: 1-10, 12-14 and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acids comprise at least three of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO:176 or SEQ ID NO: 229.
In embodiments, provided herein is a nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising: (i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding said THBD protein is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOs 1-10, 12-14 and 159-162;
(b) An innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence encoding said CD47 protein is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOs 1-10, 12-14 and 159-162;
(c) A complement regulatory cassette comprising: (i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding said CD46 protein is selected from the group consisting of SEQ ID NOs 71-76, 185, 200, 253-258, 262-264; (ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence encoding said CD55 protein is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOs 1-10, 12-14 and 159-162; and
(d) An inflammatory and apoptotic cassette comprising: (i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (ii) A second cistron encoding HO1 protein, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (iii) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250; (iv) A poly a sequence selected from the group consisting of SEQ ID nos. 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 NOS: 1-10, 12-14 and 159-162.
In embodiments, the aforementioned nucleic acids comprise at least two of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acids comprise at least three of the aforementioned polycistronic cassettes. In embodiments, the aforementioned nucleic acid comprises four of the aforementioned polycistronic cassettes. In embodiments, the nucleic acid comprises the sequence of SEQ ID NO. 177 or SEQ ID NO. 230.
Provided herein is a nucleic acid comprising or consisting of a CD46 cistron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 72, 73, 185 and 200. Provided herein is a nucleic acid comprising or consisting of a THBD cistron comprising 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, the CD46 cistron comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 72, 73, 185 and 200. In an embodiment, provided herein is a cell comprising a nucleic acid comprising or consisting of a THBD cistron comprising a nucleic acid sequence selected from any one of SEQ ID NOs 99-102 and 166. In embodiments, provided herein is a tissue, organ or animal comprising a cell comprising or consisting of a nucleic acid comprising a CD46 cistron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 72, 73, 185 and 200. In embodiments, provided herein is a tissue, organ or animal comprising a cell comprising or consisting of a nucleic acid comprising a THBD cistron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS 99-102 and 166.
In embodiments, the nucleic acids described herein comprise a 5'loxP site and a 3' loxP site selected from the group consisting of SEQ ID NOS 146-150 and 244. In embodiments, the nucleic acids described herein comprise 5 'and 3' insulator sites selected from the group consisting of SEQ ID NOS 49-58 and 163-164. In embodiments, the nucleic acids described herein comprise a 5 'guide RNA (gRNA) target sequence having a sequence selected from the group consisting of SEQ ID NOS: 47-48 and a 3' gRNA target sequence, and combinations thereof. In embodiments, the nucleic acids described herein comprise a Ubiquitous Chromatin Opening Element (UCOE) having the sequence of any one of SEQ ID NOs 19-22, 157 and 193. In embodiments, a nucleic acid described herein comprises: (i) A first Inverted Terminal Repeat (ITR) located at the 5 'end of the 5' polycistronic cassette and having the sequence of SEQ ID NO:17 or 18, and (ii) a second ITR located at the 3 'end of the 3' polycistronic cassette and having the sequence of SEQ ID NO:15 or 16. In embodiments, a nucleic acid described herein comprises at least two polycistronic cassettes. In embodiments, a nucleic acid described herein comprises at least three polycistronic cassettes. In embodiments, a nucleic acid described herein comprises at least four polycistronic cassettes. In embodiments, the nucleic acids described herein comprise fluorescent proteins. In embodiments, the nucleic acids described herein comprise a green fluorescent protein having the sequence of SEQ ID NO. 111, 242 or 246.
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 pig cells comprising any of the foregoing nucleic acids. In embodiments, provided herein are organs or tissues comprising any of the foregoing nucleic acids. In embodiments, provided herein are animals comprising any of the foregoing nucleic acids. In embodiments, provided herein are pigs comprising any of the foregoing nucleic acids. In embodiments, provided herein are pigs comprising any of the foregoing nucleic acids, wherein the pigs are free of PERV. In embodiments, provided herein are animals comprising any of the foregoing nucleic acids, wherein the animals comprise at least one inactivated carbohydrate antigen producing gene. In embodiments, provided herein is an animal comprising any of the foregoing nucleic acids, wherein the animal comprises at least one inactivated carbohydrate antigen producing gene, and wherein the inactivated carbohydrate antigen producing gene is glycoprotein α -1, 3-galactosyltransferase (GGTA 1), cytidine monophosphate-N-acetylneuraminidase (CMAH), β -1, 4-N-acetylgalactosamine transferase 2 (β 4GALNT 2), or a combination thereof.
In embodiments, provided herein are contiguous nucleic acid sequences 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 1kb. In embodiments, the contiguous nucleic acid sequence is at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 35kb, at least 40kb, at least 45kb, at least 50kb, at least 55kb, at least 60kb, or at least 65kb in length. In embodiments, the contiguous nucleic acid sequence encodes one or more of the foregoing polycistronic cassettes. In embodiments, the contiguous nucleic acid sequence encodes at least two of the foregoing polycistronic cassettes. In embodiments, the contiguous nucleic acid sequence encodes at least three of the foregoing polycistronic cassettes. In embodiments, the contiguous nucleic acid sequence encodes at least four of the foregoing polycistronic cassettes.
In embodiments, provided herein are chromosomal integration forms having any of SEQ ID NOs 23-46, 172-177 and 201-230.
In embodiments, provided herein are contiguous nucleic acid sequences 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 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 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 embodiments, provided herein are contiguous nucleic acid sequences that are at least 99% identical to any one of SEQ ID NOS.23-46, 172-177 and 201-230. In embodiments, provided herein are contiguous nucleic acid sequences at least 95% identical to any one of SEQ ID NOS.23-46, 172-177 and 201-230, wherein the sequences encode at least two polycistronic cassettes. In embodiments, provided herein are contiguous nucleic acid sequences at least 95% identical to any one of SEQ ID NOS.23-46, 172-177 and 201-230, wherein the sequences encode at least three polycistronic cassettes. In embodiments, provided herein are contiguous nucleic acid sequences at least 95% identical to any one of SEQ ID NOS.23-46, 172-177 and 201-230, wherein the sequences encode at least four polycistronic cassettes. In embodiments, provided herein are contiguous nucleic acid sequences that are 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.
In embodiments, provided herein are contiguous nucleic acid sequences encoding polycistronic gene product polypeptides expressed by any one of the polycistronic cassettes. In embodiments, provided herein is a polypeptide comprising one or more proteins encoded by a cistron of any of the polycistronic cassettes described herein.
In embodiments, provided herein are nucleic acid sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the non-coding sequence of any one of SEQ ID NOS.23 to 46, 172-177, and 201-230 and 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 to the coding sequence of any one of SEQ ID NOS.23 to 46, 172-177, and 201-230.
In embodiments, provided herein is a method of genetically modifying a cell, the method comprising: (i) knockout of Porcine Endogenous Retrovirus (PERV) elements; (ii) Knockout glycoprotein α -1, 3-galactosyltransferase gene (GGTA 1), cytidine monophosphate-N-acetylneuraminic acid hydrolase (CMAH), β -1, 4-N-acetylgalactosamine transferase 2 (β 4GALNT 2), or any combination thereof; and (iii) knock-in any one or more polycistronic cassettes according to any one of claims 1 to 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 the genetically modified cells. In embodiments, the animal is a pig or a human.
In embodiments, provided herein are landing gear (landing pad), wherein the landing gear is a nucleic acid comprising a first loxP site, a second loxP site, a promoter, and a poly a sequence. In embodiments, the landing gear comprises an insulator sequence. In embodiments, the landing gear comprises a fluorescent protein. In embodiments, the landing gear comprises a nucleic acid of SEQ ID NO. 248 or 249. In embodiments, provided herein are genetically modified cells comprising landing gear. In embodiments, provided herein are tissues, organs, or animals comprising genetically modified cells comprising a landing gear.
In embodiments, provided herein are nucleic acids comprising a first loxP site, a second loxP site, and one or more of the polycistronic cassettes described herein. In embodiments, provided herein are genetically modified cells comprising the foregoing nucleic acids. In embodiments, provided herein are tissues, organs, or animals comprising the foregoing genetically modified cells. In embodiments, the nucleic acid comprises the sequence of any one of SEQ ID NOS.201-230 or a nucleic acid that is 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 to any one of SEQ ID NOS.201-230. In embodiments, provided herein is a genetically modified cell comprising a nucleic acid of any one of SEQ ID NOS: 201-230 or a nucleic acid that is 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 to any one of SEQ ID NOS: 201-230. In embodiments, provided herein is a tissue, organ or animal comprising a genetically modified cell comprising a nucleic acid of any one of SEQ ID NOS: 201-230 or 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 to any one of SEQ ID NOS: 201-230.
Drawings
FIG. 1A illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI, THBD and CD39 cistron under the control of the mmEF 1a 1 (denoted mEF 1a 1) promoter, a complement regulatory cassette comprising CD46, CD55 and CD59 cistron under the control of the hEF 1a 1 promoter, and an innate immune and inflammatory and apoptotic cassette comprising B2M HLA-E fusion protein, CD47, a20, HO1 and PD-L1 cistron under the control of the CAG promoter. The genes within each cassette are separated by a nucleic acid sequence encoding a 2A peptide (E2A, T2A, F2A, P2A) or an Internal Ribosome Entry Site (IRES). Each cassette also contains a poly a sequence (e.g., HBB, bGH, or SPAActB). An exemplary nucleic acid having the cassette of FIG. 1A is SEQ ID NO.23.
FIG. 1B illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI and THBD under the control of the mmEF1 α1 promoter, a complement regulatory cassette comprising cd46_ll (denoted CD 46) and CD59 cistron under the control of the hef1 α1 (denoted hsEF1 α1) promoter, and an innate immune cassette comprising nucleic acid encoding B2M HLA-E fusion protein and CD47 cistron under the control of the CAG promoter. The genes within each cassette are separated by a nucleic acid or IRES encoding a 2A peptide (P2A or F2A). Each cassette also contains a poly a sequence (e.g., HBB, btGH, or SPAActB). An exemplary nucleic acid having the cassette of FIG. 1B is SEQ ID NO.25.
FIG. 1C illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI and THBD cistron under the control of the mmEF1 a 1 (denoted mEF a 1) promoter, a complement regulatory cassette comprising CD46 and CD59 cistron under the control of the hEF1 a 1 promoter, and an innate immune and inflammatory and apoptotic cassette comprising nucleic acid encoding B2M HLA-E fusion proteins and CD47, a20 and HO1 cistron under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids or IRES encoding a 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly a sequence (e.g., HBB, bGH, or SPAActB). An exemplary nucleic acid having the cassette of FIG. 1C is SEQ ID NO.26.
FIG. 1D illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI and THBD cistron under the control of the mmEF1 a 1 promoter, a complement regulatory cassette comprising CD46 and CD59 cistron under the control of the hEF1 a 1 (denoted hsEF1 a 1) promoter, and an innate immune cassette comprising nucleic acid encoding B2M HLA-E fusion protein and CD47 cistron under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids or IRES encoding a 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly a sequence (e.g., HBB, btGH, or SPAActB). An exemplary nucleic acid having the cassette of FIG. 1D is SEQ ID NO.24.
FIG. 2A illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI, THBD and CD39 cistron under the control of the mmEF1 a 1 promoter, a complement regulatory cassette comprising CD46i, CD59 and CD55 cistron under the control of the ssEF1 a 1 promoter, and a innate immune and inflammatory and apoptotic cassette comprising nucleic acid encoding B2M HLA-E fusion proteins and CD47, a20, HO1 and PD-L1 cistron under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids or IRES encoding a 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (e.g., HBB, btGH, or SPA/hsActB). An exemplary nucleic acid having the cassette of FIG. 2A is SEQ ID NO.27.
FIG. 2B illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI, THBD and CD39 under the control of the ssUBC promoter, a complement regulatory cassette comprising CD46i, CD59 and CD55 cistron under the control of the ssEF1 a 1 promoter, and a innate immune and inflammatory and apoptotic cassette comprising nucleic acid encoding B2M HLA-E fusion proteins and CD47, a20, HO1 and PD-L1 cistron under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (e.g., HBB, btGH, or SPA/hsActB). An exemplary nucleic acid having the cassette of FIG. 2B is SEQ ID NO.28.
FIG. 2C illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI and THBD under the control of the mmEF1 α1 promoter, a complement regulatory cassette comprising cd46_ll (denoted CD 46) and CD59 under the control of the hef1 α1 (denoted hsEF1 α1) promoter, and an innate immune cassette comprising 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 the 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or SPAACTB). An exemplary nucleic acid having the cassette of FIG. 2C is SEQ ID NO.29.
FIG. 2D illustrates an exemplary nucleic acid comprising: a clotting cassette comprising PROCR and THBD under the control of the ssUBC promoter, an inflammatory and apoptotic cassette comprising a20 and HO1 under the control of the ssEF1 a1 promoter, and a complement regulatory and innate immune cassette comprising CD46i, CD55 and CD47 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or SPA hsActB). An exemplary nucleic acid having the cassette of FIG. 2D is SEQ ID NO.30.
FIG. 2E illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI, PROCR and THBD (denoted THBDda) under the control of the ssUBC promoter, an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssHSPA8 (denoted ssHSPA8 (ATACseq)) promoter, and a complement regulatory cassette comprising CD46da1 (denoted CD46 da) and CD55 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 2E is SEQ ID NO.31.
FIG. 2F illustrates an exemplary nucleic acid comprising: a innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of ssUBC promoter, a clotting cassette comprising TFPI, PROCR and THBDda under the control of ssHSPA8 promoter, and a complement regulatory cassette comprising CD46da1 (denoted CD46 da) and CD55 under the control of CAG promoter. The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 2F is SEQ ID NO.32.
FIG. 2G illustrates an exemplary nucleic acid comprising: a innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of ssUBC promoter, a clotting cassette comprising TFPI, PROCR and THBDda under the control of ssHSPA8 promoter, and a complement regulatory cassette comprising CD46da1 (denoted CD46 da) and CD55 under the control of CAG promoter. The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 2G is SEQ ID NO.33.
FIG. 2H illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI, PROCR and THBDda under the control of the ssUBC promoter, a innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssEF1 a1 (denoted as ssEEF1 a1 (ATACseq)) promoter, and a complement regulatory cassette comprising CD46da1 (denoted as CD46 da) and CD55 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, T2A, F2A, P2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 2H is SEQ ID NO.34.
FIG. 2I illustrates an exemplary nucleic acid comprising: a clotting and innate immune cassette comprising CD47-2, PROCR and THBDda under the control of the ssUBC promoter, an apoptosis and cellular immune cassette comprising ssA and ssHO1 under the control of the ssHSPA8 promoter (denoted "ssHSPA8 (ATAC seq)"); and a 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 (e.g., IRES (FMDV) or IRES (EMCV)). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 2I is SEQ ID NO.174.
FIG. 2J illustrates an exemplary nucleic acid comprising: a clotting and innate immunity box comprising CD47-2, PROCR and THBDda under the control of the ssUBC promoter, an apoptosis and cellular immunity box comprising ssA and LEA29Y (denoted as "LEA 29Y-Ig") under the control of the ssHSPA8 promoter (denoted as "ssHSPA8 (ATAC seq)"); and a 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 (e.g., IRES (FMDV) or IRES (EMCV)). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 2J is SEQ ID NO.175.
FIG. 3A illustrates an exemplary nucleic acid comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the mmEF1 a 1 (denoted mEF a 1) promoter, a clotting cassette comprising TFPI, THBD and CD39 under the control of the hEF1 a 1 promoter, and a complement regulatory and inflammatory and apoptotic cassette comprising CD46i, CD55, CD59, a20, HO1 and PD-L1 (denoted PDL 1) under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids or IRES encoding 2A peptides (P2A, P2Amut1, E2A, F2A, P2Amut2, P2Amut3, T2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or SPAACTB). An exemplary nucleic acid having the cassette of FIG. 3A is SEQ ID NO.35.
FIG. 3B illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI and THBD under the control of the mmEF1 a 1 promoter, a complement regulatory cassette comprising CD46i and CD55 under the control of the hEF1 a 1 (denoted hsEF1 a 1) promoter, and a innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the CAG promoter. The genes within each cassette are separated by a nucleic acid encoding a 2A peptide (T2A, F2A, P a). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or SPAACTB). An exemplary nucleic acid having the cassette of FIG. 3B is SEQ ID NO.36.
FIG. 3C illustrates an exemplary nucleic acid comprising: a clotting cassette comprising PROCR and THBD under the control of the mmEF1 a1 promoter, a complement regulatory cassette comprising CD46i and CD55 under the control of the hEF1 a1 (denoted hsEF1 a 1) promoter, an innate immune and inflammatory and apoptotic cassette comprising B2M HLA-E fusion protein, CD47, a20 and HO1 under the control of the CAG promoter. The genes within each cassette are separated by a nucleic acid or IRES encoding a 2A peptide (P2A, T2A, F A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or SPAACTB). An exemplary nucleic acid having the cassette of FIG. 3C is SEQ ID NO.37.
FIG. 4A illustrates an exemplary nucleic acid comprising: a innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a clotting cassette comprising TFPI, THBD and CD39 under the control of the hsHSPA8 promoter, and a complement regulatory and inflammatory and apoptotic cassette comprising CD46da1 (denoted CD46 da), CD55, CD59, a20, HO1 and PD-L1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids or IRES encoding the 2A peptides (E2A, P2A, P2Amut1, P2Amut2, P2Amut3, T2A, F2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 4A is SEQ ID NO.38.
FIG. 4B illustrates an exemplary nucleic acid comprising: a innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a clotting cassette comprising TFPI, THBD and CD39 under the control of the hsHSPA8 promoter, and a complement regulatory and inflammatory and apoptotic cassette comprising CD46da2 (denoted as CD46 da), CD55, CD59, a20, HO1 and PD-L1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids or IRES encoding the 2A peptides (E2A, P2A, P2Amut1, P2Amut2, P2Amut3, T2A, F2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 4B is SEQ ID NO.39.
FIG. 4C illustrates an exemplary nucleic acid comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a clotting 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 a nucleic acid encoding a 2A peptide (E2A, P2A, F a). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or SPA/ACTB). An exemplary nucleic acid having the cassette of FIG. 4C is SEQ ID NO.40.
FIG. 4D illustrates an exemplary nucleic acid comprising: a innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a clotting cassette comprising TFPI and THBD under the control of the hsHSPA8 promoter, and a complement regulatory and inflammatory and apoptotic cassette comprising CD46da1 (denoted as CD46 da), CD55, a20 and HO1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 4D is SEQ ID NO.41.
FIG. 4E illustrates an exemplary nucleic acid comprising: a innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter, a clotting 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 CD46 da), CD55, a20 and HO1 under the control of the CAG promoter. The genes within each cassette are separated by nucleic acids or IRES encoding the 2A peptide (E2A, P2A, P Amut1, F2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 4E is SEQ ID NO.42.
FIG. 4F illustrates an exemplary nucleic acid comprising: a innate immune cassette comprising B2M HLA-E fusion protein and CD47 (denoted as cd47_2) under the control of ssUBC promoter, a clotting cassette comprising TFPI, THBD and CD39 under the control of ssHSPA8 promoter, and a complement regulatory and inflammatory and apoptotic cassette comprising CD46da2 (denoted as CD46 da), CD55, CD59, a20, HO1 and PD-L1 under the control of CAG promoter. The genes within each cassette are separated by nucleic acids or IRES encoding the 2A peptides (P2A, P2Amut1, E2A, F2A, P2Amut2, PTAmut3, T2A). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 4F is SEQ ID NO.173.
FIG. 4G illustrates an exemplary nucleic acid comprising: a coagulation cassette comprising TFPI (denoted "TFPI x") and THBDda under the control of the ssUBC promoter, a innate immune cassette comprising B2M HLA-E fusion protein and CD47-2 under the control of the ssHSPA8 promoter (denoted "ssHSPA8 (ATAC seq)"; and a 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 (e.g., IRES (FMDV) or IRES (EMCV)). Each cassette also contains a poly A sequence (e.g., hsHBB, btGH, or hsGH). An exemplary nucleic acid having the cassette of FIG. 4G is SEQ ID NO.176.
FIG. 4H illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI (TFPI x) and THBDda under the control of the ssUBC promoter; an innate immune cassette comprising a B2M HLA-E fusion protein and CD47-2 under the control of a ssHSPA8 promoter (denoted "ssHSPA8 (ATAC seq)"); a complement regulatory cassette comprising CD46da and CD55 under the control of the CAG promoter; and inflammatory and apoptotic cassettes under the control of the ssef1α1 promoter (denoted as "ssEEF1 A1"). The genes within each cassette are separated by a nucleic acid encoding a 2A peptide (P2A ) or an IRES (e.g., IRES (FMDV) or IRES (EMCV)). Each cassette also contains a poly A sequence (e.g., hsHBB, ocHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 4H is SEQ ID NO.177.
FIG. 5A illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI (Δp-Sel) and THBDda under the control of ssHSPA8 promoter, an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of ssUBC promoter, a cellular immune and clotting cassette comprising CTLA-4 (e.g., LEA 29Y) and CD39 under the control of ssEF 1a1 promoter, and a complement regulatory cassette comprising CD46da1 (denoted as CD46da (BC 1)) and CD55 under the control of CAG promoter. The nucleic acid further comprises a cassette 5' end GFP (labeled d2 GFP). The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly a sequence (e.g., SPA/PTCH2 pA, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5A is SEQ ID NO.43.
FIG. 5B illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI (Δp-Sel) and THBDda under the control of ssHSPA8 promoter, an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of ssUBC promoter, an inflammatory and apoptotic cassette comprising a20 and PD-L1 under the control of ssEF1 a1 promoter, and a complement regulatory cassette comprising CD46da1 (denoted CD46da (BC 1)) and CD55 under the control of CAG promoter. The nucleic acid also contains GFP at the 5' end of the cassette (labeled d2 GFP). The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (e.g., SPA/PTCH2, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5B is SEQ ID NO.44.
FIG. 5C illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI (Δp-Sel) and THBDda under the control of ssHSPA8 promoter, an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of ssUBC promoter, an apoptosis and clotting cassette comprising XIAP and CD39 under the control of ssEF1 a1 promoter, and a complement regulatory cassette comprising CD46da1 (denoted CD46da (BC 1)) and CD55 under the control of CAG promoter. The nucleic acid also contains GFP at the 5' end of the cassette (labeled d2 GFP). The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (e.g., SPA/PTCH2, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5C is SEQ ID NO.45.
FIG. 5D illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI (Δp-Sel) and THBDda under the control of ssHSPA8 promoter, an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of ssUBC promoter, an apoptosis and cellular immune cassette comprising CTLA-4 (e.g., LEA 29Y) and a20 under the control of ssEF1 a1 promoter, and a complement regulatory cassette comprising CD46da1 (denoted CD46da (BC 1)) and CD55 under the control of CAG promoter. The nucleic acid also contains GFP at the 5' end of the cassette (labeled d2 GFP). The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (e.g., SPA/PTCH2, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5D is SEQ ID NO.46.
FIG. 5E illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI (Δp-Sel) and THBDda under the control of ssHSPA8 promoter, an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of ssUBC promoter, a cellular immune and clotting cassette comprising LEA29Y and CD39 under the control of ssEF1 a1 promoter, and a complement regulatory cassette comprising CD46da1 (denoted CD46da (BC 1)) and CD55 under the control of CAG promoter. The nucleic acid also contains GFP at the 5' end of the cassette (labeled d2 GFP). The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (e.g., SPA/PTCH2 pA, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5A is SEQ ID NO.43.
FIG. 5F illustrates an exemplary nucleic acid comprising: a clotting cassette comprising TFPI (Δp-Sel) and THBDda under the control of ssHSPA8 promoter, an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of ssUBC promoter, an apoptosis and cellular immune cassette comprising LEA29Y and a20 under the control of ssEF1 a1 promoter, and a complement regulatory cassette comprising CD46da1 (denoted CD46da (BC 1)) and CD55 under the control of CAG promoter. The nucleic acid also contains GFP at the 5' end of the cassette (labeled d2 GFP). The genes within each cassette are separated by nucleic acids encoding the 2A peptide (E2A, P2A, P2Amut1, F2A). Each cassette also contains a poly A sequence (e.g., SPA/PTCH2, hsHBB, btGH or hsGH). An exemplary nucleic acid having the cassette of FIG. 5D is SEQ ID NO.46.
FIG. 6 illustrates an exemplary nucleic acid comprising: an innate immune cassette comprising B2M HLA-E fusion protein and CD47 (denoted as CD 47-2) under the control of the ssUBC promoter, an inflammatory and apoptotic cassette comprising a20, HO1 and PD-L1 under the control of the hshshspa 8 promoter, a complement regulatory cassette comprising CD46da2 (denoted as CD46da (BC 2)), CD55 and CD59 under the control of the CAG promoter, and a clotting cassette comprising TFPI (denoted as TFPI), CD39 and THBDda under the control of the ssICAM2 promoter. The genes within each cassette were separated by nucleic acids encoding the 2A peptides (P2A, P2Amut3, T2A, F2A, P2Amut2, P2Amut1 and E2A). Each cassette also contains a poly A sequence (e.g., 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 arterial and portal venous blood flow combined in a pig liver xenograft comprising nucleic acid containing the polycistronic cassette of fig. 2D, treated with or without anti-GPIb fragmenting murine antibody and the GPIIb/IIIa inhibitor eptifibatide (ab/eptifibatide).
FIG. 7B shows lactate clearance in porcine liver xenografts comprising nucleic acid comprising the polycistronic cassette of FIG. 2D treated with or without anti-GPIb fragmented murine antibody and GPIIb/IIIa inhibitor eptifibatide (ab/eptifibatide).
Fig. 8A shows platelet counts over time in porcine liver xenografts treated with or without anti-GPIb fragmented murine antibodies and the GPIIb/IIIa inhibitor eptifibatide (ab/eptifibatide), as measured by a cytometer.
Fig. 8B shows platelet counts over time in porcine liver xenografts treated with or without anti-GPIb fragmented murine antibodies and the GPIIb/IIIa inhibitor eptifibatide (ab/eptifibatide), as measured by flow cytometry.
Fig. 9A shows an image of a porcine liver xenograft. The superior and inferior hepatic vena cava (IVC) and portal vein are indicated in the image.
Fig. 9B shows lactate clearance in a pig liver xenograft during ex vivo normothermic perfusion of the pig liver xenograft.
Fig. 10A shows the average troponin I concentration (ng/mL) over time in cardiac xenografts that were either stored in a Chilled Solution (CS) or perfused with an oxygenated Steen solution with erythrocytes (IM). The following cardiac xenografts were studied: (a) Cardiac xenografts ("TKOs") knocked out of GGTA1, CMAH and B4GALNT2 and with variable expression of human complement and thrombomodulin genes; (b) Cardiac xenografts ("gtko.hdaf") knocked out of GGTA1 and expressing CD 55; and (c) wild-type cardiac xenograft "WT".
Fig. 10B shows the average troponin I concentration (ng/mL) in cardiac xenografts stored in a Chilled Solution (CS) or perfused with an oxygenated Steen solution with erythrocytes (IM) over 1 hour. The following cardiac xenografts were studied: (a) Cardiac xenografts ("TKOs") knocked out of GGTA1, CMAH and B4GALNT2 and with variable expression of human complement and thrombomodulin genes; (b) Cardiac xenografts ("gtko.hdaf") knocked out of GGTA1 and expressing CD 55; and (c) wild-type cardiac xenograft "WT".
Fig. 10C is a graph of troponin I concentration (ng/mL) at 1 hour and final time points in cardiac xenografts stored in Chilled Solution (CS) or perfused with oxygenated Steen solution (IM) with erythrocytes.
Fig. 11A is a graph showing cardiac output over time of TKO cardiac xenografts stored in a Chilled Solution (CS).
Fig. 11B is a graph showing cardiac output over time in TKO cardiac xenografts perfused (IM) with an oxygenated Steen solution with erythrocytes.
Fig. 11C is a graph showing cardiac output over time of TKO cardiac xenografts stored in a Chilled Solution (CS).
Fig. 11D is a graph showing cardiac output over time in TKO cardiac xenografts perfused (IM) with an oxygenated Steen solution with erythrocytes.
FIG. 12A is an exemplary polycistronic cassette comprising a poly A (pA) sequence, an untranslated region (UTR), an intron, an IRES, a 2A peptide, and three cistrons (labeled "1", "2", and "3").
FIG. 12B is an exemplary nucleic acid comprising multiple polycistronic cassettes (termed "transcription unit 1", "transcription unit 2", "transcription unit 3" and "transcription unit 4"). Each transcription unit contains a promoter, a poly A (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 "lox 66"). A Ubiquitous Chromatin Opening Element (UCOE) separates the second and third polycistronic cassettes.
Fig. 13A shows expression of nucleic acids comprising the polycistronic cassette of fig. 2D in donor cells, porcine fetal cells, and adult porcine cells by flow cytometry. The circled cells contained the polycistronic cassette of figure 2D. Fig. 13B shows the expression of nucleic acids comprising the polycistronic cassette of fig. 2D in donor cells, porcine fetal cells, and adult porcine cells by immunohistochemistry.
Fig. 14 shows that ear punch derived cells (ear punch derived cell, EPDC) comprising the polycistronic cassette of fig. 2D express CD46 and CD55.
Figure 15A shows surface protein expression of human CD46 and CD55 on Ear Punch Derived Cells (EPDCs) or isotype control stained cells from PL15S pigs or from GGTA1 and B4GALNT2 Double Knockout (DKO) pigs as controls. Fig. 15B evaluates complement activation in PL15S pigs or DKO pigs. PL15S pigs express the polycistronic cassette of fig. 2D.
Figures 16A-16C illustrate that the nucleic acids described herein provide protection against complement deposition in islet cells. Fig. 16A shows that cells comprising nucleic acids containing the polycistronic cassette of fig. 2D provide protection against complement deposition in islet cells in the absence of anti-CD 46 and/or anti-CD 55 blocking antibodies. In the absence of anti-CD 46 and/or anti-CD 55 blocking antibodies, cells comprising nucleic acids containing the polycistronic cassette of fig. 4D provided protection against complement deposition in islet cells, as demonstrated by the percent decrease in C3B (fig. 16C) and the decrease in average fluorescence intensity of C3B (fig. 16B).
FIGS. 17A-17B show the 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 upon ligation to the 2A peptide. FIG. 17B shows that CD46da (labeled "CD 46") is expressed when linked to the 2A peptide. Fig. 17C shows that nucleic acids comprising CD46da ("CD 46") and CD55 reduce complement deposition.
FIGS. 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) hsCD46da 2-F2A-hsCD 55; (v) hsCD46da 2-F2A-hsCD 59; (vi) hsCD 55-P2A-hsCD 59; or (viii) hsCD46da 2-F2A-hsCD 55-P2A-hsCD 59. Fig. 18A-18C show the respective expression of CD46 (fig. 18A), CD55 (fig. 18B), and CD59 (fig. 18C). Fig. 18D shows that expression of each of these nucleic acids reduces complement deposition. In each of fig. 18A-18D, CD46da2 is labeled "46", CD55 is labeled "55", and CD59 is labeled "59".
FIG. 19A shows the expression of CD46 isoforms BC-Cyt1 ("SG_Bc1"), bc-Cy2 ("SG_Bc2"), and ABC1 ("SG_ABC1"). FIG. 19B shows the ability of CD46 isoforms BC-Cyt1 ("SG_Bc1"), bc-Cy2 ("SG_Bc2"), and ABC1 ("SG_ABC1") to reduce complement deposition. Expression of nucleic acids comprising the polycistronic cassette of fig. 2D also reduced complement deposition.
FIG. 20A shows that AEC, KEC, PUVEC and HUVEC cells transfected with nucleic acid comprising the polycistronic cassette of FIG. 2D expressed CD46 and CD55 (FIG. 20A). FIG. 20B shows that AEC, KEC, PUVEC and HUVEC cells transfected with nucleic acid comprising the polycistronic cassette of FIG. 4C expressed CD46 and CD55 (FIG. 20B). FIG. 20C shows that AEC, KEC and PUVEC cells containing nucleic acids containing the polycistronic cassette of FIG. 2D reduced complement deposition in AEC, KEC and PUVEC cells. FIG. 20D shows that AEC, KEC and PUVEC cells containing nucleic acids containing the polycistronic cassette of FIG. 4C reduced complement deposition in AEC, KEC and PUVEC cells.
Fig. 21A shows that expression of nucleic acids containing the polycistronic cassette ("15S") of fig. 2D reduced complement deposition even in the presence of Donor Specific Alloantibodies (DSA). Fig. 21B shows the concentration of DSA after implantation. "POD" refers to the number of days after implantation.
Fig. 22 shows a timeline of the experiment of example 1.
FIG. 23 shows images of CD46, CD55, CD59, HLA-E, CD47 and PD-L1 expression in TKO-A and TKO-B xenografts as compared to wild type xenografts in animals of example 1A.
FIG. 24 shows images of CD46, CD55, CD47, EPCR, THBD, A20 and HO1 expression in TKO-C animals of example 1A.
FIG. 25 shows images of CD46, CD55, B2M, CD47, TFPI and THBD expression in TKO-D animals of example 1A.
Fig. 26 shows serum creatinine (labeled Cr) concentrations up to 313 days post-implantation in the animals of example 1A.
Fig. 27 shows images of kidney tissue stained with hematoxylin and eosin or complement component C4d 217 days ("POD 217"), 230 days ("POD 230"), or 105 days ("POD 105") after implantation. Animal B6 received TKO-B xenografts. Animal C9 received TKO-F xenografts. Animal D2 received TKO-G xenografts.
Fig. 28A shows an exemplary landing gear. The landing gear contains an insulator sequence (HS 4), loxP sites (lox 71 and lox 2272), poly A sequences (SPAACT 1 pA and hsHBBpA), CAG promoter and marker gene (d 2 BFP). Fig. 28B shows the incorporation of the 15S5 payload of fig. 2I at a genomic safe harbor locus. Fig. 28C shows an exemplary landing gear. Landing gear contains loxP sites (loxP and lox 2272), hsEF 1. Alpha.1, poly A sequence (SPAC 2 pA) and Blue Fluorescent Protein (BFP). The landing gear is integrated between exon 1 and exon 2 of AAVS1 genomic safety harbor.
Fig. 29A-29E are graphs showing Blood Urea Nitrogen (BUN) and creatinine in five monkeys containing TKO-F xenografts.
Fig. 30A-30B are graphs showing BUN and creatinine levels in two monkeys containing TKO-G xenografts.
Fig. 31 and 32 are graphs showing BUN and creatinine levels in two monkeys containing xenografts containing the polycistronic cassette of fig. 2H.
FIG. 33 shows expression of a nucleic acid comprising the polycistronic cassette of FIG. 2E in fibroblasts from approximately Kxia (Yorkshire) pigs.
FIG. 34 (see circled cells) shows expression of nucleic acids comprising the polycistronic cassette of FIG. 2E in fibroblasts from Ukatan (Yucatan) pigs.
FIG. 35 shows expression of a nucleic acid comprising the polycistronic cassette of FIG. 2D in fibroblasts from Ukatan pigs.
FIG. 36 (see circled cells) shows expression of nucleic acids comprising the polycistronic cassette of FIG. 2D in fibroblasts containing PERV knockouts from Ukatan pigs.
Fig. 37A shows the expression of a20 in islet cells comprising the polycistronic cassette of fig. 4A. Fig. 37B shows that expression of a20 in islet cells comprising the polycistronic cassette of fig. 4B protects cells from tnfα -mediated apoptosis.
FIGS. 38A-38C show the expression of the transgene of TKO-F xenografts of example 1A in tissues, glomeruli and tubules.
FIG. 39 shows expression of the transgene of 15S4 in tissues, glomeruli and tubules of monkeys containing nucleic acid comprising the polycistronic cassette of FIG. 2H.
FIG. 40 shows immunohistochemical images of transgene expression in monkeys containing nucleic acid containing the polycistronic cassette of FIG. 2H.
Fig. 41A-41D show comparative expression of the polycistronic cassettes of fig. 2H ("15S 4") or fig. 2E ("15S 1") in monkeys containing kidney xenografts. Fig. 41A shows transgene expression by immunohistochemistry. FIGS. 41B-41D show transgene expression in the entire tissue (FIG. 41B), glomeruli (FIG. 41C) and tubular (FIG. 41D).
FIG. 42A shows expression of the 15S1 transgene in porcine fetal kidney relative to expression in wild type Ukatan pigs. FIG. 42B shows expression of the 15S1 transgene in porcine neonatal kidney relative to expression in wild type Ukatan pigs. Fig. 42C shows expression of the 15S1 transgene in porcine adult kidney relative to expression in wild type ukant pigs.
FIG. 43A shows expression of 15S1 transgenic TFPI, EPCR and THBD in porcine fetal, neonatal and adult kidney tissue. FIG. 43B shows expression of 15S1 transgenic B2M HLA-E fusion proteins and CD47 in porcine fetal, neonatal and adult kidney tissue. FIG. 43C shows expression of 15S1 transgenic CD46 and CD55 in porcine fetal, neonatal and adult kidney tissue. FIG. 43D shows the expression of each of the 15S1 transgenes in two different fetal porcine kidney tissues.
FIG. 44 shows the expression of CD47 by islet cells comprising nucleic acids comprising the polycistronic cassette of FIG. 2H ("15S 4") or FIG. 4A ("17M 1").
Fig. 45 shows that dissociated islet cells comprising the polycistronic cassette ("15S 4") of fig. 2H are superior to islet cells comprising the polycistronic cassette ("17M 1") of fig. 4A in protecting against phagocytosis.
FIG. 46 provides an example of how to produce a transgenic pig comprising a nucleic acid as described herein.
FIG. 47A is a schematic representation of a standard promoter. FIG. 47B is a schematic representation of a promoter comprising CpG islands.
FIGS. 48A-48C show the expression of the CD47 cistron in fibroblasts (FIG. 48A), beta islet cells (FIG. 48B) and pAEC-SV40 cells (FIG. 48C) using promoters containing or lacking CpG islands. FIGS. 48D-48F show the expression of CD46 in fibroblasts (FIG. 48D), beta islet cells (FIG. 48E) and pAEC-SV40 cells (FIG. 48F) using promoters containing or lacking CpG islands. The x-axis of each of FIGS. 48A-48F represents the SEQ ID NO for the cistron expressing promoter.
Fig. 49 shows an immunohistochemical image of islet cells containing nucleic acid containing the polycistronic cassette of fig. 4A transplanted in a diabetic mouse. Images show staining for insulin, glucagon and nuclei in individual mice.
Fig. 50 shows the experimental design of the experiment of example 16.
Fig. 51 shows blood glucose levels of NSG mice receiving neonatal porcine islet grafts comprising nucleic acids comprising the polycistronic cassette of fig. 4A.
Fig. 52A-52B show fasting blood glucose concentration, total insulin, and porcine C-peptide in two cynomolgus monkeys receiving a neonatal porcine islet graft comprising nucleic acid comprising the polycistronic cassette of fig. 4A.
FIG. 53A shows that cells expressing 14P nucleic acid are protected from NK cell mediated lysis. FIG. 53B shows that cells expressing 14P and 14S nucleic acids are protected from NK cell mediated lysis as compared to Wild Type (WT) cells or K562 cancer cells that do not express 14P and 14S nucleic acids.
Fig. 54A shows surface expression of human EPCR and THBD proteins on Wild Type (WT), GGTA1, B4GALNT2 and CMAH Triple Knockout (TKO) aortic derived endothelial cells (AEC), human umbilical cord derived endothelial cells (Hu UVEC) and umbilical cord, aortic or renal (KEC) derived payload 15S porcine endothelial cells (porcine UVEC) as shown in the left histogram. Payload 15S cells contained nucleic acid containing the polycistronic cassette of fig. 2D. FIG. 54B shows that various cells expressing nucleic acid containing the polycistronic cassette "15S" of FIG. 2D have functional THBD and PROCR, as indicated by elevated activated protein C concentrations compared to cells not expressing 15S. The following cell types were evaluated: alveolar Epithelial Cells (AEC), renal epithelial cells (KEC), human umbilical vein endothelial cells ("Hu UVEC"), porcine umbilical vein endothelial cells ("porcine UVEC"), and epicardial derived cells (EPDC).
FIG. 55 is a diagram illustrating an exemplary method of preparing a genetically modified animal comprising a nucleic acid as described herein. In a first step (round 1), CRISPR-Cas9 mediated non-homologous end joining (NHEJ) and homology directed repair are performed to knock out the GGTA1, CMAH and B4GALNT2 genes (e.g., TKO) from the genome of a cell (e.g., a fibroblast) and introduce a landing gear at a genomic safe harbor site (e.g., intron 1 of the genomic safe harbor AAVS1 site or the 3' flanking region of the AAVS1 site) in the cell. The introduction of the landing gear results in the introduction of loxP sites in the genome of the cell. In the second step (round 2), a recombinase-mediated cassette exchange is used to introduce nucleic acids comprising polycistronic cassettes (e.g., payload KI). In the third step (round 3), the porcine endogenous retrovirus element (PERV) was knocked out using CRISPR-Cas9 NHEJ.
Fig. 56A shows expression of human CD47 protein on Wild Type (WT) and GGTA1 plus B4GALNT2 Double Knockout (DKO) Ear Punch Derived Cells (EPDCs) and Fetal Fibroblasts (FFs) from PL15S pigs. Fig. 56B shows decreased phagocytosis in PL15S pigs. The phagocytosis index is determined by the following equation: the total phagocytic signal divided by the sum of the total phagocytic signal plus the non-phagocytic signal. Fig. 56C shows that PL15S cells are protected from phagocytosis. PL15S pigs express the polycistronic cassette of fig. 2D.
Detailed Description
I. Definition of the definition
The terms "pig", "pig" and "pig" are used interchangeably herein to refer to any animal associated with various breeds of the domestic pig species wild boar (Sus scrofa).
When used in reference to a fragment or derivative of a protein or polypeptide, the term "biological activity" means that the 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 to a gRNA, sometimes also referred to herein as a single guide RNA (sgRNA), binding to a target DNA sequence when complexed with a guide RNA, and/or cleaving one or more DNA strands.
The terms "treatment", "treating", "alleviating" and the like when used in the context of a disease, injury or disorder are used herein to generally mean obtaining a desired pharmacological and/or physiological effect, and may also be used to refer to ameliorating, reducing and/or reducing the severity of one or more symptoms of the condition being treated. The effect may be prophylactic in terms of a complete or partial delay of onset or recurrence of a disease, disorder, or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of the disease or disorder and/or adverse effects attributable to the disease or disorder. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, particularly a human, and includes: (a) Preventing the occurrence of the disease or disorder in a subject who may be predisposed to the disease or disorder but has not yet been diagnosed as having the disease or disorder; (b) Inhibiting (e.g., preventing the progression of) the disease or disorder; or (c) alleviating the disease or disorder (e.g., causing regression of the disease or disorder, thereby ameliorating one or more symptoms).
The term "simultaneously" as used herein refers to an event occurring simultaneously with another event, such as within seconds, milliseconds, microseconds, or less as compared to the occurrence of another event.
The term "knockout" or "knockout" as used herein refers to the deletion, inactivation or ablation of a gene or defective gene in a pig or other animal or any cell in a pig or other animal. As used herein, KO may also refer to a method of performing or having performed deletion, inactivation, or ablation of a gene or portion thereof.
The term "knock in" or "knock in" as used herein refers to the addition, substitution or mutation of one or more nucleotides of a gene in a pig or other animal or any cell in a pig or other animal. As used herein, KI may also refer to a method that performs or has performed the addition, substitution, or mutation of one or more nucleotides of a gene or portion thereof.
The term "PERV-free" with respect to porcine tissue, organ or cell refers to a tissue, organ or cell having at least 75% inactive Porcine Endogenous Retrovirus (PERV) elements. The PERV element may be the gag, pol or env genes. 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% free of PERV.
In the context of two or more nucleic acid or polypeptide sequences, the term "percent identity" refers to a specified percentage of identical nucleotide or amino acid residues that two or more sequences or subsequences have when compared. The percent identity can be calculated using the online available tool CLUSTALW 2. The following default parameters may be used for the CLUSTALW2 alignment: protein weight matrix = Gonnet; vacancy open = 10; vacancy extension = 0.1.
The term "conservative substitution" refers to the exchange of one amino acid for another in the following amino acid group: (i) Aliphatic amino acids (alanine, valine, leucine, and isoleucine); (ii) Amino acids having hydroxyl groups (serine and threonine); (iii) acidic amino acids (glutamic acid and aspartic acid); (iv) Amino acids having amide side chains (asparagine and glutamine); (v) basic amino acids (lysine and arginine); (vii) Amino acids with aromatic side chains (phenylalanine, tyrosine and tryptophan). In embodiments, provided herein are nucleic acids encoding proteins containing conservative substitutions. For example, a protein may have a conservative substitution of alanine for valine. In some aspects, the number of amino acid changes of the encoded protein may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
As used herein, the term "polycistronic cassette" refers to a nucleic acid encoding two or more "cistrons" or genes under the control of a single promoter and a poly a sequence. The term "polycistronic cassette" is used interchangeably herein with "transcription unit". In embodiments, the polycistronic cassette comprises a cDNA. In embodiments, the polycistronic cassette comprises DNA. In embodiments, the polycistronic cassette comprises one or more introns. An exemplary polycistronic cassette is depicted in fig. 12A. An exemplary nucleic acid comprising a plurality of polycistronic cassettes is depicted in fig. 12B.
As used herein, the term "control element" refers to a nucleic acid sequence that controls cistron expression. Exemplary control elements include promoters, enhancers, poly-a signals, and terminators. The cistron whose expression is regulated by the control element is referred to as being operably controlled by the control element.
As used herein, the term "isolated" in reference to, for example, a nucleic acid or cell refers to a nucleic acid or cell that has been purified to separate it from other components; for example, the isolated nucleic acid may be purified from bacterial cells used to produce the nucleic acid, or the cells may be isolated from animals used for therapeutic purposes.
As used herein, the term "heterologous" refers to modulating the expression of nucleic acid in an organism as a result of manipulation of recombinant DNA in contrast to the expression of endogenous nucleic acid of the host organism. Typically, the cistron and/or one or more control elements that regulate the expression of the cistron are artificial or derived from a different species than the host organism. In some aspects, the cistron and control elements can be from the same species as the host organism, but introduced by recombinant methods that alter the control and expression of the cistron as compared to an equivalent native cistron.
Nucleic acids comprising one or more polycistronic cassettes
Porcine xenografts are widely compatible with human organ size and physiology and are ethically accepted by the general population in the united states. However, xenograft porcine tissue initiates a complex series of events, leading to graft rejection reactions, including: hyperacute rejection due to the presence of preformed antibodies to porcine antigens, complement activation and hypercoagulability, and enhancement of innate and adaptive immune responses due to molecular incompatibility. The present disclosure uses genetic engineering methods to address the shortcomings of current xenografts.
The present disclosure provides nucleic acids comprising one or more polycistronic cassettes for the genetic engineering of cells, organs, tissues and animals.
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 comprises 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 (e.g., LEA 29Y), XIAP, and combinations thereof. In embodiments, the polycistronic cassette is flanked by insulator sequences, loxP sites, or a combination thereof.
In embodiments, each cistron of the polycistronic cassette is separated by a nucleic acid encoding a 2A polypeptide or a sequence comprising an Internal Ribosome Entry Site (IRES). In embodiments, 1, 2, 3, 4, or 5 polycistronic cassettes do not comprise a nucleic acid encoding a 2A polypeptide. Each polycistronic cassette is under the control of a promoter. Exemplary promoters are described in table B. Each polycistronic cassette further comprises one or more poly a (also referred to as "polyA") and/or terminator sequences. Exemplary poly a sequences and/or terminator sequences are provided in table C.
When the name of the cistron, promoter or poly-A sequence contains a lowercase letter before the name of the cistron, promoter or poly-A sequence, the lowercase letter refers to the genus and species from which the cistron is isolated. For example, ssA refers to a20 from wild boars (Sus scrofa), hsCD46 refers to CD46 from Homo sapiens (Homo sapiens), mmA refers to a20 from mice (Mus museuus), and btA refers to a20 from cattle (Bos taurus). When the lower case letter "s" is used before the cistron, promoter or poly-A sequence name, s refers to the wild boar. When the lowercase "h" is used before the cistron, promoter or poly-a sequence name, h refers to homo-or homo-. When the lower case letter "m" is used before the cistron, promoter or poly-a sequence name, m refers to the mouse.
In embodiments, the nucleic acid sequences of the cistrons are provided in table a. The NCBI reference sequences, gene IDs, and protein IDs of table a are incorporated herein by reference. In embodiments, the cistron is a porcine cistron. In embodiments, the cistron is a human cistron. In embodiments, the cistron is a mouse cistron.
Table A
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In embodiments, the polycistronic cassette comprises a nucleic acid encoding a beta-2 microglobulin (B2M). The B2M gene encodes a serum protein that is found to associate with the Major Histocompatibility Complex (MHC) class I heavy chain on the surface of almost all nucleated cells. In embodiments, the polycistronic cassette comprises a nucleic acid encoding a major histocompatibility complex class 1E (HLA-E). HLA-E belongs to the class I heavy chain paralogs of HLA. The class I molecule is a heterodimer comprising a heavy chain and a light chain (beta-2 microglobulin). In embodiments, the polycistronic cassette comprises nucleic acids encoding B2M and HLA-E proteins. In embodiments, the polycistronic cassette comprises a nucleic acid encoding a fusion protein of B2M and HLA-E (which is referred to as a "B2M HLA-E fusion protein"). In embodiments, the B2MHLA-E fusion protein is encoded by a nucleic acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO. 62, 66 or 105. In embodiments, the B2M HLA-E fusion protein comprises a peptide epitope. In embodiments, the peptide epitope is encoded by a nucleic acid having the sequence of SEQ ID NO. 260 or SEQ ID NO. 261. B2M HLA-E fusion proteins comprising peptide epitopes are referred to herein as "single chain trimers". In embodiments, the amino acid sequence of the epitope is VMAPRTLIL (SEQ ID NO: 197) or VMAPRTLFL (SEQ ID NO: 198). In embodiments, the amino acid sequence of the epitope is MAPRTLIL (SEQ ID NO: 251) or MAPRTLFL (SEQ ID NO: 252). In embodiments, the epitope is located at the N-terminus of the B2M-HLA-E fusion protein. The nucleic acids having the sequences represented by SEQ ID NOS.23, 24, 26, 201, 202 and 204 encode a B2M HLA-E fusion protein comprising a peptide epitope having the amino acid sequence represented by SEQ ID NO. 251. The nucleic acid having the sequence represented by 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, 229 and 230 encodes a B2MHLA-E fusion protein comprising a peptide epitope having the amino acid sequence represented by SEQ ID NO. 198. The nucleic acids having the sequences represented by SEQ ID NOS.25, 27-29, 35-37, 40, 203, 205-207, 213-215 and 218 encode a B2M HLA-E fusion protein comprising a peptide epitope having the amino acid sequence represented by SEQ ID NO. 197.
In an embodiment, the polycistronic cassette comprises a nucleic acid encoding heme oxygenase-1 (HO 1). HO1 is an essential enzyme in heme catabolism that cleaves heme to form biliverdin and carbon monoxide (a putative neurotransmitter), which is subsequently converted to bilirubin by a biliverdin reductase. Heme oxygenase activity is induced by its substrate heme and various non-heme substances. In embodiments, the nucleic acid encoding HO1 is isolated from a wild boar. In embodiments, HO1 is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding an a20 protein. The A20 protein is a zinc finger protein and ubiquitin editing enzyme, and has been shown to inhibit NF- κB activation and TNF-mediated apoptosis. The A20 protein has ubiquitin ligase and deubiquitinase activities and participates in cytokine mediated immune and inflammatory reactions. In embodiments, the nucleic acid encoding the a20 protein is isolated from a wild boar. In embodiments, the A20 protein is encoded by a nucleic acid having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding a cluster of differentiation 39 (CD 39) protein. The protein encoded by the gene is plasma membrane protein, and extracellular ATP and ADP can be hydrolyzed into AMP. In embodiments, CD39 is encoded by a nucleic acid having a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOS: 67-70, 106, 253-258 and 262-264.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding a cluster of differentiation 46 (CD 46) protein. The protein encoded by this gene is a type I membrane protein and is the regulatory part of the complement system. The encoded protein has cofactor activity and can inactivate complement components C3b and C4b by serum factor I, thereby protecting the host cell from complement. In embodiments, CD46 is encoded by a nucleic acid having a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from SEQ ID NOS: 71-76 and 185. In embodiments, CD46 comprises splice site mutations in which both the acceptor site and the donor site are removed. CD46_LL (SEQ ID NO:71 or SEQ ID NO:258 or SEQ ID NO: 254), CD46da1 (SEQ ID NO: 72), CD46da2 (SEQ ID NO: 73), CD46da (SEQ ID NO:72, 73, 185, 200 or 256), and CD46i (SEQ ID NO:74-75, SEQ ID NO: 255) refer to CD46 proteins with splice site mutations. In embodiments, the polycistronic cassette comprising a gene encoding a CD46 protein encodes a CD46 isoform selected from the group consisting of CD 46C 1, CD 46C 2, CD46 BC1, CD46 BC2, and CD46 ABC 1. The cistron encoding the CD46 BC1 protein isoform has the nucleic acid sequence of SEQ ID NO. 263. The cistron encoding the CD46 BC2 protein isoform has the nucleic acid sequence of SEQ ID NO. 264. These isoforms are described in the following references, which are incorporated herein in their entirety: human Genetics volume 136, pages 421-435 (2017).
In embodiments, the polycistronic cassette comprises a nucleic acid encoding a cluster of differentiation 47 (CD 47) protein. CD47 protein is a membrane protein that is involved in the increase in intracellular calcium concentration that occurs when cells adhere to the extracellular matrix. The CD47 protein is also a receptor for the C-terminal cell binding domain of thrombospondin and can play a role in membrane transport and signal transduction. The CD47 protein comprises an IgV domain, a transmembrane domain and a C-terminal domain. There are four isoforms of the C-terminal domain of CD47 protein: 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 CD 47. Isotype 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, intestines and testes. In embodiments, CD47 is encoded by a nucleic acid having a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259. In embodiments, CD47 is isoform 1, 2, 3 or 4. In embodiments, CD47 is CD47 isoform 2 (CD 47-2). In an embodiment, CD47-2 is encoded by a nucleic acid having a sequence selected from any one of SEQ ID NOS: 78, 80, 83 and 180. In embodiments, CD47 is CD47 isoform 1 (CD 47-1). In an embodiment, CD47-1 is encoded by a nucleic acid having the sequence of SEQ ID NO. 259. In embodiments, the polycistronic cassette comprises a nucleic acid encoding a CD47 protein having an S64A mutation, an S79A mutation, or a combination thereof, as compared to SEQ ID NO. 201. The following articles describe the S64A and S79A mutations and are incorporated herein by reference in their entirety: j Biol chem.2011, 4, 29; 286 (17):14991-5002.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding a cluster of differentiation 55 (CD 55) protein. The gene encodes a glycoprotein involved in the regulation of the complement cascade. Binding of the encoded protein to the complement protein accelerates their decay, thereby disrupting the cascade and preventing damage to the host cell. In embodiments, the CD55 protein is encoded by a nucleic acid having a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding a cluster of differentiation 59 (CD 59) protein. CD59 protein is a cell surface glycoprotein that regulates complement-mediated cell lysis and is involved in lymphocyte signaling. CD59 protein is a potent inhibitor of the complement membrane attack complex, where it binds complement C8 and/or C9 during assembly of the complex, thereby inhibiting the incorporation of multiple copies of C9 into the complex, which is necessary for the formation of a permeate pore. In embodiments, CD59 is encoded by a nucleic acid having a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from SEQ ID NO. 86 or SEQ ID NO. 108.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding a CTLA-4 protein. In embodiments, the nucleic acid encodes an extracellular domain of a CTLA-4 protein. In embodiments, the extracellular domain of the CTLA-4 protein comprises an L104E mutation, an A29Y mutation, or both, relative to SEQ ID NO 199. In embodiments, the nucleic acid encoding CTLA-4 includes nucleic acid encoding an immunoglobulin or fragment thereof. In embodiments, the fragment of an immunoglobulin is a kappa light chain or a lambda light chain. In embodiments, the fragment thereof is an immunoglobulin heavy chain. In embodiments, the fragment thereof is a CH1, CH2, CH3 or CH4 domain of an immunoglobulin heavy chain. In embodiments, the fragment thereof is a variable heavy domain or a variable light domain of an immunoglobulin. In embodiments, the nucleic acid encoding a CLTA-4 protein or fragment thereof and an immunoglobulin or fragment thereof is LEA29Y. In embodiments, the nucleic acids encoding CTLA-4 protein or fragment thereof and immunoglobulin or fragment thereof comprise the extracellular domain of CTLA-4 (SEQ ID NO: 199), which has an 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 having the amino acid sequence: MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 incorporated herein by reference in their entirety: international publication No. 2001/092337 and Wolf-van Buerck et al Scientific reports.7:3572 (2017). In embodiments, CTLA-4 is encoded by a nucleic acid having a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:88 or SEQ ID NO: 186.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding a programmed cell death 1 ligand 1 (PD-L1) protein. PD-L1 proteins are immunosuppressive receptor ligands expressed by hematopoietic and non-hematopoietic cells (e.g., T cells and B cells) and by various types of tumor cells. PD-L1 proteins are type I transmembrane proteins with immunoglobulin V-like and C-like domains. The interaction of the ligand with its receptor inhibits T cell activation and cytokine production. In embodiments, PD-L1 is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from SEQ ID NOS: 89-91.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding an Endothelial Protein C Receptor (EPCR) protein. The gene encoding EPCR is called PROCR. EPCR binds activated protein C. In embodiments, the EPCR is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 92, 93 and 181.
In embodiments, the polycistronic cassette comprises a 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 embodiments, TFPI is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187. In embodiments, TFPI is a fusion between TFPI, a CD4 transmembrane domain (nm_000616) or fragment thereof, and the P-selectin cytoplasmic tail (nm_ 003005) or fragment thereof. In embodiments, TFPI lacks a P-selectin tail and is referred to herein as TFPI (Δp-Sel). In embodiments, the CD4 transmembrane domain comprises amino acids 166 to 435 of CD4 with NCBI RefSeq ID of nm_000616. In embodiments, the P-selectin cytoplasmic tail comprises amino acids 797 to 830 of P-selectin with NCBI RefSeq ID of nm_ 003005.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding a Thrombomodulin (THBD). THBD protein is an endothelial specific type I membrane receptor that binds thrombin. This binding results in activation of protein C, degrading blood coagulation factors Va and VIIIa and reducing the amount of thrombin produced. In embodiments, the THBD protein is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166, 265-266. In embodiments, the gene encoding THBD protein is "THBDda". THBDda is a cistron encoding THBD containing splice site mutations. In embodiments, the THBDda cistron has a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOS: 99-102, 166 and 266.
In embodiments, the polycistronic cassette comprises a nucleic acid encoding an X-chromosome linked inhibitor of apoptosis (XIAP) protein. In embodiments, XIAP is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID No. 110. The gene encodes a protein belonging to the family of apoptosis-inhibiting proteins.
In embodiments, any of the cistrons described herein comprises an initiation codon or a termination codon. The polycistronic cassette is organized such that transcription proceeds from the 5 'priming end of the cassette all the way to the 3' end to facilitate expression of each cistron. A separate cistron may be added or removed from the start or stop codon to facilitate polycistronic expression.
In embodiments, the polycistronic cassette comprises two or more cistrons sharing functions, e.g., genes involved in complement regulation, coagulation, innate immunity, inflammation and apoptosis, and cellular immunity. In embodiments, the polycistronic cassette comprises two or more cistrons having different functions.
In embodiments, the nucleic acids described herein comprise one or more polycistronic cassettes selected from the group consisting of: coagulation cassettes, complement regulatory and inflammatory and apoptotic cassettes, innate immune cassettes, innate immunity and inflammatory and apoptotic cassettes, complement regulatory and innate immune cassettes, cellular immunity and coagulation cassettes, apoptosis and coagulation cassettes, and apoptosis and cellular immunity cassettes.
In embodiments, the clotting cassette comprises one or more cistrons selected from THBD, TFPI, CD39 and PROCR. In embodiments, the clotting cassette comprises one or more cistrons selected from THBD and TFPI. In embodiments, the clotting cassette comprises one or more cistron selected from THBD, TFPI and CD 39. In embodiments, the clotting cartridge comprises one or more cistrons selected from PROCR and THBD. In embodiments, the clotting cassette comprises one or more cistrons selected from TFPI, PROCR, and THBD.
In embodiments, the complement regulatory cassette comprises one or more cistrons selected from the group consisting of CD46, C59, and CD 55. In embodiments, the complement regulatory cassette comprises one or more cistrons selected from CD46 and CD 59. In embodiments, the complement regulatory cassette comprises one or more cistrons selected from CD46 and CD 55. In embodiments, the complement regulatory cassette comprises one or more cistrons selected from the group consisting of CD46, CD55, and CD 59. In embodiments, the complement regulatory cassette comprises one or more cistrons selected from the group consisting of CD55 and CD 59. In embodiments, the nucleic acid comprising a complement regulatory cassette prevents complement deposition. For example, fig. 18D shows that expression of nucleic acids comprising cistron CD46, CD55, CD59, or a combination thereof reduces deposition of complement protein C3b as compared to expression of nucleic acids lacking cistron CD46, CD55, and CD 59.
In embodiments, the complement regulatory and inflammatory and apoptotic cassettes comprise one or more cistrons selected from the group consisting of CD46, CD55, CD59, a20, HO1, and PD-L1. In embodiments, the complement regulatory and inflammatory and apoptotic cassettes comprise one or more cistrons selected from the group consisting of CD46, CD55, CD59, a20, HO1, and PD-L1. In embodiments, the complement regulatory and inflammatory and apoptotic cassettes comprise one or more cistrons selected from the group consisting of CD46, CD55, a20, and HO 1.
In embodiments, the innate immune cassette comprises one or more cistrons selected from the group consisting of B2M, HLA-E, B2MHLA-E fusion proteins, CD47, and combinations thereof. In embodiments, the innate immune cassette comprises a cistron encoding a B2M HLA-E fusion protein and a CD47 cistron.
In embodiments, the innate immune and inflammatory and apoptotic cassettes comprise one or more cistrons selected from the group consisting of B2M, HLA-E, B M HLA-E fusion proteins, CD47, a20, HO1, PD-L1, and combinations thereof. In embodiments, the innate immune and inflammatory and apoptotic cassettes comprise one or more of B2M cistron, HLA-E cistron, cistron encoding B2M HLA-E fusion protein, CD47 cistron, a20 cistron, HO1 cistron and PD-L1 cistron. In embodiments, the innate immune and inflammatory and apoptotic cassettes comprise one or more cistrons selected from the group consisting of B2M, HLA-E, B2M HLA-E fusion protein, CD47, a20 and HO 1. In embodiments, the innate immune and inflammatory and apoptotic cassettes comprise one or more cistrons selected from the group consisting of cistrons encoding B2MHLA-E fusion proteins, CD47, a20, and HO 1.
In embodiments, the inflammatory and apoptotic cassettes comprise one or more cistrons selected from a20, HO1, and PD-L1. In embodiments, the inflammatory and apoptotic cassettes comprise one or more cistrons selected from a20 and HO 1. In embodiments, the inflammatory and apoptotic cassettes comprise one or more cistrons selected from a20 and PD-L1.
In embodiments, the complement regulatory and innate immune cassette comprises one or more cistrons selected from the group consisting of CD46, CD55, CD47, B2M, HLA-E, B M HLA-E fusion proteins, and combinations thereof. In embodiments, the complement-modulating and innate immune cassette comprises one or more cistrons selected from the group consisting of CD46, CD55, and CD 47.
In embodiments, the cellular immune and coagulation cassette comprises one or more cistrons selected from LEA29Y, CD39, TFPI, THBD, and PROCR. In embodiments, the cellular immunity and coagulation cassette comprises a CTLA-4 (e.g., LEA 29Y) cistron and a CD39 cistron.
In embodiments, the apoptosis and coagulation cassette comprises one or more cistrons selected from THBD, TFPI, CD39, PROCR and XIAP. In embodiments, the apoptosis and clotting cassette comprises an XIAP cistron and a CD39 cistron.
In embodiments, the apoptosis and cellular immune cassettes comprise one or more cistrons selected from a20, PD-L1, HO1, and CTLA-4 (e.g., LEA 29Y). In embodiments, the apoptosis and cellular immune cassettes comprise a20 and CTLA-4 (e.g., LEA 29Y) cistron. In embodiments, the apoptosis and cellular immune cassettes comprise a20 and HO1 cistron. In embodiments, the a20 cistron, HO1 cistron, or both are from wild boars.
In embodiments, the clotting and innate immune 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, encoding B2M HLA-E fusion protein cistron and CD47 cistron. In embodiments, the clotting and innate immune cassette comprises THBD cistron, CD47 cistron and PROCR cistron.
In embodiments, the nucleic acid comprises a first polycistronic cassette, a second polycistronic cassette, and a third polycistronic cassette, wherein the first polycistronic cassette is located 5 'to the second polycistronic cassette and the second polycistronic cassette is located 5' to the third polycistronic cassette. In embodiments, the coagulation cassette is a first, second or third polycistronic cassette. In embodiments, the complement regulatory cassette is a first, second, or third polycistronic cassette. In embodiments, the complement regulatory and inflammatory and apoptotic cassette is a first, second, or third polycistronic cassette. In embodiments, the innate immune cassette is a first, second or third polycistronic cassette. In embodiments, the innate immune and inflammatory and apoptotic cassette is a first, second or third polycistronic cassette. In embodiments, the inflammatory and apoptotic cassette is a first, second or third polycistronic cassette. In embodiments, the complement-modulating and innate immune cassette is a first, second or third polycistronic cassette. In embodiments, the cellular immune and coagulation cassette is a first, second or third polycistronic cassette. In embodiments, the apoptosis and coagulation cassette is a first, second or third polycistronic cassette. In embodiments, the apoptosis and cellular immune cassette is a first, second or third polycistronic cassette. In embodiments, the clotting and innate immune cassette is a first, second or third polycistronic cassette.
In embodiments, 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 5' to the second polycistronic cassette, the second polycistronic cassette is located 5' to the third polycistronic cassette, and the third polycistronic cassette is located 5' to 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 a first, second, third, or fourth polycistronic cassette. In embodiments, the complement regulatory and inflammatory and apoptotic cassette is a first, second, third or fourth polycistronic cassette. In embodiments, the innate immune cassette is a first, second, third or fourth polycistronic cassette. In embodiments, the innate immune and inflammatory and apoptotic cassette is a first, second, third or fourth polycistronic cassette. In embodiments, the inflammatory and apoptotic cassette is a first, second, third, or fourth polycistronic cassette. In embodiments, the complement-modulating and innate immune cassette is a first, second, third or fourth polycistronic cassette. In embodiments, the cellular immune and coagulation cassette is a first, second, third or fourth polycistronic cassette. In embodiments, the apoptosis and coagulation cassette is a first, second, third or fourth polycistronic cassette. In embodiments, the apoptosis and cellular immune cassette is a first, second, third or fourth polycistronic cassette. In embodiments, the clotting and innate immune cassette is a first, second, third or fourth polycistronic cassette.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a coagulation cassette, a complement regulatory cassette, and an innate immune cassette. In embodiments, the coagulation cassette is located 5' to the complement regulatory cassette. In embodiments, the complement regulatory cassette is located 5' to the innate immune cassette. In embodiments, the coagulation cassette is located at the 5 'end of the complement regulatory cassette and the complement regulatory cassette is located at the 5' end of the innate immune cassette. In embodiments, the coagulation cassette is located at the 5 'end of the innate immune cassette and the innate immune cassette is located at the 5' end of the complement regulatory cassette. In embodiments, the innate immune cassette is located at the 5 'end of the clotting cassette and the clotting cassette is located at the 5' end of the complement regulatory cassette.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a coagulation cassette, a complement regulatory cassette, and an innate immune and inflammatory and apoptotic cassette. In embodiments, the coagulation cassette is located at the 5 'end of the complement regulatory cassette, and the complement regulatory cassette is located at the 5' end of 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 5' to the complement regulatory cassette.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a clotting cassette, an inflammatory and apoptotic cassette, and a complement regulatory and innate immune cassette. In embodiments, the coagulation cassette is located 5 'to the inflammatory and apoptotic cassettes, and the inflammatory and apoptotic cassettes are located 5' to the complement regulatory and innate immune cassettes. In embodiments, the coagulation cassette is located 5' to the inflammatory and apoptotic cassettes. In embodiments, the inflammatory and apoptotic cassettes are located 5' to the complement regulatory and innate immune cassettes.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a clotting cassette, a complement regulatory and inflammatory and apoptotic cassette, and an innate immune cassette. In embodiments, the innate immune cassette is located at the 5 'end of the clotting cassette and the clotting cassette is located at the 5' end of the complement regulatory and inflammatory and apoptotic cassette.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a clotting cassette, a complement regulatory cassette, an innate immune cassette, and a cellular immune and clotting cassette. In embodiments, the clotting cassette is located 5' to the innate immune cassette. In embodiments, the innate immune cassette is located 5' of the cellular immune and coagulation cassette. In embodiments, the cellular immunity and coagulation cassette is located 5' to the complement regulatory cassette. In embodiments, the cassette is located at the 5' end of the innate immune cassette, the innate immune cassette is located at the 5' end of the cellular immunity and cassette, and the cellular immunity and cassette are located at the 5' end of the complement regulatory cassette.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a clotting cassette, a complement regulatory cassette, an innate immune cassette, and an apoptosis and inflammation cassette. In embodiments, the coagulation cassette is located at the 5' end of the innate immune cassette, the innate immune cassette is located at the 5' end of the apoptosis and inflammation cassette, and the apoptosis and inflammation cassette is located at the 5' end of the complement regulatory cassette. In embodiments, the coagulation cassette is located at the 5' end of the innate immune cassette, the innate immune cassette is located at the 5' end of the complement regulatory cassette, and the complement regulatory cassette is located at the 5' end of the apoptosis and inflammation cassette.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a clotting cassette, a complement regulatory cassette, an innate immune cassette, and an apoptosis and clotting cassette. In embodiments, the cassette is located at the 5' end of the innate immune cassette, the innate immune cassette is located at the 5' end of the apoptosis and clotting cassette, and the cellular immunity and clotting cassette is located at the 5' end of the complement regulatory cassette.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a clotting cassette, a complement regulatory cassette, an innate immune cassette, and an apoptosis and cellular immune cassette. In embodiments, the coagulation cassette is located at the 5' end of the innate immune cassette, the innate immune cassette is located at the 5' end of the apoptosis and cellular immune cassette, and the apoptosis and cellular immune cassette is located at the 5' end of the complement regulatory cassette.
In embodiments, the nucleic acid comprising a plurality of polycistronic cassettes comprises a clotting and innate immune cassette, an apoptosis and cellular immune cassette, and a complement regulatory cassette. In embodiments, the clotting and innate immunity cassette is located 5 'to the apoptosis and cellular immunity cassette, and the apoptosis and cellular immunity cassette is located 5' to the complement regulatory cassette.
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. The promoter is a DNA sequence that defines the starting position for transcription of the polycistronic cassette by RNA polymerase. In embodiments, the promoter is derived from a porcine gene. In embodiments, the promoter is derived from a mouse gene. In embodiments, the promoter is derived from a human gene. In embodiments, the promoter is a eukaryotic translation elongation factor 1α1 (abbreviated as "EF1 α1" or "eef1α1") promoter. The EF1 a 1 promoter is a constitutive promoter. In embodiments, the promoter is a CAG promoter. The CAG promoter comprises the cytomegalovirus enhancer fused to the chicken β -actin promoter and the splice acceptor of the rabbit β -globin gene. In embodiments, the promoter is an actin γ1 (ACTG 1) promoter. In embodiments, the promoter is a heat shock protein family a member 8 (HSPA 8) promoter. In embodiments, the promoter is a ubiquitin C (UBC) promoter. In embodiments, the promoter is an intracellular adhesion molecule 2 (ICAM 2) promoter. In embodiments, the promoter comprises an EF1 a 1, CAG, ACTG1, HSPA8, UBC, or ICAM2 promoter. In embodiments, the promoter comprises an extension at the 5 'or 3' end. In embodiments, the extension is an intron or fragment thereof. Introns may be derived from endogenous promoter genes (e.g., EF1 a 1, CAG, ACTG1, HSPA8, UBC, or ICAM 2) or provided from other sources.
In embodiments, the promoter comprises a CpG island. CpG islands are regions of elevated GC content and high density CpG dinucleotides. In embodiments, the CpG island is 5' to the exon sequence of the promoter. In embodiments, a 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 500bp to about 5kb. In embodiments, the CpG island comprises from about 1kb to about 3kb. For example, a CpG island can comprise at least about 500bp, at least about 1kb, at least about 1.5kb, at least about 2kb, at least about 2.5kb, at least about 3kb, at least about 3.5kb, at least about 4kb, at least about 4.5kb, or at least about 5kb. In contrast, standard promoters are about 100-200 base pairs in length. In embodiments, the CpG island contains a ratio of observed to expected number of CpG dinucleotides greater than 0.60. In embodiments, the CpG island comprises a non-methylated CpG dinucleotide. In embodiments, the CpG island comprises a methylated CpG dinucleotide. The following references describe CpG islands: j Mol Bio.1987, 7, 20; 196 261-82; nat Rev Mol Cell biol.2018, 10 months; 19 (10) 621-637; biochem Biophys Res Commun.2009, 5 month 15; 382 (4):643-645. Each of these references is incorporated herein in its entirety.
Non-limiting examples of promoters contained in the nucleic acids or polycistronic cassettes described herein are shown in table B. In embodiments, the promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS 126-145, 167, 168, 178, 179, 231-238, 247 and 250.
Table B
Name of the name SEQ ID NO.
CAG 126-128,247,250
hsACTG1 129
hsEF1 alpha 1, also known as "hEF1 alpha 1" 130-132,231-232
hsHSPA8 133
mmEF1 alpha 1, also known as "mEF a1" 134-135
ssEEF1A1, also known as "ssEEF1A1 (ATACseq)", 136-138,179,233-235
ssHSPA8, also known as "ssHSPA8 (ATACseq)"; 139-140,142-145,236-238
ssUBC 141,168,178
ssICAM2 167
in embodiments, each promoter within a nucleic acid is different. In embodiments, the first promoter and the second promoter within the nucleic acid are the same. In embodiments, each polycistronic cassette of nucleic acid comprises a different promoter. In embodiments, the promoter is a forward promoter located on the sense strand. In embodiments, the promoter is a reverse promoter located on the antisense strand.
In FIGS. 1A-1D, 2A-2J, 3A-3C, 4A-4H, 5A-5F and 6, the forward promoters are indicated by forward arrows, and the reverse promoters are indicated by reverse arrows.
In embodiments, any of the cassettes described herein may comprise any promoter from table B. Exemplary arrangements of promoters and polycistronic cassettes are shown in FIGS. 1A-1D, 2A-2J, 3A-3C, 4A-4H, 5A-5F, and 6.
In embodiments, provided herein are nucleic acids comprising, from 5 'to 3', a first polycistronic cassette, a second polycistronic cassette, and a third polycistronic cassette. In embodiments, the first polycistronic cassette and the second polycistronic cassette are under the control of promoters that initiate transcription of the cassettes on different chains. For example, a first polycistronic cassette may comprise a forward promoter and a second polycistronic cassette may comprise a reverse promoter. Alternatively, the first polycistronic cassette may comprise a reverse promoter and the second polycistronic cassette may comprise a forward promoter. In embodiments, the second polycistronic cassette and the third polycistronic cassette are under the control of promoters that initiate transcription of the cassettes on different chains. For example, the second polycistronic cassette may comprise a forward promoter and the third polycistronic cassette may comprise a reverse promoter. Alternatively, the second polycistronic cassette may comprise a reverse promoter and the third polycistronic cassette may comprise a forward promoter. In embodiments, the first polycistronic cassette is under the control of a forward promoter, the second polycistronic cassette is under the control of a reverse promoter, and the third polycistronic cassette is under the control of a forward promoter. In embodiments, the first polycistronic cassette is under the control of a reverse promoter, the second polycistronic cassette is under the control of a forward promoter, and the third polycistronic cassette is under the control of a reverse promoter.
In embodiments, provided herein are nucleic acids comprising from 5 'to 3' a first polycistronic cassette, a second polycistronic cassette, a third polycistronic cassette, and a fourth polycistronic cassette. In embodiments, the first polycistronic cassette and the second polycistronic cassette are under the control of promoters that initiate transcription of the cassettes on different chains. For example, a first polycistronic cassette may comprise a forward promoter and a second polycistronic cassette may comprise a reverse promoter. Alternatively, the first polycistronic cassette may comprise a reverse promoter and the second polycistronic cassette may comprise a forward promoter. In embodiments, the second polycistronic cassette and the third polycistronic cassette are under the control of promoters that initiate transcription of the cassettes on different chains. For example, the second polycistronic cassette may comprise a forward promoter and the third polycistronic cassette may comprise a reverse promoter. Alternatively, the second polycistronic cassette may comprise a reverse promoter and the third polycistronic cassette may comprise a forward promoter. In embodiments, the third polycistronic cassette and the fourth polycistronic cassette are under the control of promoters that initiate transcription of the cassettes on different chains. For example, the third polycistronic cassette may comprise a forward promoter and the fourth polycistronic cassette may comprise a reverse promoter. Alternatively, the third polycistronic cassette may comprise a reverse promoter and the fourth polycistronic cassette may comprise a forward promoter. In embodiments, the first polycistronic cassette is under the control of a reverse promoter, the second polycistronic cassette is under the control of a forward promoter, the third polycistronic cassette is under the control of a reverse promoter, and the fourth polycistronic cassette is under the control of a forward promoter. In embodiments, the first polycistronic cassette is under the control of a forward promoter, the second polycistronic cassette is under the control of a reverse promoter, the third polycistronic cassette is under the control of a forward promoter, and the fourth polycistronic cassette is under the control of a reverse promoter.
In embodiments, the nucleic acid comprising at least one polycistronic cassette comprises one or more poly-a sequences. In embodiments, the poly a sequence is from a human Growth Hormone (GH) gene. In embodiments, the poly a sequence is from the beta globin (HBB) gene. In embodiments, the poly a is from SV40 virus. In embodiments, the poly a sequence is a synthetic poly a (pA). Exemplary poly a sequences are shown in table C. In embodiments, any of the cassettes described herein may comprise any poly a sequence from table C. In embodiments, the poly A sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 159-162, 190-192 and 239-240.
Exemplary arrangements of poly a sequences within the polycistronic cassette are shown in fig. 1A-1D, fig. 2A-2J, fig. 3A-3C, fig. 4A-4H, fig. 5A-5F, and fig. 6. In embodiments, the nucleic acids described herein comprise one or more poly a sequences from table C located 5' to the polycistronic cassette. In embodiments, the poly a sequence comprises a terminator. In embodiments, the nucleic acids described herein comprise one or more poly a sequences from table C located 3' to the polycistronic cassette.
Table C
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In embodiments, the poly A 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 poly-a sequence comprises synthetic pA and a transcription terminator. In embodiments, the transcription terminator is ACTB. In an embodiment, ACTB has the nucleic acid sequence of SEQ ID NO. 118. In embodiments, the presence of the poly a sequence and terminator sequence terminates transcription of the polycistronic cassette. In embodiments, transcription of the polycistronic cassette is more effectively 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 embodiments, the terminator is selected from any terminator sequence described in the following documents, which are incorporated herein by reference in their entirety: nojima et al Cell Reports 3,1080-1092 (2013).
In embodiments, the nucleic acid comprises a poly a sequence and a transcription terminator (e.g., ACTB) at the 5 'or 3' end of the first or last polycistronic cassette within the nucleic acid. The transcription termination is contained at the 5 'or 3' end of the first or last polycistronic cassette to terminate transcription mediated by the upstream genomic promoter. Thus, only polycistronic cassettes are transcribed.
In embodiments, the cistrons within the 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 jump during protein translation in a cell. In embodiments, the polycistronic cassette comprises one or more 2A polypeptides selected from the group consisting of E2A, F2A, P2A and T2A polypeptides. In embodiments, the polycistronic cassette encodes an E2A polypeptide from a nucleic acid sequence selected from SEQ ID nos. 1 and 9. In an embodiment, the polycistronic cassette comprises an F2A polypeptide encoded by a nucleic acid having a sequence selected from the group consisting of 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 the group consisting of 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. Liu et al describe additional 2A polypeptides 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.
In embodiments, when a polycistronic cassette containing cistrons separated by 2A peptides is expressed, the expressed cistrons may comprise an N-terminal proline, a C-terminal fusion, or a combination thereof of the 2A peptide or fragment thereof. In embodiments, the expressed cistron comprises a C-terminal fusion of a 2A peptide, wherein the 2A peptide lacks a C-terminal proline.
In embodiments, when a cistron located at the 5' end of the 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 embodiments, when a cistron located at the 3' end of the 2A peptide is expressed, the expressed cistron comprises an N-terminal proline. In embodiments, when the cistron is located at the 5 'end of the first 2A peptide and the 3' end of the second 2A peptide, the cistron expressed comprises the N-terminal proline of the 2A peptide and the 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.
In embodiments, the cistrons within the polycistronic cassette are separated by an Internal Ribosome Entry Site (IRES). In embodiments, the nucleic acids described herein comprise one or more IRES having the nucleic acid sequence of any one of SEQ ID NOs 11, 194 and 195. In embodiments, when a polycistronic cassette containing cistrons separated by IRES is expressed, the expressed cistron is a wild-type protein.
In embodiments, the nucleic acids provided herein comprise a first insulator sequence at the 5 'end of a 5' polycistronic cassette and a second insulator sequence at the 3 'end of a 3' polycistronic cassette. In an embodiment, the insulator is HS4. In embodiments, HS4 has a sequence selected from any one of SEQ ID NOS.49-58, 163 and 164. In embodiments, HS4 prevents transcriptional interference from a promoter at the genomic integration site.
In embodiments, a nucleic acid provided herein comprises: a first Inverted Terminal Repeat (ITR) located at the 5 'end of the 5' polycistronic cassette and having the sequence of SEQ ID NO:17 or 18, and a second ITR at the 3 'end of the 3' polycistronic cassette having the sequence of SEQ ID NO:15 or 16. The inclusion of ITRs in the nucleic acids provided herein enables integration of the nucleic acids into the genome (e.g., porcine genome) via PiggyBac transposon-mediated gene transfer.
In embodiments, the nucleic acids provided herein comprise a first loxP site at the 5 'end of a 5' polycistronic cassette and a second loxP site at the 3 'end of a 3' polycistronic cassette. The loxP site is a nucleic acid sequence that is recombined by recombinase mediated mitosis. The inclusion of loxP sites in the nucleic acids provided herein enables integration of the nucleic acids into the genome (e.g., porcine genome) via a recombinase. Maizels et al describe genome engineering using loxP and recombinase and are incorporated herein by reference in their entirety: maizels et al J immunol.2013;191 (1). In embodiments, the first or 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.
In embodiments, the nucleic acids provided herein comprise 5 'and 3' guide RNA (gRNA) target sequences (e.g., fos_sg1 or aavs_sg3 of fig. 5A-5F). The gRNA target sequence is recognized by a guide RNA and a nuclease (e.g., cas 9). The region between the 5 'guide RNA target sequence and the 3' grna sequence is cleaved by guide RNA and nuclease. In embodiments, the nucleic acid portion comprising a plurality of polycistronic cassettes described herein between 5 'and 3' grna target sequences is integrated into a cell, tissue, organ, or animal. In embodiments, 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 non-homology dependent targeted integration (HITI). Suzuki et al describe in vivo genome editing using HITI and are incorporated herein by reference in their entirety: suzuki et al Nature 2016, 12/1; 540 (7631):144-149. In embodiments, a nucleic acid sequence located between 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 incorporated herein by reference in their entirety: hsu et al Cell 157,1262-1278 (2014). In embodiments, the 5' gRNA target sequence is FOS (SEQ ID NO: 47) or AAVS (SEQ ID NO: 48). In embodiments, the 3' gRNA target sequence is FOS (SEQ ID NO: 47) or AAVS (SEQ ID NO: 48).
Exemplary arrangements of loxP sites, insulator sequences, guide RNA target sequences, and polycistronic cassettes are presented in Table D.
Table D
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 embodiments, the stop codon is found 3' to each polycistronic cassette. In embodiments, the nucleic acids described herein comprise a Kozak sequence. In embodiments, the Kozak sequence is a nucleic acid of SEQ ID NO. 59 or 61.
In embodiments, the nucleic acids described herein comprise one or more Ubiquitous Chromatin Opening Elements (UCOEs). The UCOE is described by Neville et al and incorporated herein by reference in its entirety: neville et al Biotechnology Advances (2017) 557-564. In embodiments, the polycistronic cassettes of the nucleic acids described herein are separated by UCOE. In embodiments, the UCOE is selected from Can 6-3, A2CBX3, and SRF1. In embodiments, the UCOE comprises any one of the sequences defined by SEQ ID NOS.19-22, 157 and 193.
In embodiments, the nucleic acids provided herein comprise fluorescent proteins. In embodiments, the fluorescent protein is located at the 5 'end of the 5' polycistronic cassette. In embodiments, the fluorescent protein is located at the 3 'end of the 3' polycistronic cassette. In embodiments, the fluorescent protein is located 5' to an ITR, a gRNA target sequence, a loxP site, or a combination thereof. In embodiments, fluorescent proteins are used for negative selection. In embodiments, the fluorescent protein is a green fluorescent protein. In an embodiment, the fluorescent protein is GFP having the nucleic acid sequence of SEQ ID NO. 111. In embodiments, the fluorescent protein is BFP having the nucleic acid sequence of SEQ ID NO. 242 or SEQ ID NO. 246.
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 a nucleic acid described herein, e.g., the fragments may be about 20%, about 25%, about 30%, about 35%, about 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% of the nucleotides of any of the nucleic acids described herein. 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 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 at least 100% of the nucleotides of any of the nucleic acids described herein.
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 at least 50 base pairs at their 5 'or 3' ends. Fragments were 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 acids
Exemplary nucleic acids comprising a plurality of 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.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a mouse EF 1a 1 promoter selected from SEQ ID NOs 134-135, and an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from SEQ ID NOs 114-116, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NOs 94-96, 103 and 187, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a human EF1 alpha 1 promoter selected from the group consisting of SEQ ID NOS: 130-132, a 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, 200, and 253-258, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOS: 86, 108; and an innate immune cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS.123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2MHLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180, and 259. 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 immune cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a mouse EF1 a 1 promoter selected from SEQ ID NOs 134-135, and an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from SEQ ID NOs 114-116, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NOs 94-96, 103 and 187, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a human EF1 alpha 1 promoter selected from the group consisting of SEQ ID NOS: 130-132, a 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, 200, and 253-258, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOS: 86, 108; and an innate immune cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS.123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2MHLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180, and 259. In embodiments, the UCOE separates the complement regulatory cassette from the innate immune cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a mouse EF1 a 1 promoter selected from SEQ ID NOs 134-135, and an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from SEQ ID NOs 114-116, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NOs 94-96, 103 and 187, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a human EF1 alpha 1 promoter selected from the group consisting of SEQ ID NOS: 130-132, a 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, 200, and 253-258, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOS: 86, 108; and an innate immunity and inflammation and apoptosis cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a third cistron encoding A20, a fourth cistron encoding HO1, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180, and 259, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS: 64-65, 104, 182, and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183, and 189. In embodiments, IRES (SEQ ID NO: 11) separates the second and third cistrons of the innate immune and inflammatory and apoptotic cassettes. In embodiments, the UCOE separates the complement regulatory cassette from the innate immune and inflammatory and apoptotic cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3UCOE having the sequence of SEQ ID NO. 20.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39, a mouse EF1 a 1 promoter selected from SEQ ID NOs 134-135, and an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from SEQ ID NOs 114-116, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NOs 94-96, 103 and 187, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NOs 67-70 and 106, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a third cistron encoding CD55, a human EF1 alpha 1 promoter selected from SEQ ID NOS 130-132, a 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 SEQ ID NOS 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NOS 86 and 108, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS 84, 85, 107, and 184; and an innate immunity and inflammation and apoptosis cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a third cistron encoding A20, a fourth cistron encoding HO1, a fifth cistron encoding PD-L1, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2MHLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180 and 259, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183 and 189, and wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NOS: 89-91. In embodiments, IRES (SEQ ID NO: 11) separates the second and third cistrons of the innate immune and inflammatory and apoptotic cassettes. In embodiments, the UCOE separates the complement regulatory cassette from the innate immune and inflammatory and apoptotic cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a mouse EF1 a 1 promoter selected from SEQ ID NOs 134-135, and an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from SEQ ID NOs 114-116, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NOs 94-96, 103 and 187, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a human EF1 alpha 1 promoter selected from the group consisting of SEQ ID NOS: 130-132, a 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, 200, and 253-258, wherein the nucleic acid sequence of CD59 is selected from the group consisting of SEQ ID NOS: 86, 108; and an innate immune cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS.123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2MHLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180, and 259. 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 immune cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE located at the 5 'or 3' end of the coagulation cassette is an SRF having the sequence of SEQ ID NO. 22.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39, a mouse EF1 a 1 promoter selected from SEQ ID NOs 134-135, and an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from SEQ ID NOs 114-116, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NOs 94-96, 103 and 187, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NOs 67-70 and 106, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a third cistron encoding CD55, a ssEF1 alpha 1 promoter selected from SEQ ID NOS: 136-138, a 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 SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NOS: 86, 108, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184; and an innate immunity and inflammation and apoptosis cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a third cistron encoding A20, a fourth cistron encoding HO1, a fifth cistron encoding PD-L1, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2MHLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180 and 259, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183 and 189, and wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NOS: 89-91. In embodiments, IRES (SEQ ID NO: 11) separates the second and third cistrons of the innate immune and inflammatory and apoptotic cassettes. In embodiments, the UCOE separates the complement regulatory cassette from the innate immune and inflammatory and apoptotic cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is a CBX 3UCOE having the sequence of SEQ ID NO. 21. In embodiments, the UCOE is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE located at the 5 'or 3' end of the coagulation cassette is a Can 6-3UCOE 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.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39, a ssUBC promoter having the sequence of SEQ ID NO:141, an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from any one of SEQ ID NOs 114-116 and 191, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187, wherein the nucleic acid sequence of CD39 is selected from the group consisting of SEQ ID NOs 67-70 and 106, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a third cistron encoding CD55, a ssEF1 alpha 1 promoter selected from SEQ ID NOS: 136-138, a btGH pA sequence selected from SEQ ID NOS: 112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NOS: 86, 108, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184; and an innate immunity and inflammation and apoptosis cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a third cistron encoding A20, a fourth cistron encoding HO1, a fifth cistron encoding PD-L1, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2MHLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180 and 259, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183 and 189, and wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NOS: 89-91. In embodiments, IRES (SEQ ID NO: 11) separates the second and third cistrons of the innate immune and inflammatory and apoptotic cassettes. In embodiments, the UCOE separates the complement regulatory cassette from the innate immune and inflammatory and apoptotic cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, 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.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding PROCR, a ssUBC promoter having the sequence of SEQ ID NO:19-22, 157 or 193, an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from any one of SEQ ID NOs 114-116 and 191, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of PROCR is selected from the group consisting of SEQ ID NOs 92, 93 and 181, (b) an inflammatory and apoptotic cassette comprising: a first cistron encoding A20, a second cistron encoding HO1, a sseF1 alpha 1 promoter selected from SEQ ID NOS: 136-138 and 179, a btGH pA sequence selected from SEQ ID NOS: 112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS: 64-65, 104, 182 and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183 and 189; and a complement-modulating and innate immune cassette comprising: a first cistron encoding CD46, a second cistron encoding CD47, a third cistron encoding CD55, a CAG promoter selected from SEQ ID NOS: 126-128, a SPAACTB poly A sequence selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180, and 259, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the UCOE separates inflammatory and apoptotic cassettes from complement-modulating and innate immune cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, 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.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding PROCR, a third cistron encoding TFPI, a ssUBC promoter having the sequence of SEQ ID NO:141, an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from any one of SEQ ID nos. 114-116 and 191, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID nos. 97-102, 166 and 265-266, wherein the nucleic acid sequence of PROCR is selected from the group consisting of SEQ ID nos. 92, 93 and 181, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID nos. 94-96, 103 and 187, (b) a innate immune cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssHSPA8 promoter selected from SEQ ID NOs 139-140, a btGH pA sequence selected from SEQ ID NOs 112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from SEQ ID NOs 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOs 77-83, 180, and 259; and (c) 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 an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the UCOE separates the innate immune cassette from the complement regulatory cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is a CBX3 UCOE having the sequence of SEQ ID NO. 21. In embodiments, the poly a is located at the 5 'or 3' end of the coagulation cassette. In an embodiment, the poly A 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 embodiments, THBD is THBDda having a sequence selected from SEQ ID NOS 99-102. In embodiments, the B2M HLA-E fusion protein comprises an HLA-G epitope. In embodiments, CD47 is CD47-2 (e.g., NM-198793) having the sequence of SEQ ID NO:80, 81 or 83. In embodiments, the poly a is located at the 5 'or 3' end of the complement regulatory cassette. In an embodiment, the poly A at the 5 'or 3' end of the complement cassette is SPAACTB pA having the sequence of SEQ ID NO: 118.
In some embodiments, provided herein are nucleic acids comprising: (a) A innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssUBC promoter having the sequence of SEQ ID NO:141, an hsHBB poly a ("hshbbpa" or "hbbpa") 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 the group consisting of SEQ ID NOs 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259, (B) a clotting cassette comprising: a first cistron encoding THBD, a second cistron encoding PROCR, a third cistron encoding TFPI, an hsHSPA8 promoter having the sequence of SEQ ID NO:133, a btGH pA sequence selected from the group consisting of SEQ ID NO.112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NO. 97-102, 166, and 265-266, wherein the nucleic acid sequence of PROCR is selected from the group consisting of SEQ ID NO. 92, 93, and 181, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NO. 94-96, 103, and 187; and (c) 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, an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, and combinations thereof, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the UCOE separates the innate immune cassette from the complement regulatory cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the poly a is located at the 5 'or 3' end of the innate immune cassette. In an embodiment, the poly A 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 embodiments, THBD is THBDda having a sequence selected from SEQ ID NOS 99-102. In embodiments, the B2M HLA-E fusion protein comprises an HLA-G epitope. In embodiments, CD47 is CD47-2 (e.g., NM-198793) having the sequence of SEQ ID NO:80, 81 or 83. In embodiments, the poly a is located at the 5 'or 3' end of the complement regulatory cassette. In an embodiment, the poly A at the 5 'or 3' end of the complement cassette is SPAACTB pA having the sequence of SEQ ID NO: 118. In embodiments, the nucleic acid comprises a gRNA target sequence selected from SEQ ID NOS.47-48.
In some embodiments, provided herein are nucleic acids comprising: (a) A innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssUBC promoter having the sequence of SEQ ID NO:141, an hsHBB poly a ("hshbbpa" or "hbbpa") 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 the group consisting of SEQ ID NOs 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259, (B) a clotting cassette comprising: a first cistron encoding THBD, a second cistron encoding PROCR, a third cistron encoding TFPI, an hsHSPA8 promoter having the sequence of SEQ ID NO:133, a btGH pA sequence selected from the group consisting of SEQ ID NO.112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NO. 97-102, 166, and 265-266, wherein the nucleic acid sequence of PROCR is selected from the group consisting of SEQ ID NO. 92, 93, and 181, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NO. 94-96, 103, and 187; and (c) 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, an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, and combinations thereof, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the UCOE separates the innate immune cassette from the complement regulatory cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22 and 157. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the poly a is located at the 5 'or 3' end of the innate immune cassette. In an embodiment, the poly A 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 embodiments, THBD is THBDda having a sequence selected from SEQ ID NOS 99-102. In embodiments, the B2M HLA-E fusion protein comprises an HLA-G epitope. In embodiments, CD47 is CD47-2 (e.g., NM-198793) having the sequence of SEQ ID NO:80, 81 or 83. In embodiments, the poly a is located at the 5 'or 3' end of the complement regulatory cassette. In an embodiment, the poly A at the 5 'or 3' end of the complement cassette is SPAACTB pA having the sequence of SEQ ID NO: 118. In embodiments, the nucleic acid comprises a gRNA target sequence selected from SEQ ID NOS.47-48.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding PROCR, a third cistron encoding TFPI, a ssUBC promoter having the sequence of SEQ ID NO:141, an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from any one of SEQ ID nos. 114-116 and 191, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID nos. 97-102, 166 and 265-266, wherein the nucleic acid sequence of PROCR is selected from the group consisting of SEQ ID nos. 92, 93 and 181, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID nos. 94-96, 103 and 187, (b) a innate immune cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssef1α1 promoter selected from SEQ ID NOs 136-138 and 179, a btGH pA sequence selected from SEQ ID NOs 112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from SEQ ID NOs 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOs 77-83, 180 and 259; and (c) 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 SPAACTB poly A sequence selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the UCOE separates the innate immune cassette from the complement regulatory cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is a CBX3 UCOE having the sequence of SEQ ID NO. 21. In embodiments, the poly a is located at the 5 'or 3' end of the coagulation cassette. In an embodiment, the poly A 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 embodiments, THBD is THBDda having a sequence selected from SEQ ID NOS 99-102. In embodiments, the B2MHLA-E fusion protein comprises an HLA-G epitope. In embodiments, CD47 is CD47-2 (e.g., NM-198793) having the sequence of SEQ ID NO:80, 81 or 83. In embodiments, the poly a is located at the 5 'or 3' end of the complement regulatory cassette. In an embodiment, the poly A at the 5 'or 3' end of the complement cassette is SPAACTB pA having the sequence of SEQ ID NO: 118. In embodiments, the nucleic acid comprises a gRNA target sequence selected from SEQ ID NOS.47-48.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding TFPI, a mouse EF1 a 1 promoter selected from SEQ ID NOs 134-135, an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from any one of SEQ ID NOs 114-116 and 191, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NOs 97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NOs 94-96, 103 and 187, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD55, a human EF1 alpha 1 promoter selected from the group consisting of SEQ ID NOS: 130-132, a btGH pA sequence selected from the group consisting of SEQ ID NOS: 112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOS: 84, 85, 107, and 184; and an innate immune cassette comprising: a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS.123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180, and 259. In embodiments, the UCOE separates the complement regulatory cassette from the innate immune cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE located at the 5 'or 3' end 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.
In some embodiments, provided herein are nucleic acids comprising: (a) A clotting cassette comprising a first cistron encoding THBD, a second cistron encoding PROCR, a mouse EF1 a 1 promoter selected from SEQ ID nos. 134-135, an hsHBB poly a ("hshbbpa" or "hbbpa") sequence selected from any one of SEQ ID nos. 114-116 and 191, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID nos. 97-102, 166 and 265-266, wherein the nucleic acid sequence of PROCR is selected from SEQ ID nos. 92, 93 and 181, (b) a complement regulatory cassette comprising: a first cistron encoding CD46, a second cistron encoding CD55, a human EF1 alpha 1 promoter selected from the group consisting of SEQ ID NOS: 130-132, a btGH pA sequence selected from the group consisting of SEQ ID NOS: 112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOS: 84, 85, 107, and 184; and (c) an innate immunity and inflammation and apoptosis cassette comprising: a first cistron encoding a B2MHLA-E fusion protein, a second cistron encoding CD47, a third cistron encoding A20, a fourth cistron encoding HO1, a CAG promoter selected from SEQ ID NOS: 126-128, and a SPAACTB poly A sequence selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein is selected from SEQ ID NOS: 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOS: 77-83, 180, and 259, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS: 64-65, 104, 182, and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183, and 1895. In embodiments, IRES having the sequence of SEQ ID NO.11 separates B2M HLA-E fusion protein and CD47 from A20 and HO 1. In some embodiments, the UCOE separates the complement regulatory cassette from the innate immune cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22 and 157. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22 and 157 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE located at the 5 'or 3' end 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.
In some embodiments, provided herein are nucleic acids comprising: (a) An innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a mouse EF1 a 1 promoter selected from SEQ ID NOs 134-135, an hsHBB poly a ("hshbbpa" or "hbbpa") 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 NOs 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from SEQ ID NOs 77-83, 180 and 259, (B) a blood coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39, a human EF1 alpha 1 promoter selected from the group consisting of SEQ ID NOS: 130-132, a btGH pA sequence selected from the group consisting of SEQ ID NOS: 112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOS: 97-102, 166, and 265-266, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NOS: 94-96, 103, and 187, wherein the nucleic acid sequence of CD39 is selected from the group consisting of SEQ ID NOS: 67-70, and SEQ ID NO:106; and (c) a complement regulatory and inflammatory and apoptotic cassette comprising: a first cistron encoding CD46, a second cistron encoding CD59, a third cistron encoding CD55, a fourth cistron encoding A20, a fifth cistron encoding HO1, a sixth cistron encoding PD-L1, a CAG promoter selected from SEQ ID NOS: 126-128, and SPAACTB poly A sequences selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NOS: 86, 108, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS: 64-65, 104, 182, and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183, and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NOS: 89-91. In embodiments, 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 immune cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22 and 157. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22 and 157 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE located at the 5 'or 3' end 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.
In some embodiments, provided herein are nucleic acids comprising: (a) A innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssUBC promoter having the sequence of SEQ ID NO:141, an hsHBB poly a ("hshbbpa" or "hbbpa") 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 the group consisting of SEQ ID NOs 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259, (B) a clotting cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, an hsHSPA8 promoter selected from SEQ ID NO. 133, a btGH pA sequence selected from SEQ ID NO.112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO. 97-102, 166, and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO. 94-96, 103, and 187; and (c) 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 SPAACTB poly A sequence selected from SEQ ID NOS: 123-125 and 154-156, and combinations thereof, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the UCOE separates the complement regulatory cassette from the coagulation cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of an innate immune cassette. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of an innate immune cassette. In embodiments, the UCOE located at the 5 'or 3' end of the innate immune cassette is an SRF with 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.
In some embodiments, provided herein are nucleic acids comprising: (a) A innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssUBC promoter having the sequence of SEQ ID NO:141, an hsHBB poly a ("hshbbpa" or "hbbpa") 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 the group consisting of SEQ ID NOs 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259, (B) a clotting cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, an hsHSPA8 promoter selected from SEQ ID NO. 133, a btGH pA sequence selected from SEQ ID NO.112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO. 97-102, 166, and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO. 94-96, 103, and 187; and (c) a complement regulatory and inflammatory and apoptotic cassette comprising: a first cistron encoding CD46, a second cistron encoding CD55, a third cistron encoding A20, a fourth cistron encoding HO1, a CAG promoter selected from SEQ ID NOS: 126-128, an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS: 64-65, 104, 182, and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183, and 189. In an embodiment, IRES having the sequence of SEQ ID NO.11 separates CD46 and CD55 from A20 and HO 1. In embodiments, the UCOE separates the complement regulatory cassette from the coagulation cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE located at the 5 'or 3' end 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.
In embodiments, provided herein are nucleic acids comprising: (a) A innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssUBC promoter having the sequence of SEQ ID NO:141, an hsHBB poly a ("hshbbpa" or "hbbpa") 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 the group consisting of SEQ ID NOs 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259, (B) a clotting cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39, an hsHSPA8 promoter selected from SEQ ID NO:133, a btGH pA sequence selected from SEQ ID NO:112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO:97-102, 166, and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO:94-96, 103, and 187, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NO:67-70, and SEQ ID NO:106; and (c) a complement regulatory and inflammatory and apoptotic cassette comprising: a first cistron encoding CD46, a second cistron encoding CD55, a third cistron encoding CD59, a fourth cistron encoding A20, a fifth cistron encoding HO1, a sixth cistron encoding PD-L1, a CAG promoter selected from SEQ ID NOS: 126-128, and an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 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 NOS: 64-65, 104, 182, and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183, and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NOS: 89-91. In embodiments, 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 regulatory cassette from the coagulation cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the UCOE is an A2CBX3 UCOE having the sequence of SEQ ID NO. 20. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22 and 157 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE having a sequence selected from the group consisting of SEQ ID NOS.19-22 and 157 is located at the 5 'or 3' end of the cassette. In embodiments, the UCOE located at the 5 'or 3' end 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.
In embodiments, provided herein are nucleic acids comprising: (a) A innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssUBC promoter having the sequence of SEQ ID No. 141, an hsHBB poly a ("hshbbpa" or "hbbpa") 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 the group consisting of SEQ ID nos. 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID nos. 77-83, 180 and 259; (b) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a third cistron encoding CD39, a ssHSPA8 promoter selected from SEQ ID NOS: 142-145, a btGH pA sequence selected from SEQ ID NOS: 112, 120-122, 159, 190, and combinations thereof, wherein the nucleic acid sequence of THBD is selected from SEQ ID NOS: 97-102, 166, and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NOS: 94-96, 103, and 187, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NOS: 67-70, and SEQ ID NO:106; and (c) a complement regulatory and inflammatory and apoptotic cassette comprising: a first cistron encoding CD46, a second cistron encoding CD55, a third cistron encoding CD59, a fourth cistron encoding A20, a fifth cistron encoding HO1, a sixth cistron encoding PD-L1, a CAG promoter selected from SEQ ID NOS: 126-128, and an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 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 NOS: 64-65, 104, 182, and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS: 63, 109, 165, 183, and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NOS: 89-91. In embodiments, 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 regulatory cassette from the coagulation cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In an embodiment, the UCOE is CBX3 UCOE having the sequence of SEQ ID No. 21. In embodiments, the nucleic acid comprises a poly a sequence located at the 5 'or 3' end of all polycistronic cassettes. In embodiments, the poly A sequence located at the 5 'or 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO:119 or SEQ ID NO: 162.
In embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a ssHSPA8 promoter selected from SEQ ID NO:142-145, a SPA PTCH2 pA sequence having the sequence of SEQ ID NO:124, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO:97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO:94-96, 103 and 187, (B) a innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a 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 NO: 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, 180 and 259; (c) A cellular immunity and coagulation cassette comprising a first cistron encoding CTLA-4 (e.g., LEA 29Y), a second cistron encoding CD39, a ssEF1 a 1 promoter selected from SEQ ID NOs 136-138 and 179, and a btGH poly a sequence having the sequences of SEQ ID NOs 112, 120-122, 159 and 190, wherein the nucleic acid sequence of CTLA-4 is selected from SEQ ID NOs 87-88 and 186, wherein the nucleic acid sequence of CD39 is selected from SEQ ID NOs 67-70 and 106; and (d) a complement cassette comprising a first cistron encoding CD46, a second cistron encoding CD55, a CAG promoter selected from SEQ ID NOS: 126-128, and an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the nucleic acid comprises a poly a sequence located at the 5 'or 3' end of all polycistronic cassettes. In an embodiment, the poly A sequence located at the 5 'or 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO: 119. In embodiments, the nucleic acid comprises a fluorescent protein located at the 5 'or 3' end of all polycistronic cassettes. In an embodiment, the fluorescent protein located at the 5 'or 3' end of all polycistronic cassettes is a 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 embodiments, THBD is THBDda having a sequence selected from SEQ ID No. 99-102. In embodiments, TFPI comprises TFPI and a CD4 transmembrane domain. In embodiments, the TFPI has the sequence of SEQ ID NO. 94. In embodiments, the UCOE separates the complement regulatory cassette from the cellular immune and coagulation cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In an embodiment, the UCOE is CBX3 UCOE having the sequence of SEQ ID No. 21.
In embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a ssHSPA8 promoter selected from SEQ ID NO:142-145, a SPA PTCH2 pA sequence having the sequence of SEQ ID NO:124, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO:97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO:94-96, 103 and 187, (B) a innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a 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 NO: 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, 180 and 259; (c) An inflammatory and apoptotic cassette comprising 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 btGH poly a sequence having the sequences of SEQ ID NOs 112, 120-122, 159, 190, wherein the nucleic acid sequence of a20 is selected from SEQ ID NOs 64-65, 104, 182 and 188, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NOs 89-91; and (d) a complement cassette comprising a first cistron encoding CD46, a second cistron encoding CD55, a CAG promoter selected from SEQ ID NOS: 126-128, and an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the nucleic acid comprises a poly a sequence located at the 5 'or 3' end of all polycistronic cassettes. In an embodiment, the poly A sequence located at the 5 'or 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO: 119. In embodiments, the nucleic acid comprises a fluorescent protein located at the 5 'or 3' end of all polycistronic cassettes. In an embodiment, the fluorescent protein located at the 5 'or 3' end of all polycistronic cassettes is a 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 embodiments, THBD is THBDda having a sequence selected from SEQ ID No. 99-102. In embodiments, TFPI comprises TFPI and a CD4 transmembrane domain. In embodiments, the TFPI has the sequence of SEQ ID NO. 94. In embodiments, the UCOE separates the complement regulatory cassette from the inflammatory and apoptotic cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In an embodiment, the UCOE is CBX3 UCOE having the sequence of SEQ ID No. 21.
In embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a ssHSPA8 promoter selected from SEQ ID NO:142-145, a SPA PTCH2 pA sequence having the sequence of SEQ ID NO:124, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO:97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO:94-96, 103 and 187, (B) a innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a 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 NO: 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, 180 and 259; (c) Apoptosis and clotting cassettes comprising 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 btGH poly a sequence having a sequence selected from any of SEQ ID NOs 112, 120-122, 159, 190, 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 an hsGH poly a sequence having the sequence of SEQ ID NO 113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOs 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOs 84, 85, 107 and 184. In embodiments, the nucleic acid comprises a poly a sequence located at the 5 'or 3' end of all polycistronic cassettes. In an embodiment, the poly A sequence located at the 5 'or 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO: 119. In embodiments, the nucleic acid comprises a fluorescent protein located at the 5 'or 3' end of all polycistronic cassettes. In an embodiment, the fluorescent protein located at the 5 'or 3' end of all polycistronic cassettes is a 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 embodiments, THBD is THBDda having a sequence selected from SEQ ID No. 99-102. In embodiments, TFPI comprises TFPI and a CD4 transmembrane domain. In embodiments, the TFPI has the sequence of SEQ ID NO. 94. In embodiments, the UCOE separates the complement regulatory cassette from the apoptosis and coagulation cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In an embodiment, the UCOE is CBX3 UCOE having the sequence of SEQ ID No. 21.
In embodiments, provided herein are nucleic acids comprising: (a) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a ssHSPA8 promoter selected from SEQ ID NO:142-145, a SPA PTCH2 pA sequence having the sequence of SEQ ID NO:124, wherein the nucleic acid sequence of THBD is selected from SEQ ID NO:97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from SEQ ID NO:94-96, 103 and 187, (B) a innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a 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 NO: 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, 180 and 259; (c) Apoptosis and cellular immune cassettes comprising a first cistron encoding a20, a second cistron encoding CTLA-4 (e.g., LEA 29Y), a ssef1α1 promoter selected from SEQ ID NOs 136-138 and 179, and a btGH poly a sequence having the sequence of any one of SEQ ID NOs 112, 120-122, 159 and 190, wherein the nucleic acid sequence of a20 is selected from SEQ ID NOs 64-65, 104, 182 and 188, wherein the nucleic acid sequence of CTLA-4 is selected from SEQ ID NOs 87-88 and 186; and (d) a complement cassette comprising a first cistron encoding CD46, a second cistron encoding CD55, a CAG promoter selected from SEQ ID NOS: 126-128, and an hsGH poly A sequence having the sequence of SEQ ID NO:113, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOS: 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOS: 84, 85, 107, and 184. In embodiments, the nucleic acid comprises a poly a sequence located at the 5 'or 3' end of all polycistronic cassettes. In an embodiment, the poly A sequence located at the 5 'or 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO: 119. In embodiments, the nucleic acid comprises a fluorescent protein located at the 5 'or 3' end of all polycistronic cassettes. In an embodiment, the fluorescent protein located at the 5 'or 3' end of all polycistronic cassettes is a 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 embodiments, THBD is THBDda having a sequence selected from SEQ ID No. 99-102. In embodiments, TFPI comprises TFPI and a CD4 transmembrane domain. In embodiments, the TFPI has the sequence of SEQ ID NO. 94. In embodiments, the UCOE separates the complement regulatory cassette from the apoptosis and cellular immune cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In an embodiment, the UCOE is CBX3 UCOE having the sequence of SEQ ID No. 21.
In embodiments, provided herein are nucleic acids comprising: (a) A innate immune cassette comprising a first cistron encoding a B2M HLA-E fusion protein, a second cistron encoding CD47, a ssUBC promoter having the sequence of SEQ ID No. 141, an hsHBB poly a ("hshbbpa" or "hbbpa") 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 the group consisting of SEQ ID nos. 62, 66, 105, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID nos. 77-83, 180 and 259; (b) An inflammatory and apoptotic cassette comprising: a first cistron encoding A20, a second cistron encoding HO1, a third cistron encoding PD-L1, a ssHSPA8 promoter selected from SEQ ID NOS 139-140, 142-145, and a btGH poly A sequence having a sequence selected from any one of SEQ ID NOS 112, 120-122, 159, 190, wherein the nucleic acid sequence of A20 is selected from SEQ ID NOS 64-65, 104, 182, and 188, wherein the nucleic acid sequence of HO1 is selected from SEQ ID NOS 63, 109, 165, 183, and 189, wherein the nucleic acid sequence of PD-L1 is selected from SEQ ID NOS 89-91; (c) A complement cassette comprising a first cistron encoding CD46, a second cistron encoding CD55, a third cistron encoding CD59, a CAG promoter selected from SEQ ID NOs 126-128, a btHBB poly a sequence having the sequence of SEQ ID NO 160, wherein the nucleic acid sequence of CD46 is selected from SEQ ID NOs 71-76, 185, 200, and 253-258, wherein the nucleic acid sequence of CD55 is selected from SEQ ID NOs 84, 85, 107, and 184, wherein the nucleic acid sequence of CD59 is selected from SEQ ID NOs 86 and 108; and (d) a coagulation cassette comprising: a first cistron encoding THBD, a second cistron encoding TFPI, a ssICAM2 promoter having the sequence of SEQ ID NO:167, and an hsGH poly A sequence having the sequence of SEQ ID NO:113 or SEQ ID NO:161, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NO:97-102, 166 and 265-266, wherein the nucleic acid sequence of TFPI is selected from the group consisting of SEQ ID NO:94-96, 103 and 187. In embodiments, the TFPI has the sequence of SEQ ID NO: 96. In embodiments, the UCOE separates the complement regulatory cassette from the inflammatory and apoptotic cassettes. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In an embodiment, the UCOE is CBX3 UCOE having the sequence of SEQ ID No. 21. In embodiments, the nucleic acid comprises a poly a sequence located at the 5 'or 3' end of all polycistronic cassettes. In embodiments, the poly A sequence located at the 5 'or 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO:119 or SEQ ID NO: 162.
In embodiments, provided herein are nucleic acids comprising: (a) A clotting and innate immune cassette comprising: (i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOS 97-102, 166 and 265-266; (ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOS 77-83 and 180; (ii) A third cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) a ssUBC promoter selected from the group consisting of SEQ ID NOs 141, 168 and 178; and (iv) an hsGH poly-A sequence selected from SEQ ID NO.113 and 161; (b) An apoptosis and cellular immune cassette comprising (i) a first cistron encoding an a20 protein, wherein the nucleic acid sequence of a20 is selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188; (ii) A second cistron encoding HO1 protein, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (iii) A ssHSPA8 promoter selected from the group consisting of SEQ ID NOS 139-140 and 142-145; and (iv) a btGH poly a sequence selected from SEQ ID nos. 112, 120-122, 159, 190; and (c) a complement regulatory cassette comprising: (i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, and 253-258; (ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) a CAG promoter selected from SEQ ID NOS.126-128; and (iv) an hsHBB poly A sequence selected from SEQ ID nos. 114-116, 191. In embodiments, the UCOE separates the apoptosis and cellular immune cassettes from the complement regulatory cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the nucleic acid comprises a poly-a sequence at the 5 "end of all polycistronic cassettes. In an embodiment, the poly A sequence at the 5 "end of all polycistronic cassettes is btHBBpA with the nucleic acid sequence of SEQ ID NO. 160. In embodiments, the nucleic acid comprises a poly a sequence at the 3' end of all polycistronic cassettes. In an embodiment, the poly A sequence at the 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO:162 or 119.
In embodiments, provided herein are nucleic acids comprising: (a) A clotting and innate immune cassette comprising: (i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD is selected from the group consisting of SEQ ID NOS 97-102, 166 and 265-266; (ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 is selected from the group consisting of SEQ ID NOS 77-83 and 180; (ii) A third cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) a ssUBC promoter selected from the group consisting of SEQ ID NOs 141, 168 and 178; and (iv) an hsGH poly-A sequence selected from SEQ ID NO.113 and 161; (b) An apoptosis and cellular immune cassette comprising (i) a first cistron encoding an a20 protein, wherein the nucleic acid sequence of a20 is selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188; (ii) A second cistron encoding a LEA29Y protein, wherein the nucleic acid sequence of LEA29Y is selected from the group consisting of SEQ ID No. 186; (iii) A ssHSPA8 promoter selected from the group consisting of SEQ ID NOS 139-140 and 142-145; and (iv) a btGH poly a sequence selected from SEQ ID nos. 112, 120-122, 159, 190; and (c) a complement regulatory cassette comprising: (i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200, and 253-258; (ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) a CAG promoter selected from SEQ ID NOS.126-128; and (iv) an hsHBB poly A sequence selected from SEQ ID nos. 114-116, 191. In embodiments, the UCOE separates the apoptosis and cellular immune cassettes from the complement regulatory cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the nucleic acid comprises a poly-a sequence at the 5 "end of all polycistronic cassettes. In an embodiment, the poly A sequence at the 5 "end of all polycistronic cassettes is btHBBpA with the nucleic acid sequence of SEQ ID NO. 160. In embodiments, the nucleic acid comprises a poly a sequence at the 3' end of all polycistronic cassettes. In an embodiment, the poly A sequence at the 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO:162 or 119.
In embodiments, provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) a coagulation cassette comprising: (i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding said THBD protein is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) a ssUBC promoter selected from the group consisting of SEQ ID NOs 141, 168, 178; (iv) an hsGH poly a sequence selected from SEQ ID nos. 113 and 161; (b) an innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence encoding said CD47 protein is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A ssHSPA8 promoter selected from the group consisting of SEQ ID NOS 139-140, 142-145; and (iv) a btGH poly a sequence selected from SEQ ID nos. 112, 120-122, 159, 190; and (c) a complement regulatory cassette comprising: (i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding said CD46 protein is selected from the group consisting of SEQ ID NOs 71-76, 185, 200, and 253-258; (ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence encoding said CD55 protein is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) a CAG promoter selected from SEQ ID NOS.126-128; and (iv) an hsHBB poly A sequence selected from SEQ ID NOS.114-116 and 191. In embodiments, the UCOE separates the innate immune cassette from the complement regulatory cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the nucleic acid comprises a poly a sequence at the 5' end of all polycistronic cassettes. In an embodiment, the poly A sequence at the 5' end of all polycistronic cassettes is btHBBpA with the nucleic acid sequence of SEQ ID NO. 160. In embodiments, the nucleic acid comprises a poly a sequence at the 3' end of all polycistronic cassettes. In an embodiment, the poly A sequence at the 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO:162 or 119.
In embodiments, provided herein are nucleic acids comprising one or more polycistronic cassettes selected from the group consisting of: (a) a coagulation cassette comprising: (i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding said THBD protein is selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266; (ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein is selected from the group consisting of SEQ ID NOs 94-96, 103 and 187; (iii) a ssUBC promoter selected from the group consisting of SEQ ID NOs 141, 168, 178; (iv) an hsGH poly a sequence selected from SEQ ID nos. 113 and 161; (b) an innate immune cassette comprising: (i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2M HLA-E fusion protein is selected from the group consisting of SEQ ID NOs 62, 66, 105; (ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence encoding said CD47 protein is selected from the group consisting of SEQ ID NOs 77-83, 180 and 259; (iii) A ssHSPA8 promoter selected from the group consisting of SEQ ID NOS 139-140, 142-145; and (iv) a btGH poly a sequence selected from SEQ ID nos. 112, 120-122, 159, 190; (c) a complement regulatory cassette comprising: (i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding said CD46 protein is selected from the group consisting of SEQ ID NOs 71-76, 185, 200, and 253-258; (ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence encoding said CD55 protein is selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184; (iii) a CAG promoter selected from SEQ ID NOS.126-128; and (iv) an ocHBB poly A sequence having the nucleic acid sequence of SEQ ID NO: 192; and (d) an inflammatory and apoptotic cassette comprising: (i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 is selected from the group consisting of SEQ ID NOS 64-65, 104, 182 and 188; (ii) A second cistron encoding HO1 protein, wherein the nucleic acid sequence of HO1 is selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189; (iii) A sseeF1α1 promoter selected from the group consisting of SEQ ID NOS 136-138, 179; and (iv) an hsHBB poly A sequence selected from SEQ ID NOS.114-116 and 191. In embodiments, the UCOE separates the innate immune cassette from the complement regulatory cassette. In embodiments, the UCOE has a sequence selected from the group consisting of SEQ ID NOS.19-22, 157 and 193. In embodiments, the nucleic acid comprises a poly a sequence at the 5' end of all polycistronic cassettes. In an embodiment, the poly A sequence at the 5' end of all polycistronic cassettes is btHBBpA with the nucleic acid sequence of SEQ ID NO. 160. In embodiments, the nucleic acid comprises a poly a sequence at the 3' end of all polycistronic cassettes. In an embodiment, the poly A sequence at the 3' end of all polycistronic cassettes is SV40 pA with the nucleic acid sequence of SEQ ID NO:162 or 119.
III, cells, tissues, organs and animals
Porcine xenografts are widely compatible with human organ size and physiology and are ethically accepted by the general population in the united states. However, xenograft porcine tissue initiates a complex series of events, leading to graft rejection reactions, including: hyperacute rejection due to the presence of preformed antibodies to porcine antigens, complement activation and hypercoagulability, and enhancement of innate and adaptive immune responses due to molecular incompatibility. The present disclosure uses genetic engineering methods to address the shortcomings of current xenografts.
In particular, there are many immunological and functional challenges related to innate and adaptive immune functions. Complement and coagulation mediated dysfunction is caused by molecular incompatibility between donor porcine tissue and human physiology and results in acute xenograft failure. Preformed antibodies to the alpha-1, 3-galactosyl-galactose (αgal) epitope activate hyperacute transplant rejection by activation of complement. Inactivation of the glycoprotein GGTA1 gene may reduce this rapid graft disruption. The protective effect is further improved by overexpression of the human complement regulator protein (hCRP) CD46 (membrane cofactor protein), CD55 (complement decay acceleration factor) and CD59 (MAC inhibitor protein) genes.
Most non-Gal xenogenous antibodies recognize sialic acid N-glycolylneuraminic acid (Neu 5 Gc) synthesized from the CMAH protein. This gene is inactive in humans, and therefore, porcine Neu5Gc is immunogenic in humans. Thus, porcine CMAH may have to be inactivated to have clinical success in xenografts. Although expression of complement regulator and knockout of GGTA1 reduced hyperacute rejection, these genetic modifications did not affect Acute Vascular Rejection (AVR).
Coagulation dysfunction, including thrombotic microangiopathy and systemic wasting coagulopathy, persists even in the case of GGTA1 knockdown and hCRP overexpression, mainly due to molecular incompatibility of the coagulation system between pigs and non-human primate (NHP).
Although others have attempted to produce transgenic pigs for safe xenografts, these transgenic pigs carry only a limited number of transgenes due to limitations in construction capacity and transcriptional interference between transgenes. These methods have proven inadequate to overcome xenograft incompatibilities. For example, U.S. patent publication No. 2018/0249688 utilizes polycistronic expression vectors with different transgene combinations. Importantly, these polycistronic vectors contain only 4 transgenes and are used to produce pigs with 6 genetic modifications, including αgal's KO (GTKO). In the present disclosure, a combination of KO, KI and genome substitution strategies is utilized. PERV-free pigs were produced for the first time that expressed more than 6 transgenes from a single locus.
The embodiments described and disclosed herein provide a platform that enables a greater number of genetic modifications in the same pig. Through this work, porcine cells were genetically modified with more than six transgenes to produce immunocompatible cells, tissues, organs, pigs, and offspring. Multiple genes, including GGTA1, CMAH and β4galnt2, were functionally knocked out using CRISPR-Cas9 to eliminate glycans recognized by preformed human anti-swine antibodies. PiggyBAC-mediated integration or CRISPR-mediated integration is used to integrate one or more polycistronic cassettes described herein (e.g., a clotting cassette, a complement regulatory cassette, an innate immune cassette, an innate immunity and inflammation and apoptosis cassette, a cellular immunity and clotting cassette, an apoptosis and cell immunity cassette, an inflammation and apoptosis cassette, a complement regulatory and innate immunity cassette, a complement regulatory and inflammation and apoptosis cassette). In addition, porcine Endogenous Retrovirus (PERV) genes of porcine cells were knocked out by CRISPR-Cas 9.
In embodiments, provided herein are cells comprising the polycistronic cassettes described herein. In embodiments, provided herein are cells comprising a nucleic acid described herein. In embodiments, the cell is a porcine cell. In embodiments, the cell is a primary porcine cell. In embodiments, the cells are clonal. In embodiments, clonal cells are selected and genotyped. Cells of the correct genotype were expanded to support porcine cloning. Cells comprising one or more polycistronic cassettes described herein at a genomic safe harbor locus are 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 offspring produced from breeding a genetically modified animal with another genetically modified animal or a non-genetically modified animal. In embodiments, provided herein are cells, tissues, or organs from offspring.
In embodiments, the porcine donor cells used to produce the transgenic pigs are substantially free of PERV, exhibit knockouts of GGTA1, CMAH, and β4galnt2, exhibit knockins of one or more polycistronic cassettes described herein, or exhibit any combination of the foregoing features.
The present disclosure provides cells, tissues, organs, and animals having multiple modified genes, and methods of producing the same. In embodiments, the cell, tissue, organ is obtained from an animal or is an animal. In embodiments, the animal is a mammal. In embodiments, the mammal is a non-human mammal, and in embodiments, the non-human mammal is, for example, equine, primate, porcine, bovine, buffalo, bison, cow, sheep, goat, canine, or feline. In embodiments, the non-human mammal is an ungulate. In embodiments, the ungulate is a horse, rhinoceros, beagle dog, cow, pig, giraffe, camel, deer, sheep, hippocampus, whale, dolphin, or murine dolphin. In embodiments, the mammal is a porcine mammal.
Modification of genes according to the present disclosure is useful for improving molecular compatibility between a donor and a recipient and reducing 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, typically minutes to hours after implantation, and is caused by complement activation and preformed antibodies to the transplanted endothelial cells, which in turn result in procoagulant changes, which lead to hemostasis and ultimately destruction of the transplanted organ. In certain embodiments, the cells, tissues, organs, and animals produce reduced hyperacute rejection.
In embodiments, the present disclosure provides one or more cells, tissues, organs, or animals having a plurality of modified genes. In embodiments, the cell, tissue, organ or animal has been genetically modified such that multiple genes have been added, deleted, inactivated, disrupted, portions thereof have been excised, or the sequence of the genes has been altered. In embodiments, the cell, tissue, organ or animal has from about 5 to about 20 modified genes, e.g., 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 modified genes. 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 embodiments, 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 modified gene. In embodiments, the cell, tissue, organ or animal has one copy of the modified gene, and in other embodiments, the cell, tissue, organ or animal has more than one copy of the 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 the modified gene. In embodiments, the cell has one or more modified genes 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 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, between about 5 copies and about 20 copies, between about 10 copies and about 15 copies, or between 1 copy and about 5 copies.
In embodiments, 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 may have 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 15, about 20, about 25, about 30, or more copies of one or more modified genes.
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 (e.g., adult ear fibroblasts). In embodiments, the one or more cells are fetal/embryonic cells (e.g., embryonic blastomeres). In embodiments, the one or more cells are germ line 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 embodiments, the one or more cells are selected from the group consisting of: epithelial cells, liver cells, granulosa cells, adipocytes. In certain embodiments, the one or more cells are fibroblasts. In embodiments, the fibroblast is a female fetal fibroblast. 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 cell colony. In embodiments, the cell is an islet cell.
In embodiments, the one or more cells are porcine cells. Non-limiting examples of pig cell-derived or derived breeds include any of the following pig breeds: long White pigs in the united states (American Landrace), about White pigs in the united states (American Yorkshire), aksai Black pigs, bigen saddle-back pigs (Angeln saddleback), abacavia British pigs (Appalachian English), alapawa Island pigs (arabawa Island), aokland Island pigs (Auckland Island), about australian summer pigs (Australian Yorkshire), babi Kampung pigs, bacchun pigs (Ba Xuyen), bang pigs (Bantu), bask pigs (Basque), bazna pigs (Bazna), beijing Black pigs (beijin Black), white russian Black pigs (Belarus Black Pied), belgian Landrace, fig palm Shannaj pigs (Bengali Brown Shannaj), ben jeam Black spot pigs (Bentheim Black Pied) Bakesha pigs (Bakesha pigs), pizza pigs (Bisaro), ban Guer pigs (Bangur), heilazania pigs (Black Slavian), heilazanli pigs (Black Canarian), breitovo pigs, british White pigs (British Landrace), british apocynum pigs (British Lop), british White shoulder pigs (British Saddleback), bulgarian pigs (Bulgarian White), cambrough pigs, guangdong pigs (Cantoniese), keltet pigs (Celtic), chatomurciano pigs (Chato Murciana), bretser White pigs (Chester White), qingmei Black pigs (Chiangmai Blackpig), 823 pigs (Choctaw Hog), croll pigs (Croole), jettk modified White pigs (Czech Improved White), danish white pig (Danish Landrace), danish protein pig (Danish protein), mottled pig (Dermansi Pied), li Yan pig, duroc pig (Duroc), dutch white pig (Dutch Landrace), east white pig (East Landrace), east Balls pig (East Balkan), ecex pig (Essex), irish Bacon pig (Estonian Bacon), maple jing pig (Fengjin), finnish white pig (Finnish Landrace), lin Shanzhu (Forest Mountain), french white pig (French Landrace), gascon, german white pig (German Landrace), grosre (Gloucestershire Old Spots), gontin mini pig (Gottingen minipig), groskin's pig (German white) glace pigs (Grice), guinea pigs (Guinea Hog), hampshire pigs (Hampshire), hanter pigs (Hante), herford pigs (herford), hezuo pigs, huo Genzhu (Hogan Hog), huntington Black pigs (Huntington Black Hog), iberian pigs (Iberian), italian long white pigs (Italian Landrace), japanese long white pigs (Japanese Landrace), jizhou Black pigs (Jeju Black), golden pigs (Jinhua), karst Black grape pigs (Kakhetian), cola pigs (Kele), kemeju Luo Wozhu (kemero), korean Native pigs (Korean Native), kezuo pigs (krskoponje), kunty pigs (Kunekune), rad pigs (Lamcombe), large Black pigs (lange Black), kunty pigs (kunekunzune), large Black White pigs (Large Black-White), large White pigs (Large White), lamia White pigs (Latvian White), leinoma pigs, litaro local pigs (Lithuanian Native), litaro White pigs (lithuan White), linken county rolled pigs (Lincolnshire Curly-Coated), li Fu inner pigs (Livny), malhado de Alcobaca pigs, mangalis (Mangalisa), meishan pigs (Meishan), middle White pigs (Middle White), minzhu, minokawa Buta pigs, open pigs (Mong Cang, mora roman pigs, mora pigs (Moura), mukota pigs, miao Erfu special pigs (Mulefoot), mu Luom pigs (Murogin), mi Erhuo Luo Dezhu (Myrhood), nero dei Nebrodi pigs, neiji (Neiji) and combinations thereof New Zealand pigs (New Zealand), ningxia pigs (Ningxiang), north Gaojia pigs (North Caucasian), north Siberian pigs (North Siberian), norway Changbai pigs (Norwegian Landrace), norway approximately Chazu pigs (Norwegian Yorkshire), ossabawa Island pigs (Ossabaw Island), oxford Sandiner pigs (Oxford Sandy and Black), pakchong 5 pigs, philippine local pigs (Philippine Native), pietrain, poland China, red pig (Red Watle), saddleback pigs, semirechesk pigs (Siberian Black Pied), small Black pigs (Smalblack), small White pigs (Small White), spot pigs (Spots), surabaya Babi, shi Weibi Shi Haer pigs (Swan-Hall), pakchon 5 pigs (Pond China), swedish white pigs (Swedish Landrace), yan Fu Swedish Manganin pigs (Swallow Belied Mangalitza), taihu pigs (Taihu pig), tamhwok pigs (Tamworth), thuoc Nhieu pigs, tibetan pigs (Tibetan), tokyo-X pigs (Tokyo-X), ji Wei Lisk pigs (Tsivilsk), turopupje pigs, wuban prairie pigs (Ukrainian Spotted Steppe), wu Bai prairie pigs (Ukrainian White Steppe), wu Erru mu pigs (Urzhhum), vietnam big belly pigs (Vietnamese Potbelly), weissels pigs (Welsh), weissex Bai Jian pigs (Wessex Saddleback), west white pigs (West French White), windser pigs, wuzhishan pigs (Wuzhishman), yanan pigs (Yanan), dakka pigs and about Xia Lanbai pigs (Yorkshire Blue and White). In embodiments, the porcine cell is an approximately gram summer and ewing porcine cell.
In embodiments, the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes have been modified by addition, deletion, inactivation, disruption, excision of portions thereof, or a portion of the gene sequence has been altered.
In embodiments, the cells, tissues, organs or animals of the present disclosure comprise 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 having the one or more mutations. In embodiments, the one or more genes are inactivated by genetic modification of one or more nucleic acids present in the cell, tissue, organ or animal. In embodiments, inactivation of one or more genes is confirmed by an assay. In embodiments, the assay is an infectious assay, a reverse transcriptase PCR assay, an RNA-seq, a real-time PCR, or a ligation PCR localization assay.
Specific genotypes
To provide cells, tissues, organs and animals that are safe and effective for clinical use in humans, the cells, tissues, organs and animals of the present disclosure (e.g., donor pigs) are genetically engineered to have an enhanced complement (i.e., complement toxicity), coagulation, inflammation (i.e., apoptosis/inflammation), immunity (i.e., cytotoxicity) and/or immune modulating system, making them compatible in humans. The novel combination of Knockout (KO), knock-in (KI) (also referred to herein as Transgene (TG)) and/or genome replacement strategies provides enhanced complement, coagulation, inflammation, immune and/or immune regulatory systems. Various methods are used to assess the expression of genes that make porcine cells and organs compatible with humans. Non-limiting examples of such methods include flow cytometry, immunohistochemistry, and RNA sequencing.
For example, cells, tissues, organs and animals lacking expression of the major xenogenous carbohydrate (xenogenic carbohydrate) antigen due to gene KO reduce or eliminate humoral rejection reactions during xenograft. Three major heterologous carbohydrate antigens include those produced by glycosyltransferases/glycosylhydrolases GGTA1, CMAH and B4GALNT 2. The purpose of the loss of function of these genes is to reduce and/or eliminate the binding of preformed anti-porcine antibodies to the porcine graft endothelium.
Insertion of key complement, coagulation, inflammation, immune and/or immunomodulatory factors into one or more genomic loci, e.g., a safe harbor genomic locus (e.g., AAVS 1), will help regulate the human complement system as well as Natural Killer (NK), macrophage and T cell function. Non-limiting examples include over-expression of hCD46, hCD55, and hCD59 by KI to inhibit the human complement cascade; humanizing vWF to prevent unregulated platelet sequestration and thrombotic microangiopathy, for example, by humanizing the A1 domain and/or flanking regions of the porcine vWF sequence; KI of B2M-HLA-E SCT to provide protection against human NK cell cytotoxicity and humanization of porcine cells; and KI of CD47, CD39, THBD, TFPI, A20 to act as immunosuppressants, immunomodulators and/or anticoagulants.
In embodiments, the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes or portions thereof have been modified by addition, deletion, inactivation, disruption, excision of portions thereof. In embodiments, the present disclosure provides isolated cells, tissues, organs, or animals having a plurality of modified genes. In embodiments, the modified gene comprises one or more of the following: αGGTA1, β4GalNT2, CMAH, THBD, TFPI, CD, 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 (e.g., LEA 29Y), and XIAP.
In embodiments, the modified gene is GGTA1, B4GalNT2, CMAH, or any combination thereof. In embodiments, GGTA1, B4GalNT2 and/or CMAH are genetically KO. In embodiments, the modified genes are MHC-I genes SLA-1, SLA-2, and SLA-3, MHC-II genes DQA and DRA, endogenous vWF, CD9, asialoglycoprotein receptors, at least one complement inhibitor gene (e.g., C3, CD46, CD55, and CD 59), and any combination thereof. In embodiments, CD46, CD55, and/or CD59 are genetically KI.
In one embodiment, the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgenic expression vector comprising CD46, CD55, CD59, THBD, TFPI, EPCR, CD39, B2M, HLA-E, CD47, a20, PD-L1, HO1, CTLA-4 (e.g., LEA 29Y), XIAP, or any combination thereof. In one embodiment, the cells, tissues, organs or animals of the present disclosure have been further genetically modified to have reduced or no expression of GGTA1, B4GalNT2, CMAH or any combination thereof, for example by gene KO.
The cells, tissues, organs or animals of the present disclosure can be genetically modified by any method. In embodiments, a cell, tissue, organ or animal of the present disclosure is modified with a nucleic acid comprising a plurality of polycistronic cassettes. Non-limiting examples of suitable methods for Knockout (KO), knock-in (KI) and/or genome substitution strategies disclosed and described herein include CRISPR-mediated genetic modification using Cas9, cas12a (Cpf 1) or other CRISPR endonucleases, argonaute endonucleases, transcription activator-like (TAL) effectors and nucleases (TALENs), zinc Finger Nucleases (ZFNs), expression vectors, transposon systems (e.g., piggyBac transposase), or any combination thereof.
The cells, tissues, organs or animals of the present disclosure may be PERV-free. For example, cells, tissues, organs or animals of the present disclosure may have a PERV copy functionally deleted from their genome. The cells, tissues, organs or animals of the present disclosure may have functionally inactive copies of PERV in their genomes. PERV represents a risk factor if porcine cells, tissues or organs are transplanted into a human recipient. PERV is released from normal porcine cells and is infectious. PERV-Sup>A and PERV-B are amphotropic viruses that infect cells of several species, including humans (e.g., they are amphotropic); whereas PERV-C is an aviphilic virus that infects only porcine cells. Non-limiting methods of functionally deleting PERV copies are disclosed and described in Niu 2017 and WIPO publication No. WO2018/195402, both of which are incorporated herein by reference in their entirety. In embodiments, the pig is genetically engineered to be free of PERV-A, PERV-B or PERV-C (or any combination thereof).
In embodiments, the additional genes of the cells, tissues, organs or animals of the present disclosure are modified by addition, deletion, inactivation, disruption, excision of portions thereof or portions of the gene sequence. In embodiments, the modification comprises the 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 expression of one or more of the following transgenes: PD-L1, exogenous vWF, HLA-E, HLA-G, B2M and CIITA-DN. In embodiments, the modification comprises the deletion of one or more of the following genes: alpha galactosyltransferase 1, GGTA1, beta 4GALN2 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 (e.g., LEA 29Y), CD73, SOD3, CXCL12, fasL, CXCR3, CD39L1, GLP-1R, M3R, IL35, IL12A and EB13. In embodiments, the modified gene is CD46, CD55, CD59, CD47, HO1, A20, TNFR1-Ig, CD39, THBD, TFPI, EPCR, PD-1, CTLA-4 (e.g., LEA 29Y), CD73, SOD3, CXCL12, fasL, CXCR3, CD39L1, GLP-1R, M3R, IL35, IL12A and EB13.
In embodiments, the cells, tissues, organs or animals of the present disclosure comprise one or more genetically modified genes. The genetically modified gene may contain one or more nucleotide additions, one or more nucleotide deletions, or one or more nucleotide mutations. In embodiments, the genetically modified gene is disrupted. In embodiments, 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 the wild-type cell, tissue, organ or animal. In embodiments, the transgene is an exogenous gene. In embodiments, the transgene is an endogenous gene. In embodiments, 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.
In embodiments, the modified gene is an MHC class I gene. In embodiments, the modified MHC class I genes include one or more of the following SLA-1, SLA-2, SLA-3, and B2M. In embodiments, the modified genes are SLA-1, SLA-2 and/or SLA-3. In embodiments, the modified gene is B2M. In embodiments, the modified MHC class I genes include one or more of the following SLA-1, SLA-2, SLA-3, and B2M. In embodiments, the modified B2M, SLA-1, SLA-2 and/or SLA-3 genes and/or parts thereof are replaced with human HLA-E genes, human HLA-G genes, human B2M genes and/or human (dominant negative mutant class II transactivator factor) CIITA-DN genes and/or parts thereof. In embodiments, the modified gene is conditionally and/or inducibly modified. In embodiments, conditional promoters and/or inducible promoters are used to conditionally and/or inductively modify the one or more modified genes. In embodiments, provided herein are isolated cells, tissues, organs, or animals comprising B2M, SLA-1, SLA-2, or SLA-3 genes, or any combination of genes thereof, that are conditionally altered and replaced with at least a portion of a human HLA-E gene, a human HLA-G gene, a human B2M gene, and/or a human CIITA-DN gene.
In embodiments, provided herein are cells, tissues, organs, or animals comprising modified MHC class II genes. In embodiments, the modified MHC class II gene is DRQ, DRA, or any combination thereof. The 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, transcription and/or translation of the genetically modified MHC class II gene is disrupted. In embodiments, the genetically modified MHC class II gene is inactivated. In embodiments, the cell, tissue, organ or animal comprises an MHC class II transgene or portion thereof that is not present in the wild-type cell, tissue, organ or animal. In embodiments, the modified gene is conditionally and/or inducibly modified. In embodiments, conditional promoters and/or inducible promoters are used to conditionally and/or inductively modify the one or more modified genes. In embodiments, the isolated cell, tissue, organ, or animal comprises a conditionally altered DRQ and/or DRA gene, or any combination thereof.
In embodiments, provided herein are cells, tissues, organs or animals comprising a modified vWF gene and/or a vWF-related gene. The genetically modified vWF gene and/or vWF-related gene may contain one or more nucleotide additions, one or more nucleotide deletions, or one or more nucleotide mutations. In embodiments, the genetically modified vWF gene and/or vWF-related gene is disrupted. In embodiments, transcription and/or translation of the genetically modified vWF gene and/or vWF-related gene is disrupted. In embodiments, the genetically modified vWF gene and/or vWF-related gene is inactivated. In embodiments, the cell, tissue, organ or animal comprises a vWF gene and/or vWF-related gene transgene or a portion thereof that is not present in the wild-type cell, tissue, organ or animal. In embodiments, the modified vWF gene and/or part thereof is replaced with a human vWF gene and/or part thereof. In embodiments, the modified vWF gene, modified vWF-related gene, and/or one or more portions thereof is replaced with a human vWF gene, one or more human vWF-related genes, and/or portions thereof. In embodiments, the modified vWF gene and/or vWF-related gene is conditionally and/or inducibly modified. In embodiments, conditional promoters and/or inducible promoters are used to conditionally and/or inductively modify the one or more modified genes. In embodiments, the isolated cell, tissue, organ or animal comprises a conditionally altered vWF, vWF-related gene, one or more parts thereof, or any combination thereof, and the conditionally altered gene is replaced with a human vWF gene, at least part of a human vWF gene, one or more other human vWF-related genes, at least part of one or more human vWF-related genes, or any combination thereof. In embodiments, the vWF gene is modified with a gRNA designed to initiate HDR substitution in the endogenous porcine genome and cut near the region to be replaced by the human sequence.
In embodiments, provided herein are cells, tissues, organs, or animals comprising one or more modified complement genes. In embodiments, the complement gene is C3. The genetically modified complement gene may contain one or more nucleotide additions, one or more nucleotide deletions, or one or more nucleotide mutations. In embodiments, the genetically modified complement gene is disrupted. In embodiments, transcription and/or translation of the genetically modified complement gene is disrupted. In embodiments, the genetically modified complement gene is inactivated. In embodiments, the cell, tissue, organ or animal comprises a complement gene or portion thereof that is not present in the wild-type cell, tissue, organ or animal. In embodiments, the modified C3 gene and/or complement gene is conditionally and/or inducibly modified. In embodiments, conditional promoters and/or inducible promoters are used to conditionally and/or inductively modify the one or more modified genes. In embodiments, the isolated cell, tissue, organ, or animal comprises conditionally altered C3, complement gene, one or more portions thereof, or any combination thereof. In embodiments, the C3 gene is modified using gRNA.
In embodiments, the modified gene is a knockout of C3. In embodiments, the modified gene is a knock-in of PD-L1. In embodiments, the modified gene is a humanized vWF of porcine vWF. In embodiments, the modified gene is a conditional knock-in of MHC-I genes SLA-1, SLA-2 and SLA-3.
In embodiments, the host does not or substantially does not elicit an immune response in a genetically modified cell, tissue, or organ.
In embodiments, the present disclosure provides nucleic acids 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 alternatively the genome of the cell is modified. In embodiments, the gene or portion thereof is genetically modified using any of the genetic modification systems known in the art and/or disclosed herein. In embodiments, the genetic modification system is a TALEN, zinc finger nuclease, and/or CRISPR-based system. In embodiments, the gene modification system is a CRISPR-Cas9 system. In embodiments, the genetic modification system is a type II CRISPR system. In embodiments, the genetic modification system is a class II V-type CRISPR system. In embodiments, the cell is genetically modified such that one or more genes or portions thereof in the cell are inactivated, and further the cell is genetically modified such that expression of one or more genes or portions thereof of the cell (which genes or portions thereof would induce an immune response if the cell (or tissue or organ cloned/derived from the cell) were transplanted into a human body) is reduced. In embodiments, the cells are genetically modified to increase expression of one or more human genes or portions thereof. In embodiments, the cells are genetically modified to increase expression of one or more humanized genes or portions thereof. In embodiments, the cell is genetically modified such that one or more genes or portions thereof in the cell are inactivated, and further the cell is genetically modified such that expression of one or more genes of the cell (which genes inhibit an immune response if the cell (or tissue or organ cloned/derived from the cell) is transplanted into a human body) is increased. In embodiments, the cell is genetically modified such that one or more genes or portions thereof in the cell are inactivated, and further the cell is genetically modified such that expression of one or more genes of the cell (which genes would induce an immune response if the cell (or tissue or organ cloned/derived from the cell) is transplanted into a human body) is reduced, and further the cell is genetically modified such that expression of one or more genes of the cell (which genes would inhibit an immune response if the cell (or tissue or organ cloned/derived from the cell) is transplanted into a human body) is increased.
In embodiments, the present disclosure provides embryos cloned from genetically modified cells. The following references describe the production of embryos from genetically modified cells and are incorporated herein by reference in their 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 embodiments, the oocyte may be enucleated by: a partial zona pellucida dissection was performed near the pole body, and then the cytoplasm was pressed out at the dissection zone. In embodiments, the genetically modified cells are injected into enucleated oocytes arrested in meiosis 2 using an injection pipette with a sharp beveled tip. Oocytes that arrest in meiosis phase 2 are commonly referred to as "eggs". In embodiments, the embryo is produced by fusing and activating an oocyte. In embodiments, the oocytes are fused by delivering an electrical pulse to the oocytes. Such embryos may be referred to herein as "genetically modified embryos". In embodiments, the genetically modified embryo is activated by treatment with 6-dimethylaminopurine (6-DMAP), ionomycin, or a combination thereof. In embodiments, the genetically modified embryo is treated with a histone deacetylase inhibitor (HDACi). In embodiments, the genetically modified embryo is transferred to the oviduct of a recipient female pig. In embodiments, the genetically modified embryo is transferred to the oviduct of a recipient female pig 12 hours to 4 days after activation. For example, the genetically modified embryo is transferred to the oviduct of a recipient female pig 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 after activation. In embodiments, the genetically modified embryo is transferred to the oviduct of a recipient female pig 20 to 24 hours after activation. See, for example, cibelli 1998 and U.S. Pat. No. 6,548,741. In embodiments, the recipient female is checked for pregnancy approximately 20-21 days after transferring the genetically modified embryo.
In embodiments, the genetically modified embryo is grown into a post-natal genetically modified animal. In embodiments, the post-natal genetically modified animal is a neonatal genetically modified animal. In embodiments, the genetically modified pig is a young genetically modified animal. In embodiments, the genetically modified animal is an adult genetically modified animal (e.g., greater than 5-6 months). In embodiments, the genetically modified animal is a female genetically modified animal. In embodiments, 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 swine is bred with another genetically modified animal having reduced or no active virus. In embodiments, the genetically modified animal is bred with a second genetically modified animal that has been genetically modified such that cells, tissues, or organs from the second genetically modified animal are less likely to induce an immune response if transplanted into a human.
In embodiments, the genetically modified animal is an animal having one or more modified genes and maintains the same or similar level of expression or inactivation of the one or more modified genes for at least one month, at least 6 months, at least 1 year, at least 5 years, at least 10 years after gestation. In embodiments, the genetically modified animal remains genetically modified as a genetically modified pig with one or more modified genes even after delivery from a non-virally inactivated surrogate or after being in a facility/space with other non-virally inactivated animals.
In embodiments, the present disclosure provides cells, tissues or organs obtained from any of the post-natal gene modified pigs described herein. In embodiments, the cell, tissue or organ is selected from the group consisting of liver, kidney, lung, heart, pancreas, muscle, blood and bone. In particular embodiments, the organ is the liver, kidney, lung or heart. In embodiments, the cells from the post-natal genetically modified pig are selected from the group consisting of: pancreatic islets, lung epithelial cells, cardiac myocytes, skeletal muscle cells, smooth muscle cells, liver cells, non-parenchymal liver cells, gall bladder epithelial cells, gall bladder endothelial cells, bile duct epithelial cells, bile duct endothelial cells, liver vascular epithelial cells, liver vascular endothelial cells, sinus cells, choroidal 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 (i.e., from the pig in which they are growing). In embodiments, separation from the natural environment means an overall physical separation from the natural environment, e.g., removal from a genetically modified donor animal, and altering the relationship of the genetically modified organ, tissue, or cell to adjacent cells that they are in direct contact with (e.g., by dissociation).
Methods for producing cells, tissues, organs or animals
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 embodiments, 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 cells a gene editing agent specific for a gene, wherein the agent disrupts 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 agent introduces an inactivating mutation into the gene. In embodiments, the agent inhibits transcription of the gene.
In embodiments, the present disclosure provides methods of altering one or more genes, or portions thereof, in vivo, comprising administering to the cell a gene editing agent specific for a gene, wherein the agent alters the sequence of the gene, such as by humanizing or otherwise altering the natural (e.g., wild-type) sequence of the gene.
In embodiments, the present disclosure provides a method of expressing one or more nucleic acids comprising one or more polycistronic cassettes or portions thereof, the method comprising administering to a cell a gene editing agent comprising one or more polycistronic cassettes or portions thereof, wherein the agent introduces a nucleic acid sequence comprising one or more polycistronic cassettes. In embodiments, the agent is a nucleic acid sequence, such as a plasmid, vector, or the like. In embodiments, the nucleic acid sequences include one or more nucleic acid sequences, such as promoters, transgenes, and/or additional genes. In embodiments, the nucleic acid sequence or portion thereof is derived from one or more species and/or one or more sources. In embodiments, the species is a species that will receive a genetically modified cell, tissue, or organ. In embodiments, the species is human. In other embodiments, the species is non-human, such as mammalian, animal, bacterial, and/or viral.
In embodiments, any of the reagents disclosed herein are polynucleotides. In embodiments, the polynucleotide encodes one or more of the nucleases and/or nicking enzymes 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 one or more cells in a manner such that the one or more cells transiently express the polynucleotide. In embodiments, the polynucleotide is introduced into one or more cells in a manner such 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 with one or more transposable elements. In embodiments, the transposable element is a polynucleotide sequence encoding a transposase. In embodiments, the transposable element is a polynucleotide sequence encoding a PiggyBac transposase. In embodiments, the transposable element is inducible. In embodiments, the transposable element is doxycycline-inducible. In embodiments, the 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 (e.g., GFP).
In embodiments, the agent is a nuclease or nickase for targeting DNA in a cell. In embodiments, the agent specifically targets and inhibits expression of a gene. In embodiments, the agent comprises a transcriptional repressor domain. In embodiments, the transcriptional repressor domain is a kruppel-associated cassette (KRAB).
In embodiments, the agent is any programmable nuclease. In embodiments, the agent is a natural homing meganuclease. In embodiments, the agent is a TALEN-based agent, ZFN-based agent, or CRISPR-based agent, or any biologically active fragment, fusion, derivative, or combination thereof. CRISPR-based agents include, for example, class II type II and type V systems, including various species variants such as Cas9 and Cpf 1. In embodiments, the agent is a deaminase or a nucleic acid encoding a deaminase. In embodiments, the cells are genetically engineered to stably and/or transiently express TALEN-based agents, ZFN-based agents, and/or CRISPR-based agents.
In embodiments, any of the nucleic acids described herein, or fragments thereof, are integrated into the genome of a host cell. Nucleic acids may be integrated into cells by recombinase-mediated cassette exchange, piggyBac transposon-mediated gene transfer, CRISPR-mediated homologous dependent recombination, or CRISPR-mediated nonhomologous dependent targeted integration.
In embodiments, any of the nucleic acids described herein, or fragments thereof, are integrated into a host cell at a genomic safe harbor locus comprising a landing gear. Fig. 28A and 28C provide an exemplary landing gear. Non-limiting examples of genomic safe harbor loci include AAVS1, CCR5, and Fos5. In embodiments, the landing gear is inserted into intron 1 of the AAVS1 site. In embodiments, the landing gear incorporates a 3' flanking region of the AAVS1 site. In embodiments, the landing gear comprises an Ank2 gene, cpsf4 gene, C-Mos gene, fos1 gene, or adeno-associated virus integration site (AAVS). U.S. patent No. 8,980,579 describes a chromosomal landing gear and is incorporated herein by reference in its entirety. In embodiments, the landing gear comprises an adeno-associated virus integration site (AAVS) or Fos1 gene. In embodiments, the landing gear comprises an insulator sequence (e.g., HS 4). In embodiments, the landing gear comprises one or more loxP sites. In embodiments, the landing gear comprises two loxP sites. In embodiments, the landing gear comprises a poly a sequence. In embodiments, the poly a sequence comprises a terminator. In embodiments, the landing gear comprises a label, such as Blue Fluorescent Protein (BFP). In embodiments, the landing gear comprises a promoter. In embodiments, the promoter comprises any one of the sequences in table B. In embodiments, the poly a sequence comprises any one of the sequences in table C. In embodiments, from 5 'to 3', the landing gear 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 embodiments, from 5 'to 3', the landing gear comprises a first loxP site, a promoter, a fluorescent protein, a poly a sequence, and a second loxP site. In embodiments, the landing gear comprises the nucleic acid sequence of SEQ ID NO 248 or 249.
In embodiments, the landing gear is integrated into the genomic safe harbor locus by recombinant enzyme-mediated cassette exchange. In embodiments, the landing gear is integrated into the genomic safe harbor locus by CRISPR-Cas9 Homology Directed Repair (HDR).
In embodiments, provided herein are cells, organs, or tissues comprising landing gear. In embodiments, provided herein are cells, organs or tissues comprising a landing gear comprising the nucleic acid sequence of SEQ ID NO 248 or 249.
In embodiments, the polycistronic cassette of any one of fig. 1A-1D, fig. 2A-2J, fig. 3A-3C, fig. 4A-4H, fig. 5A-5F, or fig. 6 is introduced into the cell after the landing gear is introduced into the cell. In embodiments, the polycistronic cassette of any of figures 1A-1D, 2A-2J, 3A-3C, 4A-4H, 5A-5F, or 6 replaces the poly a sequence and marker gene of the landing gear. Integration of the polycistronic cassette can be performed using recombinase-mediated cassette exchange or CRISPR-Cas9 HDR. Fig. 28B provides an illustration of the integration of the 15S5 payload (see fig. 2I) into the landing gear of fig. 28A. In embodiments, the nucleic acid integrated into the genomic safe harbor locus comprises sequences homologous to genomic integration sites at the 5 'and 3' ends of the nucleic acid. In embodiments, the nucleic acid comprising the polycistronic cassette of any one of figures 1A-1D, 2A-2J, 3A-3C, 4A-4H, 5A-5F, or 6 comprises sequences homologous to the genomic integration sites at the 5 'and 3' ends of the nucleic acid.
In embodiments, provided herein is a method of producing a genetically modified cell, the method comprising (i) knocking out GGTA1, CMAH, and β4galnt2; (ii) Knocking out one or more PERV elements (e.g., PERV pol, env, gag genes, or a combination thereof); (iii) Knocking in any one or more polycistronic cassettes described herein. The foregoing process may be performed in any order of: (i), (ii), (iii); (i), (iii), (ii); (ii), (i), (iii); (ii), (iii), (i); (iii), (i), (ii); or (iii), (ii), (i). In embodiments, the tissue, organ or animal comprises cells produced according to any of the preceding methods. In embodiments, the cell is a porcine cell. In embodiments, the cell is a primary porcine cell. In embodiments, the cell is a human cell.
V. therapeutic methods
In embodiments, any of the genetically modified cells, tissues, or organs disclosed herein can be used to treat a subject of a different species than the genetically modified cells. 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, marmoset, chimpanzee, bonobo, gorilla, gibbon, cow, horse, bird, sheep, or gorilla.
In embodiments, the genetically modified organ used in any of the methods disclosed herein may be selected from the group consisting of heart, lung, liver, eye, pituitary, thyroid, parathyroid, esophagus, thymus, adrenal, appendix, bladder, gall bladder, small intestine, large intestine, small intestine, kidney, pancreas, spleen, stomach, skin, and/or prostate of a genetically modified pig. In embodiments, the genetically modified tissue for use in any of the methods disclosed herein may be selected from genetically modified porcine cartilage (e.g., esophageal cartilage, knee cartilage, ear cartilage, nose cartilage), muscle (e.g., without limitation, smooth muscle and heart (e.g., heart valve)), tendons, ligaments, bone (e.g., 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, and/or stem cells. The compositions of the present disclosure may also be administered to any portion of an organ or tissue (e.g., a portion of an eye such as the cornea).
In embodiments, the heart, lung, liver, kidney, pancreas, or spleen is isolated from a pig that has been genetically modified to comprise (a) a deletion or disruption of GGTA1, CMAH, and B4GALNT 2; (b) Addition of CD46, CD55, CD59, THBD, TFPI, PROCR, CD39, B2M, HLA-E, CD47, a20, PD-L1, HO1, CTLA-4 (e.g., LEA 29Y), XIAP, and combinations thereof (e.g., human or humanized copies thereof) expressed from a single multiple transgene cassette in the pig genome; and (c) a functional absence of all PERV copies. In embodiments, the heart, lung, liver, kidney, pancreas, or spleen is isolated from a pig that has been genetically modified to comprise (a) a functional disruption of GGTA1, CMAH, and B4GALNT 2; (b) Addition of CD46, CD55, CD59, THBD, TFPI, PROCR, CD39, B2M, HLA-E, CD47, a20, PD-L1, HO1, CTLA-4 (e.g., LEA 29Y), XIAP transgene, and combinations thereof (e.g., humanized copies thereof) expressed from a single multiple transgene cassette in the pig genome; 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.
In embodiments, the organ is stored at a temperature between 2 ℃ and about 37 ℃ prior to transplantation. For example, in embodiments, the organ is stored at a temperature of about 2 ℃, about 3 ℃, about 4 ℃, 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 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, or about 37 ℃. In embodiments, the organ is stored at a temperature of about 2 ℃ to about 8 ℃, about 4 ℃ to about 8 ℃, or about 20 ℃ to about 25 ℃. In embodiments, organs are stored on ice prior to transplantation.
In embodiments, the organ is stored in an organ preservation solution prior to transplantation. In embodiments, the organ preservation solution contains histidine, tryptophan, ketoglutarate, sodium chloride, human serum albumin, dextran 40, glucose, mannitol, sucrose, citrate, hydroxyethyl-piperazine-ethane sulfonic acid (HEPES), sodium ions, potassium ions, lactobionic acid salts, raffinose, glutathione, allopurinol, magnesium ions, trehalose, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, or combinations thereof. In embodiments, the organ preservation solution is oxygen-containing. In an embodiment, the organ preservation solution comprises 0.9% sodium chloride. The organ was stored in histidine-tryptophan ketoglutarate.
In embodiments, the organ is perfused with a perfusate prior to transplantation. When referring to an organ, the term "perfusate" refers to pumping a fluid (e.g., perfusate) through the organ. In embodiments, the perfusate is delivered to the organ through the circulatory system or lymphatic system. In embodiments, the perfusate is delivered to a vessel of the organ. In embodiments, the perfusate is an organ preservation solution. In embodiments, the perfusate comprises plasma, whole blood, red blood cells, hemoglobin, human serum albumin, or a combination thereof. In embodiments, the perfusate comprises heparinized human whole blood. In embodiments, the perfusate is oxygenated.
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, a variable light domain, a constant light chain, a constant heavy chain domain (CH 1, CH2, CH3 or CH 4), or any combination thereof. In embodiments, 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).
In embodiments, the organ is perfused at a pressure between 30mmHg and about 100 mmHg. For example, the organ is perfused at a pressure of about 30mmHg, about 35mmHg, about 40mmHg, about 45mmHg, about 50mmHg, about 55mmHg, about 60mmHg, about 65mmHg, about 70mmHg, about 75mmHg, about 80mmHg, about 85mmHg, about 90mmHg, about 95mmHg, or about 100 mmHg. In embodiments, the organ is perfused at a pressure of about 40mmHg to about 75 mmHg.
In embodiments, the organ is perfused at a temperature between about 2 ℃ and about 37 ℃. For example, in embodiments, the organ is perfused at a temperature of about 2 ℃, about 3 ℃, about 4 ℃, 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 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, or about 37 ℃. In embodiments, the organ is perfused at a temperature of about 20 ℃ to about 25 ℃.
In embodiments, the organ is perfused for about 5 minutes to about 2 days. For example, the number of the cells to be processed, the organ is perfused 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 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 days.
In embodiments, the organ is perfused and then stored at a temperature of about 4 ℃ to about 8 ℃.
In embodiments, 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 reduction in troponin I levels of between about 50% to about 100% compared to a subject receiving a transplant of an organ that has not been stored and/or perfused according to one of the methods described previously. For example, 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 troponin I level that is 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% lower than a troponin I level of a subject receiving a transplant of an organ that has not been stored and/or perfused according to one of the methods described above.
In embodiments, a subject receiving a transplant of an organ that has been stored and/or perfused according to any of the methods described herein does not exhibit thrombocytopenia. In embodiments, a subject receiving a transplant of an organ that has been stored and/or perfused according to any of the methods described herein exhibits thrombocytopenia after a patient receiving a transplant of an organ that has not been stored and/or perfused according to one of the methods described herein. In embodiments, 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 platelet count that is 15% to about 500% higher than a subject receiving a transplant of an organ that has not been stored and/or perfused according to one of the methods described previously. For example, a subject receiving a transplant of an organ that has been stored and/or perfused according to any of the methods described herein exhibits about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% of the platelet count. Platelet count is measured according to any method known in the art. For example, platelet counts are measured using a cytometer or flow cytometry.
In embodiments, the induction therapy is administered to the subject prior to the transplant, at substantially the same time as the organ transplant (e.g., within 1 hour of the transplant), or after the organ transplant. The term "induction therapy" when referring to organ transplantation refers to any substance that reduces the risk of organ rejection. In embodiments, the induction therapy comprises an anti-thymocyte globulin (ATG), rituximab, an anti-CD 154 antibody or antibody fragment thereof, mycophenolate mofetil, rapamycin, tacrolimus, prednisone, or a combination thereof.
In embodiments, once xenografted into a human or non-human primate, the xenografted organ (e.g., heart, lung, liver, kidney, pancreas, spleen) exhibits sustained function for more than about 300 days, more than about 1 year, more than about 1.5 years, more than about 2 years, more than about 2.5 years, more than about 3 years, more than about 3.5 years, more than about 4 years, more than about 4.5 years, more than about 5 years, more than about 5.5 years, more than about 6 years, more than about 6.5 years, more than about 7 years, more than about 7.5 years, more than about 8 years, more than about 8.5 years, more than about 9 years, more than about 9.5 years, or more than about 10 years.
In embodiments, the present disclosure provides treatment of a subject suffering from a disease, disorder, or injury that results in impaired, insufficient, or absent function of an organ, tissue, or cell. In embodiments, the subject is suffering from a injury or trauma (e.g., car accident) that results in damage to one or more cells, tissues or organs of the subject. In embodiments, the subject suffers from fire or acid burn. In embodiments, the subject suffers from a disease or disorder that results in impaired, deficient, or absent function of an organ, tissue, or cell. In embodiments, the subject has an autoimmune disease. In embodiments, the subject has organ failure. In embodiments, the disease is selected from: heart disease (e.g., atherosclerosis), dilated cardiomyopathy, severe coronary artery disease, scar heart tissue, congenital defects of the heart, type I or type II diabetes, hepatitis, cystic fibrosis, cirrhosis, renal failure, lupus, scleroderma, igA nephropathy, polycystic kidney disease, myocardial infarction, emphysema, chronic bronchitis, bronchiolitis obliterans, pulmonary hypertension, congenital diaphragmatic hernias, congenital surfactant protein B deficiency and congenital emphysema-type lung disease, primary cholangitis, sclerosing cholangitis, biliary tract closure, alcoholism, wilson's disease, hemochromatosis and/or alpha-1 antitrypsin deficiency.
In embodiments, any genetically modified cell, tissue, and/or organ of the disclosure is isolated from a genetically modified donor and a non-donor subject host is administered. As used herein, "administration" or "administration" includes, but is not limited to, introducing, applying, injecting, implanting, transplanting, suturing, and transplanting. According to the present disclosure, the genetically modified cell, tissue, and/or organ may be administered by a method or route that results in the localization of the organ, tissue, cell, or composition of the present disclosure at a desired site. The organ, tissue, cell, or composition of the present disclosure can be administered to a subject by any suitable route that results in delivery of the cell to a desired location in the subject, wherein at least a portion of the cell remains viable. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (whether alone or as part of a tissue or organ) of the cells remain viable after administration to a subject. Methods of administering the organs, tissues, cells or compositions of the present disclosure are well known in the art. In embodiments, the cells, tissues and/or organs are transplanted into a host. In embodiments, the cells, tissues and/or organs are injected into a host. In embodiments, the cells, tissues and/or organs are transplanted onto a host surface (e.g., bone or skin).
In embodiments, hearts, lungs, livers, kidneys, pancreas, or spleen that have been genetically modified to contain: deletion or disruption of GGTA1, CMAH and B4GALNT 2; expression of CD46, CD55, CD59, THBD, TFPI, PROCR, CD39, B2M, HLA-E, CD47, a20, PD-L1, HO1, CTLA-4 (e.g., LEA 29Y), XIAP, and combinations thereof from a single multiple transgene cassette in the pig genome; and the absence of all PERV copies. In embodiments, hearts, lungs, livers, kidneys, pancreas, or spleen that have been genetically modified to contain: deletions of GGTA1, CMAH and B4GALNT 2; expression of one or more of CD46, CD55, CD59, THBD, TFPI, PROCR, CD39, B2M, HLA-E, CD47, a20, PD-L1, HO1, CTLA-4 (e.g., LEA 29Y), XIAP, and combinations thereof from a single multiple transgene cassette in the pig genome; and functional inactivation of all PERV copies. In embodiments, the transplanted heart, lung, liver, kidney, pancreas, spleen, or portion thereof survives and functions for a period of about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more.
In embodiments, it will be necessary to protect the genetically modified cell(s), tissue(s), or organ(s) from the immune system of the host to which the genetically modified cell(s), tissue(s), or organ(s) is administered. For example, in embodiments, the genetically modified cell(s), tissue(s), or organ(s) are administered with a matrix or coating (e.g., gelatin) to protect the genetically modified cell(s), tissue(s), or organ(s) from an immune response from the host. In embodiments, the matrix or coating is a biodegradable matrix or coating. In embodiments, the matrix or coating is natural. In other embodiments, the matrix or coating is synthetic.
In embodiments, the genetically modified cell or cells, tissue or tissues or organ or organs are administered with an immunosuppressive compound. In embodiments, the immunosuppressive compound is a small molecule, peptide, antibody, and/or nucleic acid (e.g., an antisense or siRNA molecule). In embodiments, the immunosuppressive compound is a small molecule. In embodiments, the small molecule is a steroid, an mTOR inhibitor, a calcineurin inhibitor, an antiproliferative agent, or an IMDH inhibitor. In embodiments, the small molecule is selected from corticosteroids (e.g., prednisone, budesonide, prednisolone), calcineurin inhibitors (e.g., cyclosporine, tacrolimus), mTOR inhibitors (e.g., sirolimus, everolimus), IMDH inhibitors (azathioprine, leflunomide, mycophenolate esters), antibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin), and methotrexate, or salts or derivatives thereof. In embodiments, the immunosuppressive compound is a polypeptide selected from the group consisting of: CTLA-4, anti-b 7 antibodies, abacavin, adalimumab, anakinra, cetuximab, etanercept, golimumab, infliximab, ai Saiji bevacizumab, natalizumab, rituximab, secukinumab, tolizumab, superior Ji Nushan antibody, vedolizumab, basophiizumab, daclizumab and moruzumab.
In embodiments, the genetically modified cell(s), tissue(s), or organ(s) of the subject to be administered have been further genetically modified such that they are less likely to induce an immune response in the subject. In embodiments, the genetically modified cell or cells, tissue or tissues or organ or organs have been further genetically modified such that they do not express a functional immunostimulatory molecule.
VI. Examples
The following examples are provided to illustrate the present disclosure, and are for illustrative purposes only, and should not be construed as limiting the scope of the present disclosure.
Example 1A: graft survival of kidney xenografts with triple xenogenic antigen knockouts and multiple human transgenes in cynomolgus monkeys
The purpose is as follows: the ability of inserted human transgenes (hTG) to affect the in vivo survival of kidney xenografts from pigs genetically modified to delete three carbohydrate xenografts GGTA1, CMAH and B4GALN2 (triple knockout, TKO) was evaluated.
The method comprises the following steps: seventeen cynomolgus monkeys received kidneys from four different TKO porcine lines (TKO-A, TKO-B, TKO-F and TKO-G) with different expression of hTG including: immunoregulatory genes (IMRG; HLA-E, CD, B2M and PDL 1), complement regulatory genes (CPRG; CD46, CD55 and CD 59) and coagulation regulatory genes (CGRG; THBD, TFPI and EPCR). The TKO-A swine strain comprises payload 10 of international publication No. 2020/228810, which is incorporated herein by reference in its entirety. TKO-B contains the payload 9 of international publication No. 2020/228810, which is incorporated herein by reference in its entirety. TKO-F contains the polycistronic cassette of FIG. 2D. TKO-G contains the polycistronic cassette of FIG. 4C. Recipients were treated with induction therapy including anti-thymocyte globulin (ATG) and rituximab 2 days prior to transplantation, then weekly with anti-CD 154 antibody (20 mg/kg) and daily with mycophenolate mofetil. Rapamycin (rapa) or tacrolimus and prednisone (tac) were also administered for the first two months. Fig. 22 shows a timeline of the experiment.
Table 1 summarizes the transgene expression for each TKO swine strain. "N/A" means a TKO pig line that does not contain a particular transgene, and "-" means that the TKO pig line contains a particular transgene but the transgene is not expressed; "+" indicates that the TKO swine line contains a particular transgene and that the transgene is expressed.
Table 1: expression of transgenes in each TKO pig line
Table 2 summarizes the induction therapies administered to each monkey.
Table 2: immunosuppression protocol in TKO plus hTG recipients and xenograft survival
Results: TKO-A expresses higher IMRG and lower CPRG, while TKO-B expresses higher CPRG and lower IMRG. TKO-F has high expression of CGRG, THBD and EPCR, while TKO-G has medium expression of CGRG, THBD and TFPI and high expression of CPRG (Table 1). In the TKO-A group, the first recipient lost his xenograft due to thrombotic microangiopathy (tmA) on day 2, while the second recipient developed rejection due to T cell-mediated rejection and antibody-mediated rejection (AMR) on day 61. Of the 6 recipients of TKO-B, 2 monkeys lost xenografts due to ureteric complications at day 15 and acute graft thrombosis at day 20. Of the other four recipients, all three survived longer (135, 265 and 316 days) despite one of them lost its graft due to TMA on day 71, and rejection occurred only after immunosuppression was reduced due to infectious complications. Of the 7 TKO-F recipients, the remaining 4 recipients were in good condition for up to 190 days without any rejection or infection complications, although 2 lost their xenografts in the early phase (day 8 and day 9) and the other lost their xenografts on day 45 due to AMR/TMA. Finally, both TKO-G recipients also performed well for > 90 days, > 103 days (table 2), with normal kidney function, and without any evidence of rejection in their procedural biopsies.
FIG. 23 shows images of CD46, CD55, CD59, HLA-E, CD47 and PD-L1 expression in TKO-A and TKO-B xenografts as compared to wild type xenografts. FIG. 24 shows images of CD46, CD55, CD47, EPCR, THBD, A, and HO1 expression in TKO-C. FIG. 25 shows images of CD46, CD55, B2M, CD47, TFPI and THBD expression in TKO-D. Fig. 26 shows the concentration of creatinine (labeled Cr) up to 313 days after implantation. Fig. 27 shows images of kidney tissue stained with hematoxylin and eosin or complement component C4 d.
Fig. 29A-29E are graphs showing Blood Urea Nitrogen (BUN) and creatinine levels in five monkeys containing TKO-F xenografts. The monkey of fig. 29A had survived 320 days after organ transplantation. The monkey of fig. 29B had survived 313 days after organ transplantation. The monkey of fig. 29C had survived for 201 days after organ transplantation. The monkey of fig. 29D had survived for 121 days after organ transplantation. The monkeys of figures 29A-29C were administered an immunosuppression regimen comprising 20mg/kg aCD40L every 7 days. On day 254, the dosing frequency of aCD40L in the monkeys of fig. 29A was switched from dosing every 7 days to dosing every 10 days. On day 240, the dosing frequency of aCD40L in the monkeys of fig. 29B was switched from dosing every 7 days to dosing every 10 days. On day 128, the dosing frequency of aCD40L in the monkeys of fig. 29C was switched from dosing every 7 days to dosing every 10 days. The monkeys of fig. 29E received TKO-F xenografts that also did not contain PERV. The data show that BUN and creatinine levels in monkeys containing TKO-F xenografts remain stable for hundreds of days after implantation.
Fig. 30A-30B are graphs showing Blood Urea Nitrogen (BUN) and creatinine levels in two monkeys containing TKO-G xenografts. Two animals were administered an immunosuppression regimen comprising 20mg/kg aCD40L every 7 days until day 200. On day 200, animals were dosed every 10 days with aCD40L.
FIGS. 38A-38C show the expression of the transgene of TKO-F xenografts in tissues, glomeruli and tubules.
This data shows that expression of human CGRG provides long term survival for porcine kidney xenografts.
Example 1B: graft survival of kidney xenografts with triple xenogenic antigen knockouts and multiple human transgenes in cynomolgus monkeys
The purpose is as follows: the ability of inserted human transgenes (hTG) to affect the in vivo survival of kidney xenografts from pigs genetically modified to delete three carbohydrate xenografts GGTA1, CMAH and B4GALN2 (triple knockout, TKO) was evaluated.
The method comprises the following steps: cynomolgus monkey receives kidneys from pigs comprising the polycistronic cassettes of fig. 2H or fig. 2E. FIG. 2H polycistronic cassette ("15S 4") comprises a clotting cassette comprising the TFPI, THBD, and PROCR transgenes under the control of the ssUBC promoter, an innate immune cassette comprising the B2MHLA-E fusion protein and CD47 under the control of the ssEEF1α1 promoter, and a complement regulatory cassette comprising the CD46da and CD55 transgenes under the control of the CAG promoter. The polycistronic cassette ("15S 1") of fig. 2E comprises a clotting cassette comprising TFPI, THBD and PROCR transgenes under the control of the ssUBC promoter, an innate immune cassette comprising B2M HLA-E fusion protein and CD47 under the control of the ssHSPA8 promoter, and a complement regulatory cassette comprising CD46da and CD55 transgenes under the control of the CAG promoter.
FIG. 39 shows the expression of the transgene of 15S4 in the tissue, glomeruli and tubules of monkeys. FIG. 40 shows expression of the transgene of 15S4 by immunohistochemistry. FIGS. 41A-41D show transgene expression in both 15S1 and 15S 4. However, the innate immune cassette of 15S1 exhibited better expression than 15S 4.
Two of the three monkeys receiving the graft containing the 15S4 transgene remained stable for at least 50 days after transplantation. Fig. 31 and 32 are graphs showing Blood Urea Nitrogen (BUN) and creatinine levels in these two monkeys. The graft of the monkey of fig. 31 was stable 68 days post-implantation and was being monitored. The grafts of the monkeys of fig. 32 were stable and were being monitored 54 days post-implantation.
Example 1C: expression of nucleic acids comprising the polycistronic cassette of FIG. 2E in fetal, neonatal and adult tissues
The purpose is as follows: expression of nucleic acids comprising the polycistronic cassette ("15S 1") of fig. 2E was evaluated at different stages of the porcine life cycle. Table 3 shows the expression of 15S1 transgene in fetal, neonatal (1 day old) and adult (4 months old) porcine tissue.
Table 3: expression of 15S1 transgenes in fetal, neonatal and adult porcine tissues
All transgenes are expressed in fetal, neonatal and adult tissues. FIG. 42A shows expression of the 15S1 transgene in porcine fetal kidney relative to expression in wild type Ukatan pigs. FIG. 42B shows expression of the 15S1 transgene in porcine neonatal kidney relative to expression in wild type Ukatan pigs. Fig. 42C shows expression of the 15S1 transgene in porcine adult kidney relative to expression in wild type ukant pigs. FIG. 43A shows expression of 15S1 transgenic TFPI, EPCR and THBD in porcine fetal, neonatal and adult kidney tissue. FIG. 43B shows expression of 15S1 transgenic B2M HLA-E fusion proteins and CD47 in porcine fetal, neonatal and adult kidney tissue. FIG. 43C shows expression of 15S1 transgenic CD46 and CD55 in porcine fetal, neonatal and adult kidney tissue. FIG. 43D shows the expression of each of the 15S1 transgenes in two different fetal porcine kidney tissues.
Example 2: GPIb and GPIIb/IIIa together inhibit delay of consumable thrombocytopenia in porcine liver xenografts
The purpose is as follows: pig liver xenograft survival is impaired by thrombocytopenia, which begins immediately after xenograft reperfusion. Non-human primate subjects surviving porcine liver xenograft recovered their platelet count 7-10 days post-transplantation. The effect of inhibition of platelet activation and adhesion via GPIb and GPIIb/IIIa mechanisms on the development of thrombocytopenia following xenograft reperfusion was evaluated in an ex vivo machine perfusion model. The liver xenografts used in this study were from wild-type pigs, or from genetically engineered pigs containing knockouts of GGTA1, β4galnt2, CMAH plus modifications targeting complement, inflammation and coagulation regulation (referred to herein as "pigs 2.F"). Pig 2.F contains the polycistronic cassette of fig. 2D.
The method comprises the following steps: the liver was subjected to normothermic machine perfusion using heparinized human whole blood and plasma perfusate. Perfusate was treated with anti-GPIb fragmented murine antibody (ab) and the GPIIb/IIIa inhibitor eptifibatide followed by pig 2.F liver intubation (n=3). Pigs 2.F perfused without drug therapy (n=3) were performed as controls. Perfusion is terminated when vascular resistance prevents portal venous blood flow. Platelet counts were measured throughout the perfusion period by a cytometer and by flow cytometry in formalin fixed samples.
Results: the average perfusion time was: pig 2.F livers perfused with ab and eptifibatide were 12 hours, and livers untreated with ab and eptifibatide (ab/eptifibatide) were 10 hours. There was no difference in arterial and portal blood flow or lactate clearance of the combination between pig 2.F liver treated with ab/eptifibatide and liver not treated with ab/eptifibatide (fig. 7A, fig. 7B). However, treatment with ab/eptifibatide unexpectedly delayed thrombocytopenia during the first 4 hours of infusion, as measured by platelet count using a cytometer (fig. 8A) and by flow cytometry (fig. 8B).
Example 3: development of ischemia-free xenograft methods for transplanting genetically porcine xenografts to non-human primates
The purpose is as follows: ischemia Reperfusion Injury (IRI) is a tissue injury that occurs when blood supply returns to tissue after an ischemic or hypoxic period. IRI can lead to rejection of organ grafts. A method of mitigating ischemia reperfusion injury was developed for transplantation of liver xenografts from genetically modified pigs to non-human primate models. The genetically modified pigs contained the polycistronic cassette of figure 2D.
The method comprises the following steps: two livers were obtained for transplantation using ischemia-free techniques. To establish arterial and portal inflow in situ, superior mesenteric artery and portal vein grafts are cannulated. With the liver fully mobilized, the hepatic superior Inferior Vena Cava (IVC) was clamped, flow was initiated with decellularized hemoglobin-based oxygen carrying perfusate, and the liver was excised and transferred on a pump to a photomedics machine (photomedics, andover, ma). Fig. 9A shows an image of the liver. Lactic acid clearance was monitored by in vitro normal temperature perfusion. Two baboons with low anti-pig triple knockout aortic endothelial cell titers were selected as recipients and subjected to standard hepatectomy with vena cava bypass. During implantation, the superior hepatic vena cava and portal vein anastomosis is completed while still on the pump, after which natural perfusion is established. Baboons recovered from anesthesia and returned to the cage for post-operative care. Blood was collected every four hours for laboratory analysis until euthanasia.
Results: during ex vivo infusion, mean perfusate lactate was reduced from 3.5 to 0.3 (fig. 9B). The average liver free time of baboons was 52 minutes and the average total implantation time (hepatectomy to bile duct completion) was 190 minutes. Both baboons required minimal phenylephrine support during the liver-free phase and were subsequently abstinent after natural circulation was established. Both baboons were extubated and returned to the cage at the time of the case, but required euthanasia after an average of 8.5 hours due to lactic acidosis, coagulation dysfunction and multisystemic organ failure. This study shows that an ischemia-free xenograft approach can be achieved using genetically modified xenografts.
Example 4: effects of refrigeration (CS) or perfusion (IM) on cardiac xenograft function
The purpose is as follows: the effect of storage temperature and perfusion on the cardiac function of genetically modified porcine cardiac xenografts was evaluated in an ex vivo model of Initial Cardiac Xenograft Dysfunction (ICXD). The genetically modified pig hearts contain (a) three specific xenogenic gene carbohydrate gene knockouts (GGTA 1, β4galnt2, CMAH: termed "TKO", i.e. triple knockouts) and have variable expression of human complement and thrombomodulin (n=13), or (b) GGTA1 knockouts and knockins CD55 (termed "gtko. Cardiac function after refrigeration (CS) or perfusion (IM) was also evaluated in wild type porcine hearts (referred to as "WTs").
The method comprises the following steps: after rinsing with a chilled solution (UW, 4 ℃) hearts from genetically modified or wild-type (WT) pigs were obtained and stored in cold saline (0.9% w/w,4 ℃ C.: chilled (CS)) for 3 hours or perfused (IM) with an oxygenated Steen solution with Red Blood Cells (RBC). IM infusion of 40mmHg was initiated at room temperature for 20 minutes to promote uniform graft infusion, after which it was cooled to 4 ℃ for the remaining shelf life. Cardiac function and laboratory parameters were assessed on working cardiac equipment at specific time points while freshly collected heparinized whole human blood was perfused. Troponin I was used as a marker of myocardial damage.
Results: a total of 19 hearts were perfused ex vivo, 10 of which employed CS (TKO n=6, gtko.hcd55n=1, wtn=3), and 9 employed IM (TKO n=7, gtko.hcd55n=1, wtn=1). After 1 hour of ex vivo infusion in the IM group, mean troponin I states a significant decrease (70.2 ng/mL relative 279.5ng/mL; p=0.038) but not at the final time point (fig. 10A-10C). Similarly, the cardiac function of TKO hearts (measured in response to cardiac output at increased filling pressure) decreases over time after CS, while IM-stored cardiac function improves over time (fig. 11A-11D). IM significantly reduced myocardial damage relative to the CS heart during the first hour of isolated working heart perfusion: cardiac injury (troponin release) is reduced and graft failure is delayed in some TKO hearts treated with IM. These results show that IM attenuated ICXD of genetically modified porcine hearts during initial exposure to human blood.
Example 5: expression of nucleic acids comprising one or more polycistronic cassettes in porcine donor cells, porcine fetal cells and cells isolated from adult pigs
The purpose is as follows: pig cells comprising the polycistronic cassette of figure 2D were evaluated for protein expression in SCNT, pig fetal cells, and cells isolated from adult pigs. Fig. 13A shows the expression of each cistron by flow cytometry. The circled cells contained the polycistronic cassette of figure 2D. Fig. 13B shows the expression of each cistron by immunohistochemistry.
Example 6: nucleic acids comprising CD46 or CD55 transgenes protect cells from complement-mediated lysis
The purpose is as follows: nucleic acids containing transgenic CD46 and CD55 were evaluated for their ability to protect cells from complement-mediated lysis by binding to complement proteins, such as complement component 3b (C3 b) and complement component 4b (C4 b). Payload 15S (PL 15S) pigs express the polycistronic cassette of figure 2D.
Method/results: FIGS. 14A-14B show the expression of CD55 (FIG. 14A) and CD46 (FIG. 14B) on the cell surface. Figure 15A shows surface protein expression of human CD46 and CD55 on Ear Punch Derived Cells (EPDCs) or isotype control stained cells from PL15S pigs or from GGTA1 and B4GALNT2 Double Knockout (DKO) pigs as controls. Fig. 15B evaluates complement activation in PL15S pigs or DKO pigs. EPDCs from the same DKO or PL15S pigs were incubated with 25% human serum (C6-)) lacking complement component 6. The C6 deficient serum was used to eliminate cell lysis by the membrane attack complex, which is the final step of complement activation, to assess the deposition of complement component C3b on the surface of porcine cells due to C3 activation by flow cytometry. Under some conditions, cells are pre-incubated with saturated amounts of blocking antibodies to human CD46 (a-CD 46) and/or human CD55 (a-CD 55) to assess the individual contribution of these proteins to complement regulation. Heat-inactivated serum (HIS) was used as a negative control under one condition because the heat inactivated complement in the serum. PL15S cells expressing human CD46 and CD55 proteins were protected from C3b deposition on the cell surface. When both human CD46 and CD55 were blocked, protection was lost and complete C3b deposition was observed.
Example 7: nucleic acids comprising CD46 or CD55 transgenes protect islet cells from complement-mediated lysis
The purpose is as follows: nucleic acids containing transgenic CD46 and CD55 were evaluated for their ability to protect islet cells from complement-mediated lysis by binding to complement proteins, such as complement component 3b (C3 b) and complement component 4b (C4 b). Islet cells were transfected with nucleic acid containing the cassette of fig. 2D ("15S") or with nucleic acid containing the cassette of fig. 4D ("17S 1").
Fig. 16A shows that in the absence of blocking antibodies (antibodies that bind CD46 or CD 55), cells expressing 15S reduced complement deposition compared to cells not expressing 15S or cells expressing CD46 and CD 55.
FIGS. 16B-16C show that cells expressing 17S reduced complement deposition in the absence of blocking antibodies. Complement deposition was not reduced in the presence of antibodies that blocked CD46, CD55, or both.
Together, this data shows that CD46 and CD55 on the islet cell surface are functional.
Example 8: nucleic acids comprising transgenic CD46, CD55 or CD59 reduce complement deposition when transgenes are linked by a 2A peptide
The purpose is as follows: the effect of the presence of the 2A peptide on the ability of CD46, CD55 and CD59 to reduce complement deposition was evaluated.
The effect of introducing a 2A peptide on the expression and function of CD46 and CD 55: KCDC cells were transfected with nucleic acid comprising CD46da, nucleic acid comprising CD55, or nucleic acid comprising CD46da and CD55 linked by an F2A peptide. Figure 17A shows CD55 expression when linked to the 2A peptide. FIG. 17B shows that CD46da (labeled "CD 46") is expressed when attached to the 2A peptide. Fig. 17C shows that nucleic acids comprising CD46da and CD55 reduce complement deposition.
The effect of the introduction of two 2A peptides on the expression and function of CD46, CD55 and CD 59: KCDC cells were transfected with one of the following nucleic acids: (i) hsCD46da2; (ii) hsCD55; (iii) hsCD59; (iv) hsCD46da 2-F2A-hsCD 55; (v) hsCD46da 2-F2A-hsCD 59; (vi) hsCD 55-P2A-hsCD 59; or (viii) hsCD46da 2-F2A-hsCD 55-P2A-hsCD 59. Fig. 18A-18C show that CD46 (fig. 18A), CD55 (fig. 18B), and CD59 (fig. 18C) are each expressed in these constructs. Each of these constructs also reduced complement deposition (fig. 18D).
Example 9: effect of CD46 isoform selection on complement deposition
The purpose is as follows: the different CD46 isoforms were evaluated for their ability to express and prevent complement protein deposition.
Method and results: fig. 19A shows robust surface expression of human CD46 on porcine cells derived from renal cortex transfected with the major isoforms of CD46 (SG-ABC 1, SG-BC2, SG-BC 1), on aortic-derived endothelial cells derived from payload 15S pigs (PL 15S AEC) and on human umbilical vein cells (HUVEC as positive control). Untransfected renal cortical derived porcine cells (empty) were used as negative controls. Surface C3b deposition on cells expressing the human CD46 isoform, PL15S AEC or untransfected cells (empty) after incubation in 25% normal human serum is shown in the right panel. Cells expressing all isoforms of human CD46 were protected from C3B deposition, whereas untransfected cells that did not express CD46 exhibited high C3B deposition (fig. 19B).
Example 10: effect of expression of nucleic acids comprising CD46 and CD55 in multiple Epithelial Cell (EC) types
The purpose is as follows: evaluation of expression and function of CD46 and CD55 in nucleic acid containing the polycistronic cassette ("15S") of FIG. 2D and nucleic acid containing the polycistronic cassette ("17P") of FIG. 4C
Method and results: alveolar Epithelial Cells (AEC), renal epithelial cells (KEC), porcine Umbilical Vein Endothelial Cells (PUVEC) and Human Umbilical Vein Endothelial Cells (HUVEC) were transfected with 15S or 17P. AEC, KEC, PUVEC and HUVEC cells transfected with 15S (FIG. 20A) or 17P (FIG. 20B) expressed CD46 and CD55, whereas TKO control cells not transfected with 15S or 17P did not. In the absence of antibodies blocking CD46, CD55, or both, both 15S (fig. 20C) and 17P (fig. 20D) reduced complement deposition in AEC, KEC, and PUVEC cells.
Example 11: protection of nucleic acid-induced organ rejection reaction comprising CD46 and CD55
The effect of expression of nucleic acids containing the cassette of fig. 2D ("15S") in the presence of Donor Specific Alloantibodies (DSA) was evaluated.
Fig. 14 shows surface expression of human CD46 and CD55 proteins on aortic-derived endothelial cells (AEC) from payload 15S (PL 15S) pigs or from control GGTA1, B4GALNT2 and CMAH triple knockout pigs (TKO AEC) or isotype control stained cells. Following incubation with 25% serum of non-human primate recipients transplanted with PL15S kidney, TKO and C3b deposition on PL15S AEC were taken at various time points post-transplantation (fig. 21A). PL15S cells were protected from C3b deposition, whereas TKO cells lacking human CD46 and CD55 expression showed significant C3b deposition over time, suggesting that human CD46 and CD55 on PL15S cells protected cells from complement activation. Cells that do not express 15S have complement deposition between 50% and 700% higher.
The same TKO cells were also used to measure the development of anti-porcine IgG or IgM antibodies after transplantation. TKO cells were incubated with the same serum samples from non-human primates (right line plot) and bound immunoglobulins were measured by flow cytometry. IgG antibodies developed over time, whereas IgM antibodies did not (fig. 21B). Complement activation is controlled by human CD46 and CD55 proteins even in the presence of pig reactive IgG antibodies.
Example 12: expression of nucleic acids comprising the polycistronic cassette of FIG. 2E in various cell types
The purpose and the method are as follows: expression of nucleic acids comprising the polycistronic cassette of fig. 2E was evaluated in various cell types. The nucleic acid of fig. 2E comprises: a clotting cassette comprising TFPI, PROCR, and THBDda under the control of the ssUBC promoter; an innate immune cassette comprising a B2M HLA-E fusion protein and CD47 under the control of ssHSPA8 promoter; and a complement regulatory cassette comprising CD46da and CD55 under the control of the CAG promoter.
Expression in fibroblasts. Figure 33 shows that each transgene was expressed in fibroblasts from approximately gram summer pigs. Figure 34 (see circled cells) shows the expression of each transgene in fibroblasts from the ewing pig.
Example 13: expression of nucleic acids comprising the polycistronic cassette of FIG. 2D in various cell types
The purpose and the method are as follows: expression of nucleic acids comprising the polycistronic cassette of fig. 2D was evaluated in various cell types. The nucleic acid of fig. 2D comprises: a clotting cassette comprising PROCR and THBD under the control of the ssUBC promoter; an inflammatory and apoptotic cassette comprising a20 and HO1 under the control of the ssef1α1 promoter; and complement regulation and innate immune cassettes comprising CD46i, CD47 and CD55 under the control of the CAG promoter.
Expression in fibroblasts. Figure 35 shows that each transgene was expressed in fibroblasts from approximately gram summer pigs.
Expression in fibroblasts containing PERV knockouts figure 36 shows that each transgene is expressed in fibroblasts containing PERV knockouts. Cells without PERV are circled in fig. 36.
Example 14: expression of nucleic acids comprising the polycistronic cassettes of FIG. 2H or FIG. 4A in islet cells
The purpose and the method are as follows: the expression of nucleic acids comprising the polycistronic cassettes of fig. 2H or fig. 4A in islet cells was evaluated. The nucleic acid of fig. 2H ("15S 4") comprises: a clotting cassette comprising TFPI, PROCR, and THBDda under the control of the ssUBC promoter; an innate immune cassette containing B2M HLA-E fusion protein and CD47 under the control of the ssef1α1 promoter; and a complement regulatory cassette comprising CD46da and CD55 under the control of the CAG promoter. The nucleic acid of fig. 4A ("17M 1") comprises: an innate immune cassette containing B2M HLA-E fusion protein and CD47 under the control of the ssUBC promoter; a coagulation cassette comprising TFPI x, CD39 and THBD under the control of hshshspa 8 promoter; and complement regulation and inflammation and apoptosis cassettes comprising CD46da, CD55, CD59, a20, HO1 and PD-L1 under the control of the CAG promoter.
Identification of human a20 transgenic protein: the activity of the human a20 transgenic protein was also evaluated in pigs containing 17M1 (PL 17M 1). FIG. 37A is a Western blot analysis of islet cells from PL17M1 or Wild Type (WT) pigs probed with antibodies specific for human A20 or beta actin (B actin), A20 or beta actin being housekeeping genes used as control for cell lysate loading. Western blot showed that PL17M1 islet cells expressed human A20 protein. Porcine cells were treated with recombinant human TNFa to induce activation of caspase 3 and caspase 7 (caspase 3/7) proteins involved in apoptosis. Caspase 3/7 activity is determined by luminescence after cleavage of a pre-luminescent probe that occurs in the presence of caspase protein. A20 protected cells from TNFa-mediated signals, including caspase protein-induced apoptosis. PL17M1 cells expressing a20 protein showed reduced luminescence, indicating protection of the cells from TNFa-mediated apoptosis (fig. 37B).
Fig. 37A shows the expression of a20 in islet cells comprising the polycistronic cassette of fig. 4A.
Islet cells expressing the polycistronic cassettes of 15S4 and 17M1 each expressed CD47. The level of CD47 expressed by 15S4 islets was higher compared to 17M1 islets (fig. 44).
Islet cells expressing 15S4 and 17M1 were evaluated for their ability to prevent phagocytosis. Briefly, islet cells labeled with a pH-sensitive fluorescent dye are incubated in the presence of monocytes. Islet cells were incubated in the absence of monocytes as a negative control. During phagocytosis, the extracellular environment pH of the phagosome becomes acidic. Thus, the amount of phagocytosis can be quantified by acidity, expressed as "total red area".
Both 15S4 islet cells and 17M1 cells prevented phagocytosis. However, dissociated 15S4 islet cells were superior to 17M1 cells in preventing phagocytosis (fig. 45).
Example 15: expression of nucleic acids comprising CpG islands upstream of a promoter
The purpose and the method are as follows: the effect of promoter selection on cistron expression was evaluated. The CD46 transgene or CD47 transgene is expressed in fibroblasts, beta islet cells and pulmonary artery endothelial cells (pAEC) -SV40 cell lines. Table 4 provides the characteristics of the promoters used to express CD 46. Table 5 provides the characteristics of the promoters used to express CD 47. Cistron expression was assessed by flow cytometry.
Table 4: promoters for expression of CD46
Promoter name SEQ ID NO: Promoter size (kb) CpG island
hsEF1α1 231 1.7 Is that
ssEF1α1 233 2 Is that
ssEF1α1 234 1.5 Is that
ssEF1α1 235 1 Whether or not
Table 5: promoters for expression of CD47
Promoter name SEQ ID NO: Promoter size (kb) CpG island
CAG 250 1.7 Is that
ssHSPA8 236 2.5 Is that
ssHSPA8 237 1.9 Is that
ssHSPA8 238 1.3 Whether or not
Results: the expression of CD46 and CD47 was stronger when controlled by the CpG island-containing promoter compared to the standard promoter lacking the CpG island (FIGS. 48A-48F)
Example 16: islet cell function in diabetic mice comprising Neonatal Porcine Islets (NPI) comprising nucleic acid comprising the polycistronic cassette of fig. 4A
Purpose/method: islet cell function in diabetic mice comprising NPI expressing nucleic acid comprising the polycistronic cassette of fig. 4A was evaluated.
Results: fig. 49 shows insulin and glucagon expression in six of eight mice.
Example 17: islet cell function in diabetic NOD Scid Gamma (NSG) mice comprising Neonatal Porcine Islets (NPI) comprising nucleic acid comprising the polycistronic cassette of fig. 4A
Purpose/method: islet cell function in diabetic NSG mice comprising NPI expressing nucleic acid comprising the polycistronic cassette of fig. 4A was evaluated. Fig. 50 shows a schematic of the experiment.
Results: fig. 51 shows blood glucose levels in NSG mice. The results show that NPI protects mice from diabetes.
Example 18: islet cell function in cynomolgus monkeys comprising nucleic acid comprising the polycistronic cassette of fig. 4A
Purpose/method: islet cell function in cynomolgus monkeys containing Neonatal Porcine Islets (NPIs) expressing nucleic acid comprising the polycistronic cassette of fig. 4A was evaluated.
Results: fig. 52A-52B show fasting blood glucose concentrations, total insulin, and porcine C-peptide for two cynomolgus monkeys. The monkey of fig. 52B exhibited porcine C-peptide. Pig C-peptide is a marker of insulin secretion. This data shows that NPI is functional for insulin production.
Example 19: nucleic acids comprising HLA-E transgenes protect cells from NK cell lysis
The purpose is as follows: nucleic acids comprising a cistron encoding HLA-E (in the form of a cistron encoding a B2M HLA-E fusion protein) were evaluated for their ability to protect cells from Natural Killer (NK) cell mediated lysis. The following nucleic acids were evaluated: (a) A nucleic acid that expresses the polycistronic cassette ("14P") of fig. 1D, and (b) a nucleic acid that expresses the polycistronic cassette ("14S") of fig. 1C. 14P and 14S each comprise a gene encoding a B2M HLA-E fusion protein. Two variants of B2M HLA-E fusion proteins were evaluated: (i) A B2MHLA-E fusion protein (labeled "Val" in fig. 53B) comprising the peptide epitope VMAPRTLIL (SEQ ID NO: 197), and (ii) a B2M HLA-E fusion protein (labeled "Val-free" in fig. 53A-53B) comprising the peptide epitope maprtil (SEQ ID NO: 251).
The method comprises the following steps: the activity of human HLA-E transgenes was determined by co-culturing payload-expressing calcein-labeled ear punch-derived porcine cells (EPDCs) with human NK cells isolated from peripheral blood for several hours, and then measuring the release of calcein from cells due to NK-mediated cell lysis using a plate reader.
Results: in FIG. 53A, representative surface protein expression of human HLA-E transgenes on porcine cells is shown. In FIG. 53B, NK cell assays were performed to evaluate porcine cells expressing payload 14P or 14S (PL 14P or PL 14S) and HLA-E transgene constructs containing sequences encoding the load peptide with or without valine (PL 14P/S Val or PL14P/S valless, respectively). Wild-type (WT) EPDC or K562 cells that do not express the human HLA-E transgene (human immortalized myelogenous leukemia cell lines known to be prone to lysis by human NK cells) are included as positive controls for the assay. Porcine cells expressing the human HLA-E transgene were protected from NK cell mediated lysis, and cells with valine-loaded peptide were better protected.
Example 20: nucleic acid expression functional THBD and PROCR transgenes
The purpose is as follows: the function of THBD and PROCR in nucleic acids containing the polycistronic cassette ("15S") of fig. 2D was evaluated.
Method and results: surface expression of human EPCR and THBD proteins on Wild Type (WT), GGTA1, B4GALNT2 and CMAH Triple Knockout (TKO) Aortic Endothelial Cells (AEC), human umbilical cord derived endothelial cells (Hu UVEC) and payload 15S porcine endothelial cells from umbilical cord (porcine UVEC), aorta or Kidney (KEC) are shown in the left histogram (fig. 54A). These cells were incubated with recombinant human protein C, activated protein C production was determined by colorimetric change, and read on a plate reader. All pig cells of PL15S pigs had functional EPCR and THBD proteins as demonstrated by the presence of activated protein C, compared to wild-type pig AEC lacking human EPCR and THBD. (FIG. 54B).
Example 21: nucleic acids expressing CD47 protein prevent phagocytosis
Surface expression of human CD47 protein on Wild Type (WT) and GGTA1 plus B4GALNT2 Double Knockout (DKO) ear punch-derived cells (EPDC) and Fetal Fibroblasts (FF) from PL15S pigs (fig. 57A). Phagocytosis was measured after co-culture of labeled pig cells with labeled primary human monocytes, and the amount of labeled pig cells phagocytosed by monocytes was assessed by flow cytometry. The decrease in phagocytosis of PL15S cells expressing CD47 transgenes was assessed by the phagocytosis index (determined by the equation: total phagocytic signal divided by the sum of total phagocytic signal plus non-phagocytic signal) (fig. 57B). The target index indicated that the amount of PL15S cells present after the co-culture period was higher, as the cells were protected from phagocytosis (fig. 57C).
Abbreviations (abbreviations)
Acute Vascular Rejection (AVR); activated Partial Thromboplastin Time (APTT); adeno-associated virus integration site 1 (AAVS 1); alanine Aminotransferase (ALT); albumin (ALB); α1, 3-galactosyl-galactose (Gal or αgal); antibody-mediated rejection (AMR); anti-thymocyte globulin (ATG); asialoglycoprotein receptor 1 (ASGR 1); aspartate Aminotransferase (AST); beta-1, 4-N-acetylgalactosamine transferase 2 (beta 4GalNT 2); beta-2 microglobulin (B2M); cluster of differentiation 39 (CD 39); cluster of differentiation 47 (CD 47); clustered Regularly Interspaced Short Palindromic Repeats (CRISPR); class II transactivator dominant negative (CIITA-DN); CMV early enhancer/chicken beta actin (CAG); complement factor 3 (C3); complement factor 3 knockout (C3-KO); whole blood count (CBC); C-X-C motif chemokine receptor 3 (CXCR 3); C-X-C motif chemokine receptor 12 (CXCR 12); cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH); deoxyribonucleic acid (DNA); dqα (DQA); DR alpha (DRA); microdroplet digital pCR (ddPCR); exo-5' nucleotidase (CD 73); elongation factor 1α (EF 1 α); endothelial Cells (EC); endothelial Protein C Receptor (EPCR); ex vivo liver xenograft infusion (EVLXP); fas ligand (FasL); fibrinogen level (FIB); fluorescence Activated Cell Sorting (FACS); fresh Frozen Plasma (FFP); green Fluorescent Protein (GFP); glomerular Filtration Rate (GFR); glucagon-like peptide 1 receptor (GLP-1R); glycoprotein IIb/IIIa (GpIIb/IIIa); glycoprotein alpha-1, 3-galactosyltransferase 1 (GGTA 1); GGTA knockout (GTKO); a guide ribonucleic acid (gRNA); hematoxylin and eosin (h+e); hepatic Arterial Thrombosis (HAT); human embryonic kidney 293 (HEK 293); heme oxygenase (HO 1); homology Directed Repair (HDR); human blood and plasma (hWB +p); human membrane cofactor protein (hCD 46); human complement decay acceleration factor (hCD 55); human complement regulatory protein (hCRP); human Leukocyte Antigen (HLA); human leukocyte antigen-E (HLA-E); human MAC inhibitory factor (hCD 59); immunoglobulin G (IgG); immunoglobulin G degrading enzymes (IdeS) of streptococcus pyogenes; immunoglobulin M (IgM); immunohistochemistry (IHC); inosine Monophosphate Dehydrogenase (IMDH); interleukin 12 (IL 12); interleukin 35 (IL 35); international Normalized Ratio (INR); intracellular adhesion molecule-2 (ICAM 2); a Killer Inhibitory Receptor (KIR); knock-in (KI); knock-out (KO); a kruppel-related cassette (KRAB); liver Function Test (LFT); long Terminal Repeat (LTR); major histocompatibility complex class I (MHC class I); major histocompatibility complex class II (MHC class II); major histocompatibility complex class I E single chain trimer (HLA-ESCT); a mechanical target of rapamycin (mTOR); messenger ribonucleic acid (mRNA); renal disease diet improvement (MDRD); mixed Lymphocyte Reaction (MLR); mycophenolate Mofetil (MMF); natural killer cells (NK); n-glycolylneuraminic acid (Neu 5 Gc); neurogenic differentiation 1 (NeuroD); non-human primate (NHP); non-homologous end joining (NHEJ); orthotopic liver xenograft (OLTx); population-reactive antibodies (PRAs); peripheral Blood Mononuclear Cells (PBMCs); porcine kidney-15 cells (PK 15); porcine Endogenous Retrovirus (PERV); porcine endogenous retrovirus knockout (PERV KO); programmed death ligand 1 (PD-L1); polymerase Chain Reaction (PCR); porcine aortic endothelial cell lines (PEC-se:Sub>A or pAEC); potassium (K); prothrombin Time (PT) and international normalized ratio (PT-NIR); quantitative reverse transcription polymerase chain reaction (qRT-PCR); recombinase-mediated cassette exchange (RMCE); red Blood Cells (RBCs); ribonucleic acid sequencing (RNAseq); reverse transcription polymerase chain reaction (RT-PCT); sgRNA (single guide RNA); small interfering ribonucleic acid (siRNA); sodium (Na); somatic Cell Nuclear Transfer (SCNT); superoxide dismutase 3 (SOD 3); porcine leukocyte antigen (SLA); t Cell Mediated Rejection (TCMR); thrombin-antithrombin III (TAT); thrombomodulin (THBD, TBM or TM); thrombotic Microangiopathy (TMA); tissue Factor Pathway Inhibitor (TFPI); topoisomerase (TOPO); total bilirubin (Tbili); transcription activator-like (TAL) effectors and nucleases (TALENs); tumor necrosis factor alpha-induced protein 3 (a 20); tumor necrosis factor receptor 1 immunoglobulin (TNFR 1-Ig); ubiquitous Chromatin Opening Element (UCOE); von willebrand factor (vWF); whole Genome Sequencing (WGS); wild Type (WT); zinc Finger Nucleases (ZFNs).
Incorporation of reference
All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, references to any references, articles, publications, patents, patent publications, and patent applications cited herein are not, and should not be taken as, an acknowledgement or any form of teaching that they form part of the active prior art or of the common general knowledge in any country in the world.

Claims (137)

1. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding the THBD protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding said CD46 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD59 protein, wherein the nucleic acid sequence encoding said CD59 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 86, 108;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195; and
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence encoding said CD47 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
2. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding the THBD protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding said CD46 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD59 protein, wherein the nucleic acid sequence encoding said CD59 protein is selected from the group consisting of SEQ ID NOs 86, 108;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195; and
(c) An innate immunity and inflammation and apoptosis cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence encoding said CD47 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A third cistron encoding an A20 protein, wherein the nucleic acid sequence encoding said A20 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(iv) A fourth cistron encoding a HO1 protein, wherein the nucleic acid sequence encoding the HO1 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(v) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(vi) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(vi) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
3. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding the THBD protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A third cistron encoding a CD39 protein, wherein the nucleic acid sequence of said CD39 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 67-70 and 106;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(vi) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD59 protein, wherein the nucleic acid sequence of CD59 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 86, 108;
(iii) A third cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(vi) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195; and
(c) An innate immunity and inflammation and apoptosis cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A third cistron encoding a20 protein, wherein the nucleic acid sequence of a20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(iv) A fourth cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(v) A fifth cistron encoding a PD-L1 protein, wherein the nucleic acid sequence of PD-L1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 89-91;
(vi) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(vii) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(viii) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
4. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 92, 93 and 181;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) An inflammatory and apoptotic cassette comprising:
(i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(ii) A second cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195; and
(c) A complement-modulating and innate immune cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A third cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(vi) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
5. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 92, 93 and 181;
(iii) A third cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(vi) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195; and
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
6. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 92, 93 and 181;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(c) An innate immunity and inflammation and apoptosis cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A third cistron encoding a20 protein, wherein the nucleic acid sequence of a20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(iv) A fourth cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(v) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(vi) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(vii) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
7. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A third cistron encoding a CD39 protein, wherein the nucleic acid sequence of CD39 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 67-70 and 106;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(vi) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory and inflammatory and apoptotic cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD59 protein, wherein the nucleic acid sequence of CD59 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 86, 108;
(iii) A third cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iv) A fourth cistron encoding a20 protein, wherein the nucleic acid sequence of a20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(v) A fifth cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(vi) A sixth cistron encoding a PD-L1 protein, wherein the nucleic acid sequence of PD-L1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 89-91;
(vii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(viii) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(ix) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
8. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 is selected from the group consisting of SEQ ID NOS: 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
9. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory and inflammatory and apoptotic cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A third cistron encoding a20 protein, wherein the nucleic acid sequence of a20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(iv) A fourth cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(v) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(vi) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(vii) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195; and
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
10. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A third cistron encoding a CD39 protein, wherein the nucleic acid sequence of CD39 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 67-70 and 106;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(vi) Optionally wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 11, 194 and 195,
(b) A complement regulatory and inflammatory and apoptotic cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD59 protein, wherein the nucleic acid sequence of CD59 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 86, 108;
(iii) A third cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iv) A fourth cistron encoding a20 protein, wherein the nucleic acid sequence of a20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(v) A fifth cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(vi) A sixth cistron encoding a PDL1 protein, wherein the nucleic acid sequence of PDL1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 89-91;
(vii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(viii) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(ix) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14, and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194, and 195, and
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
11. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 11, 194 and 195,
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 11, 194 and 195,
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(d) A cellular immunity and coagulation cassette comprising:
(i) A first cistron encoding a CTLA-4 protein, wherein the nucleic acid sequence of CTLA-4 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS 87-88 and 186,
(ii) A second cistron encoding a CD39 protein, wherein the nucleic acid sequence of CD39 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 67-70 and 106;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
12. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) Optionally, a third cistron encoding a CD39 protein, wherein the nucleic acid sequence of CD39 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 67-70 and SEQ ID NO: 106;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(vi) Optionally wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 11, 194 and 195,
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) Optionally, a third cistron encoding a CD59 protein, wherein the nucleic acid sequence of CD59 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 86, 108;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(vi) Optionally wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 11, 194 and 195,
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(d) An inflammatory and apoptotic cassette comprising:
(i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(ii) A second cistron encoding a PD-L1 protein, wherein the nucleic acid sequence of PD-L1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS 89-91;
(iii) Optionally, a third cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(v) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(vi) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
13. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 11, 194 and 195,
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO 11, 194 and 195,
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(d) An apoptosis and clotting cassette comprising:
(i) A first cistron encoding XIAP protein, wherein the nucleic acid sequence of XIAP has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID No. 110;
(ii) A second cistron encoding a CD39 protein, wherein the nucleic acid sequence of CD39 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID No. 106;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
14. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(d) An apoptosis and cellular immune cassette comprising:
(i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(ii) A second cistron encoding a CTLA-4 protein, wherein the nucleic acid sequence of CTLA-4 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from SEQ ID NOs 87-88 and 186;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
15. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) At least one 2A polypeptide having a sequence selected from the group consisting of SEQ ID NOS 1-10, 12-14 and 159-162,
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) At least one 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162,
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(d) A cellular immunity and coagulation cassette comprising:
(i) A first cistron encoding a LEA29Y protein which 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 SEQ ID NO. 186,
(ii) A second cistron encoding a CD39 protein, wherein the nucleic acid sequence of CD39 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 67-70 and 106;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 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 having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 159-162.
16. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence of TFPI has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(c) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide, which 2A polypeptide is encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195, and
(d) An apoptosis and cellular immune cassette comprising:
(i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(ii) A second cistron encoding a LEA29Y protein, wherein the nucleic acid sequence of said LEA29Y cistron has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID No. 186;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
17. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A clotting and innate immune cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83 and 180;
(iii) A third cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(v) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) An apoptosis and cellular immune cassette comprising:
(i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(ii) A second cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 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 having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 159-162; and
(c) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192, and 239-240, and combinations thereof;
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
18. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A clotting and innate immune cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence of THBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence of CD47 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83 and 180;
(iii) A third cistron encoding an EPCR protein, wherein the nucleic acid sequence of EPCR has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iv) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(v) A poly A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof,
(vi) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) An apoptosis and cellular immune cassette comprising:
(i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(ii) A second cistron encoding a CTLA-4 protein, wherein the nucleic acid sequence of CTLA-4 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from SEQ ID NOs 87-88, 174 and 186;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof, and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195; and
(c) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence of CD55 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
19. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding the THBD protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof;
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence encoding said CD47 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(c) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence encoding said CD46 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence encoding said CD55 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
20. A nucleic acid comprising one or more polycistronic cassettes selected from the group consisting of:
(a) A coagulation cassette comprising:
(i) A first cistron encoding a THBD protein, wherein the nucleic acid sequence encoding the THBD protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
(ii) A second cistron encoding a TFPI protein, wherein the nucleic acid sequence encoding the TFPI protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(b) An innate immune cassette comprising:
(i) A first cistron encoding a B2M HLA-E fusion protein, wherein the nucleic acid sequence encoding said B2MHLA-E fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
(ii) A second cistron encoding a CD47 protein, wherein the nucleic acid sequence encoding said CD47 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250; and
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195;
(c) A complement regulatory cassette comprising:
(i) A first cistron encoding a CD46 protein, wherein the nucleic acid sequence of CD46 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
(ii) A second cistron encoding a CD55 protein, wherein the nucleic acid sequence encoding said CD55 protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, wherein one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195; and
(d) An inflammatory and apoptotic cassette comprising:
(i) A first cistron encoding an A20 protein, wherein the nucleic acid sequence of A20 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64-65, 104, 182 and 188;
(ii) A second cistron encoding a HO1 protein, wherein the nucleic acid sequence of HO1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
(iii) A promoter having a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 126-145, 167-168, 178-179, 231-238 and 250;
(iv) A poly-A sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192 and 239-240, and combinations thereof; and
(v) Optionally, one or more of the encoded proteins are separated by a 2A polypeptide encoded by a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 1-10, 12-14 and 169, or by an IRES sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 11, 194 and 195.
21. The nucleic acid of any one of claims 1-20, wherein the promoter comprises a CpG island.
22. The nucleic acid of claim 21, wherein the CpG island comprises at least 500 base pairs, comprises a GC content of at least 50%, comprises CpG dinucleotides, or any combination thereof.
23. The nucleic acid of any one of claims 1-22, comprising a transcription terminator.
24. The nucleic acid of claim 23, wherein the transcription terminator is located 3' to the poly a sequence.
25. The nucleic acid of any one of claims 1-24, wherein the nucleic acid sequence of the CD46 cistron is selected from 72, 73, 185 and 200.
26. The nucleic acid of any one of claims 1-24, wherein the nucleic acid sequence of the CD46 cistron is selected from the group consisting of SEQ ID No. 74 and SEQ ID No. 75.
27. The nucleic acid of any one of claims 1-24, wherein the nucleic acid sequence of the CD46 cistron is SEQ ID No. 71.
28. The nucleic acid of any one of claims 1-27, wherein the CD47 cistron encodes CD47 isoform 2.
29. The nucleic acid of claim 28, wherein the cistron encoding CD47 isoform 2 has a nucleic acid sequence selected from any one of SEQ ID NOs 78, 83 and 180.
30. The nucleic acid of any one of claims 1-29, wherein the THBD cistron comprises or consists of a nucleic acid sequence selected from any one of SEQ ID NOs 99-102 and 166.
31. The nucleic acid of claim 15 or 16, wherein LEA29Y is a nucleic acid encoding a protein having the amino acid sequence of SEQ ID No. 174.
32. The nucleic acid of claim 15 or 16, wherein LEA29Y is a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 186.
33. The nucleic acid of any one of claims 1-32 comprising a 5'loxp site and a 3' loxp site selected from SEQ ID NOs 146-150 and 244.
34. The nucleic acid of any one of claims 1-33, comprising a 5 'insulator site and a 3' insulator site selected from the group consisting of SEQ ID NOs 49-58 and 163-164.
35. The nucleic acid of any one of claims 1-34, comprising a 5 'guide RNA (gRNA) target sequence having a sequence selected from the group consisting of SEQ ID NOs 47-48 and a 3' gRNA target sequence, and combinations thereof.
36. The nucleic acid of any one of claims 1-35, comprising a Ubiquitous Chromatin Opening Element (UCOE) having the sequence of any one of SEQ ID NOs 19-22, 157 and 193.
37. The nucleic acid of any one of claims 1-36, comprising
(i) A first Inverted Terminal Repeat (ITR) located at the 5 'end of the 5' polycistronic cassette and having the sequence of SEQ ID NO:17 or 18, and
(ii) A second ITR located at the 3 'end of the 3' polycistronic cassette and having the sequence of SEQ ID NO. 15 or 16.
38. The nucleic acid of any one of claims 1-37, comprising a polycistronic cassette.
39. The nucleic acid of any one of claims 1-37, comprising at least two polycistronic cassettes.
40. The nucleic acid of any one of claims 1-37, comprising at least three polycistronic cassettes.
41. The nucleic acid of any one of claims 1-37, comprising four polycistronic cassettes.
42. The nucleic acid of any one of claims 1-41, comprising a fluorescent protein.
43. The nucleic acid of claim 42, wherein said fluorescent protein has the sequence of SEQ ID NO. 111, 242 or 246.
44. The nucleic acid of claim 1 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 24, 25, 29, 202, 203 and 207.
45. The nucleic acid according to claim 2, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS 26 and 204.
46. The nucleic acid of claim 3, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 23, 27, 28, 201, 205 and 206.
47. The nucleic acid of claim 4, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 30 and 208.
48. The nucleic acid of claim 5 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS.31-34 and 209-212.
49. The nucleic acid of claim 6 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 37 and 215.
50. The nucleic acid of claim 7 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 35 and 213.
51. The nucleic acid of claim 8 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 36, 40, 214 and 218.
52. The nucleic acid of claim 9 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 41, 42, 219 and 220.
53. The nucleic acid of claim 10 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 38, 39, 173, 216, 217 and 226.
54. The nucleic acid of claim 11 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 43 and 221.
55. The nucleic acid of claim 11, wherein CTLA-4 is LEA29Y.
56. The nucleic acid of claim 14, wherein CTLA-4 is LEA29Y.
57. The nucleic acid of claim 12 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 44, 172, 222 and 225.
58. The nucleic acid of claim 13 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 45 and 223.
59. The nucleic acid of claim 14 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 46 and 224.
60. The nucleic acid of claim 17 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 174 and 227.
61. The nucleic acid of claim 18 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 175 and 228.
62. The nucleic acid of claim 19 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 176 and 229.
63. The nucleic acid of claim 20 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 177 and 230.
64. A nucleic acid comprising a CD46 cistron, said CD46 cistron comprising or consisting of a nucleic acid sequence selected from 72, 73, 185, 200 and 256.
65. A nucleic acid comprising or consisting of a THBD cistron comprising a nucleic acid sequence selected from any one of SEQ ID NOs 99-102, 166 and 266.
66. A vector comprising the nucleic acid of any one of claims 1-65.
67. A genetically modified cell comprising the nucleic acid of any one of claims 1-65.
68. The genetically modified cell of claim 67, wherein said cell is an islet cell.
69. The genetically modified cell of claim 67, wherein said cell is a porcine cell.
70. The genetically modified cell of any one of claims 67 to 69, wherein said cell is PERV-free.
71. The genetically modified cell of any one of claims 67 to 70, wherein said cell comprises at least one inactivated saccharide antigen producing gene.
72. The genetically modified cell of claim 71, wherein said at least one inactivated saccharide antigen producing gene is glycoprotein α -1, 3-galactosyltransferase (GGTA 1), cytidine monophosphate-N-acetylneuraminidase (CMAH), β -1, 4-N-acetylgalactosamine transferase 2 (β 4GALNT 2), or a combination thereof.
73. An organ or tissue comprising the nucleic acid of any one of claims 1-65 or the cell of any one of claims 67-72.
74. An animal comprising the nucleic acid of any one of claims 1-65, the cell of any one of claims 67-72, or the organ or tissue of claim 73.
75. The animal of claim 74, wherein the animal is a porcine mammal or a human.
76. The animal of claim 74 or 75, wherein the animal is a porcine mammal that does not contain PERV.
77. The animal of any one of claims 74-76, comprising at least one inactivated carbohydrate antigen producing gene.
78. The animal of claim 77, wherein the at least one inactivated saccharide antigen producing gene is glycoprotein α -1, 3-galactosyltransferase (GGTA 1), cytidine monophosphate-N-acetylneuraminidase (CMAH), β -1, 4-N-acetylgalactosamine transferase 2 (β 4GALNT 2), or a combination thereof.
79. A contiguous nucleic acid sequence having any one of SEQ ID NOs 23-46, 172-177 or 201-230.
80. The contiguous nucleic acid sequence of claim 79 comprising a contiguous nucleotide sequence of at least about 1 kb.
81. The contiguous nucleic acid sequence of claim 80, wherein the sequence is at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, or at least 30kb in length.
82. The contiguous nucleic acid sequence of any one of claims 79-81 wherein the sequence encodes one or more polycistronic cassettes of any one of claims 1-20.
83. The contiguous nucleic acid sequence of any one of claims 79-82 wherein the sequence encodes at least two polycistronic cassettes of any one of claims 1-16.
84. The continuous nucleic acid sequence of claim 57, wherein the sequence encodes at least three polycistronic cassettes of any one of claims 1-16.
85. The continuous nucleic acid sequence of claim 57, wherein the sequence encodes at least four polycistronic cassettes of any one of claims 1-16.
A chromosomal integrated version of any of SEQ ID NOs 23-46, 172-177 and 201-230.
87. A contiguous nucleic acid sequence which is 95% identical to any one of SEQ ID NOs 23-46, 172-177 and 201-230.
88. The continuous nucleic acid sequence of claim 87, wherein the sequence is at least 96% identical to any one of SEQ ID NOs 23-46, 172-177 and 201-230.
89. The contiguous nucleic acid sequence of claim 65 wherein said sequence is at least 97% identical to any of SEQ id nos 23-46, 172-177 and 201-230.
90. The continuous nucleic acid sequence of claim 65, wherein the sequence is at least 98% identical to any one of SEQ ID NOs 23-46, 172-177 and 201-230.
91. The continuous nucleic acid sequence of claim 65, wherein the sequence is at least 99% identical to any one of SEQ ID NOs 23-46, 172-177 and 201-230.
92. The continuous nucleic acid sequence of claim 65, wherein the sequence encodes at least two polycistronic cassettes of any one of claims 1-20.
93. The contiguous nucleic acid sequence of claim 65, wherein said sequence encodes at least three polycistronic cassettes of any one of claims 1-20.
94. The contiguous nucleic acid sequence of claim 65, wherein said sequence encodes at least four polycistronic cassettes of any one of claims 1-20.
95. The continuous nucleic acid sequence of any one of claims 92-94, wherein the sequence is at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs 23-46, 172-177 and 201-230.
96. A continuous nucleic acid sequence encoding a polycistronic gene product polypeptide expressed from any one of the polycistronic cassettes of claims 1-20.
97. A polypeptide comprising one or more proteins encoded by the cistron of any one of the polycistronic cassettes of claims 1-20.
98. A method of genetically modifying a cell, comprising:
(i) Knocking out Porcine Endogenous Retrovirus (PERV) elements;
(ii) Knockout glycoprotein α -1, 3-galactosyltransferase gene (GGTA 1), cytidine monophosphate-N-acetylneuraminic acid hydrolase (CMAH), β -1, 4-N-acetylgalactosamine transferase 2 (β 4GALNT 2), or any combination thereof; and is also provided with
(iii) Knocking in any one or more polycistronic cassettes according to any one of claims 1-20.
99. The method of claim 98, wherein step (i) is performed first, step (ii) is performed second, and step (iii) is performed last.
100. The method of claim 98, wherein step (i) is performed first, step (iii) is performed second, and step (ii) is performed last.
101. The method of claim 98, wherein step (ii) is performed first, step (i) is performed second, and step (iii) is performed last.
102. The method of claim 98, wherein step (iii) is performed first, step (i) is performed second, and step (ii) is performed last.
103. The method of claim 98, wherein step (ii) is performed first, step (iii) is performed second, and step (i) is performed last.
104. The method of claim 98, wherein step (iii) is performed first, step (ii) is performed second, and step (i) is performed last.
105. A genetically modified cell produced by the method of any one of claims 98-104.
106. A tissue, organ, or animal comprising the genetically modified cell of claim 105.
107. The animal of claim 106, wherein the animal is a human or porcine mammal.
108. A nucleic acid comprising a first loxP site, a second loxP site, a promoter, and a poly a sequence.
109. The nucleic acid of claim 108, which has at least 90% identity to the nucleic acid sequence of SEQ ID No. 248 or 249.
110. A genetically modified cell comprising the nucleic acid of claim 108 or 109.
111. A tissue, organ or animal comprising the cell of claim 110.
112. A nucleic acid comprising a first loxP site, a second loxP site, and one or more polycistronic cassettes of claims 1-20.
113. A genetically modified cell comprising the nucleic acid of claim 112.
114. A tissue, organ or animal comprising the cell of claim 113.
115. The nucleic acid of claim 112, wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs 201-230.
116. A nucleic acid having at least 80% identity to a nucleic acid of any one of SEQ ID NOS.201-230.
117. A nucleic acid having at least 85% identity to a nucleic acid of any one of SEQ ID NOS.201-230.
118. A nucleic acid having at least 90% identity to a nucleic acid of any one of SEQ ID NOS.201-230.
119. A nucleic acid having at least 95% identity to any one of SEQ ID NOS.201-230.
120. A nucleic acid having at least 97% identity to any one of SEQ ID NOS.201-230.
121. A nucleic acid having at least 99% identity to any one of SEQ ID NOS.201-230.
122. A nucleic acid comprising any one of SEQ ID NOs 201-230.
123. A genetically modified cell comprising the nucleic acid of claim 122.
124. A tissue, organ or animal comprising the cell of any one of claims 67-72, 105, 110 or 123.
125. A non-human mammal comprising the cell of any one of claims 67-72, 105, 110, or 123.
126. A nucleic acid comprising any one of SEQ ID NOs 1-195 and 205-250 or a combination thereof.
127. A genetically modified cell comprising the nucleic acid of claim 126.
128. The genetically modified cell of claim 127, wherein said cell is PERV-free.
129. The genetically modified cell of claim 127 or 128, comprising at least one inactivated saccharide antigen producing gene.
130. The genetically modified cell of claim 129, wherein said at least one inactivated saccharide antigen producing gene is glycoprotein α -1, 3-galactosyltransferase (GGTA 1), cytidine monophosphate-N-acetylneuraminidase (CMAH), β -1, 4-N-acetylgalactosamine transferase 2 (β 4GALNT 2), or a combination thereof.
131. The genetically modified cell of any one of claims 127 to 130, wherein said cell recombinantly expresses:
(a) One or more proteins that modulate coagulation;
(b) One or more proteins that modulate innate immunity;
(c) One or more proteins that modulate complement deposition, and optionally
(d) One or more proteins that modulate inflammation, and optionally
(e) One or more proteins that regulate apoptosis; and wherein
(f) The cells optionally comprise a knockout of 1, 2 or 3 of alpha-1, 3-galactosyltransferase (GGTA 1), cytidine monophosphate-N-acetylneuraminic acid hydrolase (CMAH), and beta-1, 4-N-acetylgalactosamine transferase 2 (beta 4GALNT 2).
132. An organ, tissue or animal comprising the genetically modified cell of any one of claims 127-131.
133. A non-human mammal comprising the genetically modified cell of any one of claims 127-131.
134. A nucleic acid recombinantly encoding one or more proteins selected from (a) - (d):
(a) THBD protein, and/or TFPI protein, and/or CD39 protein, and/or EPCR protein; and/or XIAP protein, and
(b) CD46 protein and/or CD55 protein and/or CD59 protein; and
(c) B2M HLA-E fusion proteins and/or CD47 proteins, and
(d) A20 protein, and/or a PD-L1 protein, and/or a CTLA-4 protein, and/or a HO1 protein.
135. The nucleic acid of claim 134, wherein expression of each protein is controlled by one or more control elements of each of (a), (b), and (c):
(a) A promoter selected from the group consisting of SEQ ID NOS.126-145, 167-168, 178-179, 231-238 and 250;
(b) A poly A sequence selected from the group consisting of SEQ ID NO.112-125, 154-156, 159-162, 190-192 and 239-240; and
(c) 2A polypeptide encoded by a sequence selected from the group consisting of SEQ ID NOS: 1-10, 12-14 and 159-162.
136. The nucleic acid of claim 134 or 135,
wherein the nucleic acid sequence encoding the THBD protein has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 97-102, 166 and 265-266;
wherein the nucleic acid sequence encoding the TFPI protein has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 94-96, 103 and 187;
wherein the nucleic acid sequence encoding said CD46 protein has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 71-76, 185, 200 and 253-258;
wherein the nucleic acid sequence encoding the B2M HLA-E fusion protein has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 62, 66, 105;
wherein the nucleic acid sequence encoding said CD47 protein has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 77-83, 180 and 259;
Wherein the nucleic acid sequence encoding the CD55 protein has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 84, 85, 107 and 184;
wherein the nucleic acid sequence encoding the CD59 protein has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 86 and 108;
wherein the nucleic acid sequence encoding the A20 protein has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs 64, 65, 104, 182 and 188;
wherein the nucleic acid sequence of the EPCR has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity with a sequence selected from the group consisting of SEQ ID NOS 92-93 and 181;
wherein the nucleic acid sequence of HO1 has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity with a sequence selected from the group consisting of SEQ ID NOs 63, 109, 165, 183 and 189;
wherein the nucleic acid sequence of CD39 has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOS 67-70 and SEQ ID NO 106;
wherein the nucleic acid sequence of XIAP has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID No. 110;
Wherein the nucleic acid sequence of CTLA-4 has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from SEQ ID NOs 87, 88, 186; and
wherein the nucleic acid sequence of PD-L1 has at least 95%, at least 97%, at least 98%, at least 99% or 100% identity with a sequence selected from the group consisting of SEQ ID NOS 89-91.
137. A genetically modified cell comprising the nucleic acid of any one of claims 134-136:
wherein the cell comprises a knockout of a gene encoding 1, 2 or 3 of alpha-1, 3-galactosyltransferase (GGTA 1), cytidine monophosphate-N-acetylneuraminic acid hydrolase (CMAH), and beta-1, 4-N-acetylgalactosamine transferase 2 (beta 4GALNT 2).
CN202180090204.7A 2020-11-13 2021-11-12 Cells, tissues, organs and animals having one or more modified genes for enhancing xenograft survival and tolerance Pending CN117157406A (en)

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US63/247,544 2021-09-23
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