Methods and constructs for increasing solid organ transplant survival

Abstract

The present disclosure relates in some aspects to constructs and methods for increasing solid organ transplant survival. More specifically, aspects of the present disclosure relate to methods and constructs for administering full-length PD-L1 and one immune modulators, such as one or more of: CTLA-4, Qa-1d, HLA-E, HLA-G, CMTM6, and Indolamine-2,3-dioxygenase (IDO), by ex vivo perfusion in order to increase solid organ transplant survival.

Description

METHODS AND CONSTRUCTS FOR INCREASING SOLID ORGAN TRANSPLANT SURVIVALRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 574065, filed on April 3, 2024, which is hereby incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled AAVAN103PRSEQLIST.XML, which was created and last modified on April 3, 2024, which is 108,862 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.BACKGROUND

[0003] While there are numerous hopeful recipients of a solid donor organ, the number of available organs is insufficient to treat every needful subject. For example, in the United States, there are more than 4,000 heart transplants performed per year, but there are more than 250,000 heart failure patients who could benefit from transplant.FIELD

[0004] The present disclosure relates in some aspects to constructs and methods for increasing solid organ transplant survival.SUMMARY

[0005] Some embodiments provided herein are described by way of the following provided embodiments and also provided as possible combinations or overlapping embodiments.

[0006] Some embodiments herein are directed to a method of increasing solid organ transplant survival. In some embodiments, the method comprises: obtaining a solid donororgan, administering PD-L1 and one or more immune system modulator to the solid donor organ. In some embodiments, the solid donor organ is selected from the group comprising: liver, pancreas, spleen, kidney, heart, and adrenal glands. In some embodiments, the PD-L1 is full-length PD-L1. In some embodiments, administering PD-L1 comprises administering a nucleotide encoding PD-L1. In some embodiments, administering the one or more immune system modulators comprises administering an adeno-associated virus (AAV) encapsidating an expression cassette for expression of the PD-L1. In some embodiments, administering the one or more immune system modulators comprises administering a nucleotide encoding the one or more immune system modulators. In some embodiments, administering the one or more immune system modulators comprises administering an adeno-associated virus (AAV) encapsidating an expression cassette for expression of the one or more immune system modulators. In some embodiments, the one or more immune system modulators is selected from the group comprising: CTLA-4, Qa-ld, HLA-E, HLA-G, CMTM6, and indolamine-2,3- dioxygenase (IDO), or an equivalent thereof. In some embodiments, the one or more immune system modulator is CTLA-4 or a CTLA-4 equivalent. In some embodiments, the one or more immune system modulator is Qa-ld or a Qa-ld equivalent. In some embodiments, the one or more immune system modulator is IDO or an IDO equivalent. In some embodiments, the one or more immune system modulator is CMTM6. In some embodiments, the one or more immune system modulator is HLA-E or an HLA-E equivalent. In some embodiments, the one or more immune system modulator is HLA-G or an HLA-G equivalent.

[0007] In some embodiments, the AAV is an AAV serotype 1 (AAV-1), AAV serotype 2 (AAV-2), AAV serotype 3 (AAV-3), AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV-7), AAV serotype 8 (AAV-8), AAV serotype 9 (AAV-9), serotype rhlO AAV, serotype rh74 AAV, or a pseudotyped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9). In some embodiments, the AAV vector comprises an AAV serotype 9, or is derived from an AAV serotype 9 vector. In some embodiments, the AAV vector comprises an AAV serotype rh74, or is derived from an AAV serotype rh74 vector. In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector comprising a capsid insertion. In some embodiments, the capsid insertion is between residues 588 and 589. In some embodiments, the insertion sequence comprises RGDLGLS (SEQ IDNO: 46). In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector comprising a capsid insertion having SEQ ID NO: 46. In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector comprising SEQ ID NO: 47.

[0008] In some embodiments, the AAV is an AAV serotype 9 vector. In some embodiments, the AAV has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an AAV serotype 9 vector, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the AAV has between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%- 85%, 75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an AAV serotype 9 vector. In some embodiments, the AAV serotype 9 vector comprises a capsid modification. In some embodiments, the capsid modification comprises an insertion of having SEQ ID NO: 46. In some embodiments, the AAV has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an AAV serotype 9 vector having SEQ ID NO: 47, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the AAV has between about 70%-100%, 70%-95%, 70%-90%, 70%- 80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-l 00% identity to an AAV serotype 9 vector having SEQ ID NO: 47.

[0009] In some embodiments, the nucleotide encoding PD-L1 comprises SEQ ID NO: 5. In some embodiments, the nucleotide encoding CTLA-4 comprises SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the nucleotide encoding CMTM6 comprises SEQ ID NO: 10. In some embodiments, the nucleotide encoding IDO comprises SEQ ID NO: 11. In some embodiments, the nucleotide encoding Qa-ld comprises SEQ ID NO: 13. In some embodiments, administering the full-length PD-L1 and one or more immune system modulators comprises administering an expression cassette for expression of full-length PD- L1 and one or more immune system modulators. In some embodiments, the expression cassette comprises one or more genetic elements selected from the group comprising one or more: ITRs, enhancers, promoters, self-cleaving peptides, protein coding sequences, or Kozak sequences. In some embodiments, the expression cassette comprises one or more ITRs. In some embodiments, the one or more ITRs comprise SEQ ID NO: 1 and / or SEQ ID NO: 15. Insome embodiments, the expression cassette comprises an enhancer. In some embodiments, the enhancer comprises a CMV enhancer. In some embodiments, the CMV enhancer comprises SEQ ID NO: 2. In some embodiments, the expression cassette comprises a promoter. In some embodiments, the promoter comprises a CMV promoter. In some embodiments, the CMV promoter comprises SEQ ID NO: 3. In some embodiments, the expression cassette comprises one or more self-cleaving peptides. In some embodiments, the one or more self-cleaving peptides comprises a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide comprises a T2A or P2A self-cleaving peptide. In some embodiments, the T2A selfcleaving peptide comprises SEQ ID NO: 6. In some embodiments, the P2A self-cleaving peptide comprises SEQ ID NO: 12. In some embodiments, the expression cassette comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises SEQ ID NO: 4. Some embodiments disclosed herein relate to an adeno-associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival.

[0010] In some embodiments, the expression cassette comprises one or more protein coding sequences. In some embodiments, the one or more protein coding sequences is selected from the group comprising: PD-L1, CTLA-4, Qa-ld, HLA-E, HLA-G, CMTM6, and Indolamine-2, 3 -di oxygenase (IDO). In some embodiments, the PD-L1 comprises a peptide comprising SEQ ID NO: 23. In some embodiments, the CTLA-4 comprises a peptide comprising SEQ ID NO: 24 or SEQ ID NO: 25. In some embodiments, the Qa-ld comprises a peptide comprising SEQ ID NO: 26 or SEQ ID NO: 27. In some embodiments, the IDO comprises a peptide comprising SEQ ID NO: 28. In some embodiments, the CMTM6 comprises a peptide comprising SEQ ID NO: 29. Some embodiments disclosed herein relate to an adeno-associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival.

[0011] In some embodiments, the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A selfcleaving peptide, a CTLA-4 coding sequence, a P2A self-cleaving peptide, a Qa-ld coding sequence, and a 3' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMVenhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15. In some embodiments, the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CMTM6 coding sequence, a P2A self-cleaving peptide, a Qa-ld coding sequence, and a 3' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM-6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15. Some embodiments disclosed herein relate to an adeno-associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A selfcleaving peptide comprising SEQ ID NO: 6, a CTLA-4 coding sequence comprising SEQ ID NO: 8, aP2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprising SEQ ID NO: 13, and a 3' ITR comprising SEQ ID NO: 15, or a percent identity that is in a range defined by any two of the preceding values. For example, in someembodiments, the expression cassette has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A self-cleaving peptide comprising SEQ ID NO: 6, a CTLA-4 coding sequence comprising SEQ ID NO: 8, aP2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprising SEQ ID NO: 13, and a 3' ITR comprising SEQ ID NO: 15.

[0012] In some embodiments, the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A selfcleaving peptide, a CTLA-4 coding sequence, a P2A self-cleaving peptide, a Qa-ld coding sequence, and a 3' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 9, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15. In some embodiments, the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a linker, an IDO coding sequence, and a 3’ ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the linker comprises SEQ ID NO: 7, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15. In some embodiments, the expression cassette comprises, from 5' to 3': a 5' ITR, aCMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A selfcleaving peptide, an IDO coding sequence, and a 3' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15. Some embodiments disclosed herein relate to an adeno-associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A self-cleaving peptide comprising SEQ ID NO: 6, a CTLA- 4 coding sequence comprising SEQ ID NO: 9, a P2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprising SEQ ID NO: 13, and a 3' ITR comprises SEQ ID NO: 15, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression cassette has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A selfcleaving peptide comprising SEQ ID NO: 6, a CTLA-4 coding sequence comprising SEQ ID NO: 9, a P2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprising SEQ ID NO: 13, and a 3' ITR comprises SEQ ID NO: 15.

[0013] In some embodiments, administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 500%, including 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, and any amount therebetween. In some embodiments, administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 100 days following transplantation of the donororgan. In some embodiments, administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by greater than 100 days following transplantation of the donor organ. In some embodiments, administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 12 months.

[0014] Some embodiments disclosed herein relate to an expression cassette. In some embodiments, the expression cassette comprises from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2 A self-cleaving peptide, a Qa- 1 d coding sequence, and a 3 ' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15. Some embodiments disclosed herein relate to an adeno-associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A self-cleaving peptide comprising SEQ ID NO: 6, a CTLA-4 coding sequence comprising SEQ ID NO: 8, a P2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprising SEQ ID NO: 13, and a 3’ ITR comprising SEQ ID NO: 15, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression cassette has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A self-cleaving peptide comprisingSEQ ID NO: 6, a CTLA-4 coding sequence comprising SEQ ID NO: 8, a P2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprising SEQ ID NO: 13, and a 3' ITR comprising SEQ ID NO: 15.

[0015] Some embodiments disclosed herein relate to an expression cassette. In some embodiments, the expression cassette comprises from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CMTM6 coding sequence, a P2A self-cleaving peptide, a Qa-ld coding sequence, and a 3' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM-6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15. Some embodiments disclosed herein relate to an adeno-associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A selfcleaving peptide comprising SEQ ID NO: 6, the CMTM6 coding sequence comprising SEQ ID NO: 10, a P2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprises SEQ ID NO: 13, and a 3' ITR comprising SEQ ID NO: 15, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression cassette has between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-l 00% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A self-cleaving peptide comprising SEQ ID NO: 6, the CMTM6 coding sequence comprising SEQ ID NO: 10, a P2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprises SEQ ID NO: 13, and a 3' ITR comprising SEQ ID NO: 15.

[0016] Some embodiments disclosed herein relate to an expression cassette. In some embodiments, the expression cassette comprises from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2 A self-cleaving peptide, a Qa- 1 d coding sequence, and a 3 ' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 9, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15. Some embodiments disclosed herein relate to an adeno- associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A self-cleaving peptide comprising SEQ ID NO: 6, a CTLA-4 coding sequence comprising SEQ ID NO: 9, a P2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprising SEQ ID NO: 13, and a 3' ITR comprises SEQ ID NO: 15, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression cassette has between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 70%- 75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-l 00% identity to an expression cassette comprising: a 5’ ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A self-cleaving peptide comprising SEQ ID NO: 6, a CTLA-4 coding sequence comprising SEQ ID NO: 9, aP2A self-cleaving peptide comprising SEQ ID NO: 12, a Qa-ld coding sequence comprising SEQ ID NO: 13, and a 3' ITR comprises SEQ ID NO: 15.

[0017] Some embodiments disclosed herein relate to an expression cassette. In some embodiments, the expression cassette comprises from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a linker, an IDO coding sequence, and a 3' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the linker comprises SEQ ID NO: 7, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15. Some embodiments disclosed herein relate to an adeno- associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a linker comprising SEQ ID NO: 7, a IDO coding sequence comprising SEQ ID NO: 11, and a 3' ITR comprising SEQ ID NO: 15, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression cassette has betweenabout 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%- 90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a linker comprising SEQ ID NO: 7, a IDO coding sequence comprising SEQ ID NO: 11, and a 3' ITR comprising SEQ ID NO: 15.

[0018] Some embodiments disclosed herein relate to an expression cassette. In some embodiments, the expression cassette comprises from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, an IDO coding sequence, and a 3' ITR. In some embodiments, the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15. Some embodiments disclosed herein relate to an adeno-associated virus (AAV) comprising the expression cassette. Some embodiments disclosed herein relate to use of the expression cassette or AAV vector in the manufacture of a medicament for increasing solid organ transplant survival. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequence comprising SEQ ID NO: 5, a T2A self-cleaving peptide comprising SEQ ID NO: 6, a IDO coding sequence comprising SEQ ID NO: 11, and a 3' ITR comprising SEQ ID NO: 15, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression cassette has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%- 100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette comprising: a 5' ITR comprising SEQ ID NO: 1, a CMV enhancer comprising SEQ ID NO: 2, a CMV promoter comprising SEQ ID NO: 3, a Kozak sequence comprising SEQ ID NO: 4, a PD-L1 coding sequencecomprising SEQ ID NO: 5, a T2A self-cleaving peptide comprising SEQ ID NO: 6, a IDO coding sequence comprising SEQ ID NO: 11, and a 3' ITR comprising SEQ ID NO: 15.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In addition to the features described above, additional features and variations will be readily apparent from the following descriptions of the drawings and exemplary embodiments. It is to be understood that these drawings depict typical embodiments and are not intended to be limiting in scope.

[0020] FIG. 1 is a flow chart depicting some embodiments of a method of increasing solid organ transplant survival.

[0021] FIG. 2A is a chart depicting some non-limiting embodiments of expression construct components that are suitable for use in the constructs and methods disclosed herein.

[0022] FIG. 2B is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0023] FIG. 2C is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0024] FIG. 3A is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0025] FIG. 3B is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0026] FIG. 4A is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0027] FIG. 4B is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0028] FIG. 5A is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0029] FIG. 5B is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0030] FIG. 6A is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0031] FIG. 6B is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0032] FIG. 7A is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0033] FIG. 7B is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0034] FIG. 8A is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0035] FIG. 8B is an illustration depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0036] FIG. 9 is a graph showing the effect of administration of full-length PD-L1 and one or more immune system modulators on tumor volume.

[0037] FIG. 10 is an illustrative representation of a method for increasing solid organ transplant survival according to some embodiments disclosed herein.

[0038] FIG. 11 is an illustrative representation of some embodiments of vectors for increasing solid organ transplant survival.

[0039] FIG. 12 is an illustrative representation of the modularity of some immune system modulators according to some embodiments disclosed herein.

[0040] FIG. 13 is a series of graphs depicting some embodiments of identity and similarity between some embodiments of PD-L1 and CMTM6 belonging to different species.

[0041] FIG. 14 is a graph showing the effect of administration of full-length PD- L1 and one or more immune system modulators on tumor volume over an extended period of time.

[0042] FIG. 15 is a graph showing the effect of administration of full-length PD- L1 and one or more immune system modulators on the probability of graft survival in an MHC- mismatched murine heterotopic heart transplant model.DETAILED DESCRIPTION

[0043] While there are numerous hopeful recipients of a donor organ, such as a solid donor organ, the number of available organs is insufficient to treat every needful subject. For example, in the United States, there are more than 4,000 heart transplants performed peryear, but there are more than 250,000 heart failure patients who could benefit from transplant. Thus, there is an unmet need for reduced allotransplant rejection, and increases in the utility of unmatched donor organs. There is also an unmet need to reduce the use of current immunosuppressants that can have lifelong consequences including high susceptibility to infectious disease, cancer, cardiac allograft vasculopathy (CAV), etc. Many of these chronic issues are associated with the ‘global’ impact of current immunosuppressive therapy. ‘Local’ delivery of immunosuppressive gene therapy to the donor organ, for example, to the heart, may ameliorate many of the consequences from current immunosuppressive therapy.

[0044] Accordingly, some embodiments herein are directed to methods and constructs for increasing solid donor organ transplant survival.

[0045] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0046] As used herein, the term “immune system modulator” or “immunomodulating agent” has its plain and ordinary meaning, including referring to any substance, agent, or molecule, that stimulates or suppresses the immune system.

[0047] The term “AAV” is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, unless otherwise indicated. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”), which refers to AAV comprising a polynucleotide sequence not of AAV origin (e.g., a transgene). The term “AAV” includes AAV serotype 1 (AAV-1), AAV serotype 2 (AAV-2), AAV serotype 3 (AAV-3), AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV-7), AAV serotype 8 (AAV-8), AAV serotype 9 (AAV-9), serotype rhlO AAV, serotype rh74 AAV, or a pseudotyped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9).

[0048] The term “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least AAV capsid protein and an encapsidated polynucleotide.

[0049] The term “AAV-SLB101” refers to a myotropic viral construct comprising a modified AAV9 having an RGDLGLS (SEQ ID NO: 46) peptide inserted between positions 588 and 589 of VPl.

[0050] The term “adeno-associated virus (AAV) capsid” refers to the three- dimensional proteinaceous shell encapsidating, or enclosing, the viral genetic material. The AAV capsid is a non-enveloped, icosahedral 60-mer of three repeating monomers: VP1, VP2, and VP3. The AAV capsid determines the properties of viral particles, including tissue tropism and antigenic properties.

[0051] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including therapeutic proteins and other peptides, e.g., linkers, tags, capsid proteins, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some respects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0052] Amino acids generally can be grouped according to the following common side- chain properties: (1) hydrophobic: norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0053] Amino acid substitutions may also refer to one or more changes in a polypeptide sequence. The changes may include replacement of one amino acid in a polypeptide with another amino acid, insertion of one or amino acids, and / or deletion of one or more amino acids, or any combination thereof. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.

[0054] A “nucleic acid” sequence refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence. The term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl- methyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1- methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy- aminomethyl- 2-thiouracil, beta-D-mannosylqueosine, 5 ’-methoxy carbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0055] The term “polynucleotide,” refers to a polymeric form of nucleotides of any length, including DNA, RNA, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0056] The term “isolated” when referring to a nucleotide sequence, means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. Thus, an “isolated nucleic acid molecule which encodes a particular polypeptide” refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not materially affect the basic characteristics of the composition.

[0057] The terms “percent (%) amino acid sequence identity” and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) refer to the percentage of amino acid residues in a candidate sequence (e.g. , the engineered AAV capsid) that are identical with the amino acid residues in the nonengineered capsid reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0058] For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated “upstream,” “downstream,” “3',” or “5'” relative to another sequence, it is to be understood that it is the position of the sequences in the “sense” or “coding” strand of a DNA molecule that is being referred to as is conventional in the art.

[0059] The term “recombinant,” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature and / or a combination of polynucleotides and viral proteins that is not found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0060] The term “gene,” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0061] The term “transgene,” as used herein, refers to a nucleic acid sequence to be positioned within a viral vector and encoding a polypeptide, protein or other product of interest. In some embodiments, one rAAV vector, or engineered rAAV vector may comprise a sequence encoding one or more transgenes (which can optionally be the same gene, or different genes). For example, one rAAV vector may comprise the coding sequence for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 transgenes.

[0062] The terms “regulatory element” or “regulatory sequence”, or variations thereof, refer to a nucleotide sequence that participates in functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulatory elements can be enhancing or inhibitory in nature, depending on the embodiment. Non-limiting examples of regulatory elements include transcriptional regulatory sequences such as promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like. These elements collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell, though not all of these sequences need always be present. It shall be appreciated that the structural components of a rAAV vector as provided for herein may be listed in individual paragraphs solely for clarity and may be used together in combination. For example, any regulatory element or other component can be used in combination with any transgene (or transgenes) provided for herein.

[0063] A “promoter” is a polynucleotide that interacts with an RNA polymerase and initiates transcription of a coding region (e.g., a transgene) usually located downstream (in the 3' direction) from the promoter.

[0064] An “enhancer” is a regulatory DNA sequence, in mammalian cells, that can be used to boost a gene’s expression levels or control transcription in specific cell types.

[0065] The term “operably linked” refers to an arrangement of elements wherein the components are configured to perform a function. For example, regulatory sequences operably linked to a coding sequence result in the expression of the coding sequence. Depending on the embodiment, a regulatory sequence need not be contiguous with the coding sequence. Thus, for example, one or more untranslated, yet transcribed, sequences can bepresent between a promoter sequence and a coding sequence, with those two sequences still being considered “operably linked”.

[0066] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0067] An “expression vector” is a vector comprising a region of nucleic acid (e.g., a transgene) which encodes a gene product (e.g., a polypeptide or protein) of interest. As disclosed herein, vectors are used for achieving expression, e.g., stable expression, of a protein in an intended target cell. An expression vector may also comprise control elements operatively linked to the transgene to facilitate expression of the encoded protein in the target cell. A combination of one or more regulatory elements and a gene or genes to which they are operably linked for expression may be referred to herein as an “expression cassette.”

[0068] A Kozak sequence is a functional sequence motif that is positioned near or at the translational initiation site of eukaryotic mRNAs. Kozak sequences mediate ribosome assembly and translation initiation and help regulate that a protein is correctly translated in the correct reading frame.

[0069] Super Optimal Broth (SOB medium) is a nutrient-rich bacterial growth medium used for microbiological culture, generally of Escherichia coli. This nutrient-rich microbial broth contains peptides, amino acids, water soluble vitamins and glucose in a low- salt formulation. It was developed by Douglas Hanahan in 1983 and is an adjusted version of the commonly used LB medium (lysogeny broth). Growth of E. coli in SOB or SOC medium results in higher transformation efficiencies of plasmids.

[0070] A “subject” refers to mammal that is the object of treatment using a method or composition as provided for herein. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and humans. In some embodiments, the subject is human.

[0071] The terms “treating,” “treatment,” “therapeutic,” or “therapy” do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of anyundesired signs or symptoms of a disease or condition, to any extent can be considered treatment and / or therapy. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0072] The term “effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect, such as reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0073] As used herein, a “composition” refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0074] As used herein, the term “kit” may be used to describe variations of the portable, self-contained enclosure that includes at least one set of components to conduct one or more of the diagnostic or therapeutic methods of the invention.

[0075] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0076] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects, embodiments, and variations described herein include “comprising,” “consisting,” and / or “consisting essentially of aspects, embodiments and variations.

[0077] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimedsubject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0078] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0079] Some embodiments herein are directed to a method of increasing solid organ transplant survival.

[0080] FIG. 1 is a flow chart depicting some embodiments of a method of increasing solid organ transplant survival.

[0081] FIG. 10 is an illustrative representation of a method for increasing solid organ transplant survival according to some embodiments disclosed herein.

[0082] In some embodiments, the method 101 comprises obtaining a solid donor organ 103. In some embodiments, the solid donor organ comprises liver, pancreas, spleen, kidney, heart, and adrenal glands. Full-length PD-L1 and one or more immune system modulators are then administered to the solid donor organ 105. In some embodiments, the PD- L1 is human PD-L1. In some embodiments, the PD-L1 is mouse PD-L1. In some embodiments, the PD-L1 is swine or pig PD-L1. In some embodiments, the PD-L1 is non-human primate (NHP), for example, chimp, PD-L1. In some embodiments, the PD-L1 and one or more immune system modulators are administered by ex vivo perfusion. In some embodiments, administering full-length PD-L1 comprises administering a nucleotide encoding full-length PD-L1. In some embodiments, administering full-length PD-L1 comprises administering an adeno-associated virus (AAV) encapsidating an expression cassette for expression of the PD- Ll. In some embodiments, administering the one or more immune system modulators comprises administering a nucleotide encoding the one or more immune system modulators. In some embodiments, an adeno-associated virus (AAV) encapsidating an expression cassette is administered for expression of the one or more immune system modulators. In someembodiments, the one or more immune system modulators comprises CTLA-4 or a CTLA-4 equivalent, Qa-ld or a Qa-ld equivalent, HLA-E or an HLA-E equivalent, HLA-G or an HLA- G equivalent, CMTM6 or a CMTM6 equivalent, and Indolamine-2, 3 -dioxygenase (IDO) or an IDO equivalent.

[0083] In some embodiments, full-length PD-L1 is administered. In some embodiments, the full-length PD-L1 comprises SEQ ID NO: 23. In some embodiments, the full-length PD-L1 comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 23, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the full-length PD-L1 comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 23. In some embodiments, full-length PD- L1 is administered. In some embodiments, the full-length PD-L1 comprises SEQ ID NO: 39. In some embodiments, the full-length PD-L1 comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 39, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the full-length PD-L1 comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 39. In some embodiments, the full-length PD-L1 comprises SEQ ID NO: 40. In some embodiments, the full-length PD-L1 comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 40, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the full-length PD-L1 comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 40. In some embodiments, the full-length PD-L1 comprises SEQ ID NO: 41. In some embodiments, the full-length PD-L1 comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 41, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the full-length PD-L1 comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 41.

[0084] Table 1 depicts some embodiments of PD-L1 amino acid sequences suitable for use in some embodiments disclosed herein. In some embodiments, the full length PD-L1 comprises a PD-L1 sequence in Table 1. In some embodiments, the full-length PD-L1 has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a PD-L1 sequence in Table 1, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the full-length PD- L1 has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-l 00% identity to a PD-L1 in Table 1.Table 1

[0085] In some embodiments, one or more immune system modulators are administered with full length PD-L1. Table 2 lists the amino acid sequence of some embodiments of immune system modulators. In some embodiments, the one or more immune system modulators comprise one or more immune system modulators in Table 2. In some embodiments, the one or more immune system modulators has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an immune system modulator in Table 2, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-l 00%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an immune system modulator in Table 2.Table 2

[0086] In some embodiments, the one or more immune system modulators comprises CTLA4. In some embodiments, the CTLA4 is human CTLA4. In some embodiments, the CTLA4is mouse CTLA4. In some embodiments, the CTLA4 is swine or pig CTLA4. In some embodiments, the CTLA4 is non-human primate (NHP), for example, chimp, CTLA4. In some embodiments, the one or more immune system modulators comprises anamino acid sequence having SEQ ID NO: 24. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 24, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 24. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 25. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 25, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%- 100%, 70%-95%, 70%-90%, 70%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 25.

[0087] In some embodiments, the one or more immune system modulators comprises Qa-ld. In some embodiments, the Qa-ld is a human equivalent or homolog of Qa- Id. In some embodiments, the Qa-ld is mouse Qa-ld. In some embodiments, the Qa-ld is a swine or pig equivalent or homolog of Qa-ld. In some embodiments, the Qa-ld is a non-human primate (NHP), for example, chimp, equivalent or homolog of Qa-ld. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 26. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 26. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 27. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQID NO: 27, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 27.

[0088] In some embodiments, the one or more immune system modulators comprises IDO. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 28. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 28, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 28.

[0089] In some embodiments, the one or more immune system modulators comprises CMTM6. In some embodiments, the CMTM6 is human CMTM6. In some embodiments, the CMTM6 is mouse CMTM6. In some embodiments, the Qa-ld is swine or pig CMTM6. In some embodiments, the CMTM6 is non-human primate (NHP), for example, chimp, CMTM6. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 29. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 29, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 29. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 34. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 34, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%,70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 34. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 36. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 36, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 36. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 37. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 37, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 37.

[0090] In some embodiments, the one or more immune system modulators comprises HLA-E. In some embodiments, the HLA-E is human HLA-E. In some embodiments, the HLA-E is mouse HLA-E. In some embodiments, the HLA-E is swine or pig HLA-E. In some embodiments, the CTLA4 is non-human primate (NHP), for example, chimp, CTLA4. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 31. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 31. In some embodiments, the one or more immune system modulators comprises HLA-E. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 32. Insome embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%- 100%, 70%-95%, 70%-90%, 70%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 32. In some embodiments, the one or more immune system modulators comprises HLA-E. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 35. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 35, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 35. In some embodiments, the one or more immune system modulators comprises HLA-E. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having SEQ ID NO: 38. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 38, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%- 100%, 70%-95%, 70%-90%, 70%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 38.

[0091] In some embodiments, one or more immune system modulators are administered with full length PD-L1. Table 3 lists the nucleic acid sequence of some embodiments of immune system modulators. In some embodiments the one or more immune system modulators comprise one or more immune system modulators in Table 3. In some embodiments, the one or more immune system modulators has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an immune system modulator in Table 3, or a percentage identity that is in a range defined by any two ofthe preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-l 00%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an immune system modulator in Table 3.Table 3

[0092] In some embodiments, the one or more immune system modulators comprises CTLA4 In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 8. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 8. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 9. In some embodiments, the one or more immune system modulators comprises an amino acidsequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ IDNO: 9, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 9. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 44. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 44, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 95%, or 95%- 100% identity to SEQ ID NO: 44.

[0093] In some embodiments, the one or more immune system modulators comprises CMTM6. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 10. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 10. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 43. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 43, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 43.

[0094] In some embodiments, the one or more immune system modulators comprises IDO. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 11. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 11.

[0095] In some embodiments, the one or more immune system modulators comprises Qa-ld. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 13. In some embodiments, the one or more immune system modulators comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 13. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 14. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 14, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 14.

[0096] In some embodiments, the one or more immune system modulators comprises HLA-E. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 31. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQID NO: 31, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%-l 00% identity to SEQ ID NO: 31. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 32. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 32. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 35. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 35, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 35. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having SEQ ID NO: 38. In some embodiments, the one or more immune system modulators comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 38, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 38.

[0097] In some embodiments, administering full-length PD-L1 and one or more immune system modulators comprises administering an expression cassette encoding or expressing the full-length PD-L1 and one or more immune system modulators. In some embodiments, the expression cassette comprises one or more genetic elements is selected fromthe group comprising one or more: ITRs, enhancers, promoters, self-cleaving peptides, protein coding sequences, Kozak sequences, linkers, or any combination thereof.

[0098] In some embodiments, the expression cassette comprises one more ITRs. In some embodiments, the expression cassette comprises a 5' ITR. In some embodiments, the expression cassette comprises a 3' ITR. In some embodiments, the expression cassette comprises a 5' ITR and a 3' ITR. In some embodiments, the one or more ITRs comprise a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 1. In some embodiments, the one or more ITRs comprise a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more ITRs has between about 70%-l 00%, 70%-95%, 70%-90%, 70%-80%, 80%-l 00%, 80%- 95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 15.

[0099] In some embodiments, the expression cassette comprises one or more enhancers. In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the one or more enhancers comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more enhancers has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 2.

[0100] In some embodiments, the expression cassette comprises one or more promoters. In some embodiments, the promoter is a CMV promoter. In some embodiments, the one or more promoters comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more promoters has between about 70%-100%,70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 2.

[0101] In some embodiments, the expression cassette comprises one or more Kozak sequences. In some embodiments, the one or more Kozack sequences comprise a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more Kozak has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 4.

[0102] In some embodiments, the expression cassette comprises one more selfcleaving peptides. In some embodiments, the one or more self-cleaving peptides comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more self-cleaving peptides has between about 70%-100%, 70%-95%, 70%-90%, 70%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 6. In some embodiments, the one or more self-cleaving peptides comprise a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more self-cleaving peptides has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 12.

[0103] In some embodiments, the expression cassette comprises one or more linker sequences. In some embodiments, the one or more linker sequences comprises a nucleic acid sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more Kozak has between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 7.

[0104] Table 4 depicts some embodiments of an expression cassette. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises an expression cassette in Table 4. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an expression cassette in Table 4, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette in Table 4.Table 4

[0105] In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises a 5' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1; an enhancer having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2; a promoter having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3; a Kozak sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6; a first immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8; a second self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12; a second immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13, and a 3' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15; or having a percentage identity that is defined by any tow of the preceding values. For example, in some embodiments,the expression cassette comprises a 5' ITR having between about 70%- 100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 1; an enhancer having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 2; a promoter having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 3; a Kozak sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 95%, or 95%-100% identity to SEQ ID NO: 6; a first immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%-100%; a second self-cleaving peptide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 12; a second immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%-l 00% identity to SEQ ID NO: 13, and a 3' ITR having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%- 95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 15.

[0106] Table 5 depicts some embodiments of an expression cassette. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises an expression cassette in Table 5. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an expression cassette in Table 5, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette in Table 5.Table 5

[0107] In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune systemmodulator, and a 3' ITR. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises a 5' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1; an enhancer having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2; a promoter having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3; a Kozak sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6; a first immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10; a second self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12; a second immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13, and a 3' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15; or having a percentage identity that is defined by any tow of the preceding values. For example, in some embodiments, the expression cassette comprises a 5' ITR having between about 70%- 100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 1; an enhancer having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 2; a promoter having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 3; a Kozak sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having between about 70%-100%,70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 95%, or 95%-100% identity to SEQ ID NO: 6; a first immune system modulator having between about 70%- 100%, 70%-95%, 70%-90% identity to SEQ ID NO: 10, 70%-80%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100%; a second self-cleaving peptide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-l 00% identity to SEQ ID NO: 12; a second immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 13, and a 3' ITR having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 15.

[0108] Table 6 depicts some embodiments of an expression cassette. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises an expression cassette in Table 6. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an expression cassette in Table 6, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette in Table 6.Table 6

[0109] In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises a 5' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1; an enhancer having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2; a promoter having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3; a Kozak sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6; a first immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9; a second self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12; a second immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13, and a 3' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15; or having a percentage identity that is defined by any tow of the preceding values. For example, in some embodiments, the expression cassette comprises a 5' ITR having between about 70%- 100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 1; an enhancer having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 2; a promoter having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 3; a Kozak sequence having between about 70%-l 00%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 95%, or 95%-100% identity to SEQ ID NO: 6; a first immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 9; a second self-cleaving peptide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-l 00% identity to SEQ ID NO: 12; a secondimmune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 13, and a 3' ITR having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 15.

[0110] Table 7 depicts some embodiments of an expression cassette. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises an expression cassette in Table 7. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an expression cassette in Table 7, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%- 100%, 80%-95%, 80%-90%, 90 %-100%, 90%-95%, or 95%-100% identity to an expression cassette in Table 7.Table 7[oni] In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises a 5' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1; an enhancer having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2; a promoter having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3; a Kozak sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6; a first immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8; a second self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identity to SEQ ID NO: 12; a second immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 14, and a 3' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15; or having a percentage identity that is defined by any tow of the preceding values. For example, in some embodiments, the expression cassette comprises a 5' ITR having between about 70%- 100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 1; an enhancer having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 2; a promoter having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 3; a Kozak sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 95%, or 95%-100% identity to SEQ ID NO: 6; a first immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 8; a second self-cleaving peptide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-l 00% identity to SEQ ID NO: 12; a second immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 14, and a 3' ITR having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 15.

[0112] Table 8 depicts some embodiments of an expression cassette. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune systemmodulator, and a 3' ITR. In some embodiments, the expression cassette comprises an expression cassette in Table 8. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an expression cassette in Table 8, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette in Table 8.Table 8

[0113] In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a first self-cleaving peptide, a first immune system modulator, a second self-cleaving peptide, a second immune system modulator, and a 3' ITR. In some embodiments, the expression cassette comprises a 5' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1; an enhancer having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2; a promoter having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3; a Kozak sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6; a first immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10; a second self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12; a second immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 14, and a 3' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15; or having a percentage identity that is defined by any tow of the preceding values. For example, in some embodiments, the expression cassette comprises a 5' ITR having between about 70%- 100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 1; an enhancer having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100%identity to SEQ ID NO: 2; a promoter having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 3; a Kozak sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 5; a first self-cleaving peptide having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 95%, or 95%-100% identity to SEQ ID NO: 6; a first immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%-l 00% identity to SEQ ID NO: 10; a second self-cleaving peptide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-l 00% identity to SEQ ID NO: 12; a second immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%- 80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 14, and a 3' ITR having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 15.

[0114] Table 9 depicts some embodiments of an expression cassette. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a linker, an immune system modulator and a 3' ITR. In some embodiments, the expression cassette comprises an expression cassette in Table 9. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an expression cassette in Table 9, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-l 00%, 80%- 95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette in Table 9. Table 9

[0115] In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a protein coding sequence, a linker, an immune system modulator and a 3' ITR. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a linker, an immune system modulator and a 3' ITR. In some embodiments, the expression cassette comprises a 5' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1; an enhancer having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2; a promoter having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3; a Kozak sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5; a linker having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7; an immune system modulatorhaving about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11; and a 3' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15; or having a percentage identity that is defined by any tow of the preceding values. For example, in some embodiments, the expression cassette comprises a 5' ITR having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 95%, or 95%-l 00% identity to SEQ ID NO: 1; an enhancer having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 2; a promoter having between about 70%-100%, 70%- 95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 3; a Kozak sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 4; a full length PD-L1 coding sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 5; a linker having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 7; an immune system modulator having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 11; and a 3' ITR having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%-l 00% identity to SEQ ID NO: 15.

[0116] Table 10 depicts some embodiments of an expression cassette. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a self-cleaving peptide, an immune system modulator and a 3' ITR. In some embodiments, the expression cassette comprises an expression cassette in Table 10. In some embodiments, the expression cassette has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an expression cassette in Table 10, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the one or more immune system modulators has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression cassette in Table 10.Table 10

[0117] In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a protein coding sequence, a self-cleaving peptide, an immune system modulator and a 3' ITR. In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a Kozak sequence, a protein coding sequence, a self-cleaving peptide, an immune system modulator and a 3' ITR. In some embodiments, the expression cassette comprises a 5' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1; an enhancer having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2; a promoter having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3; a Kozak sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ IDNO: 4; a full lengthPD-Ll coding sequence having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identity to SEQ ID NO: 5; a self-cleaving peptide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6; an immune system modulator having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11; and a 3' ITR having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15; or having a percentage identity that is defined by any tow of the preceding values. For example, in some embodiments, the expression cassette comprises a 5' ITR having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%- 95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 1; an enhancer having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 2; a promoter having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 3; a Kozak sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%- 100% identity to SEQ ID NO: 4; a full length PD- L1 coding sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 5; a self-cleaving peptide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 6; an immune system modulator having between about 70%-l 00%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 11; and a 3' ITR having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to SEQ ID NO: 15.

[0118] Table 11 depicting some non-limiting embodiments of expression construct components that are suitable for use in the constructs and methods disclosed herein. Any combination of the elements listed therein are used, depending on the embodiment provided for herein. In some embodiments, the expression construct comprises an enhancer, promoter, PD-L1, for example, full-length PD-L1, and one or more immune modulators. In some embodiments, the expression construct does not comprise an enhancer. In some embodiments, the expression construct comprises any combination of enhancer, promoter, PD-L1, forexample, full-length PD-L1, and one or more immune modulators depicted in Table 11. In some embodiments, the expression construct comprises any combination of enhancer equivalents, promoter equivalents, PD-L1, for example, full-length PD-L1, and one or more immune modulator equivalents depicted in Table 11.Table 11

[0119] FIG. 2A is a chart depicting some non-limiting embodiments of expression construct components that are suitable for use in the constructs and methods disclosed herein. Any combination of the elements listed therein are used, depending on the embodiment provided for herein. FIG. 2B and FIG. 2C are illustrations depicting some embodiments of an expression construct for increasing solid organ transplant survival. In some embodiments, an expression construct that is an expression constructs in FIGs. 2A-2C are administered. In some embodiments, the expression constructs comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to an expression construct in any one of FIGs. 2A-2C, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression construct comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%,80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression construct in any one of FIGs. 2A-2C.

[0120] FIG. 3A and FIG. 3B are illustrations depicting some embodiments of an expression construct for increasing solid organ transplant survival. In some embodiments, an expression construct that is an expression construct in FIG. 3A or FIG. 3B is administered. In some embodiments, the expression constructs comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to an expression construct in FIG. 3 A or FIG. 3B, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression construct comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression construct in FIG. 3A or FIG. 3B.

[0121] FIG. 4A and FIG. 4B are illustrations depicting some embodiments of an expression construct for increasing solid organ transplant survival. In some embodiments, an expression construct that is an expression construct in FIG. 4A or FIG. 4B is administered. In some embodiments, the expression constructs comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to an expression construct in FIG. 4A or FIG. 4B, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression construct comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression construct in FIG. 4A or FIG. 4B.

[0122] FIG. 5A and FIG. 5B are illustrations depicting some embodiments of an expression construct for increasing solid organ transplant survival. In some embodiments, an expression construct that is an expression construct in FIG. 5A or FIG. 5B is administered. In some embodiments, the expression constructs comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to an expression construct in FIG. 5A or FIG. 5B, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression construct comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%,80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression construct in FIG. 5 A or FIG. 5B.

[0123] FIG. 6A and FIG. 6B are illustrations depicting some embodiments of an expression construct for increasing solid organ transplant survival. In some embodiments, an expression construct that is an expression construct in FIG. 6A or FIG. 6B is administered. In some embodiments, the expression constructs comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to an expression construct in FIG. 6A or FIG. 6B, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression construct comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression construct in FIG. 6A or FIG. 6B.

[0124] FIG. 7A and FIG. 7B are illustrations depicting some embodiments of an expression construct for increasing solid organ transplant survival. In some embodiments, an expression construct that is an expression construct in FIG. 7A or FIG. 7B is administered. In some embodiments, the expression constructs comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to an expression construct in FIG. 7A or FIG. 7B, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression construct comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression construct in FIG. 7A or FIG. 7B.

[0125] FIG. 8A and FIG. 8B are illustrations depicting some embodiments of an expression construct for increasing solid organ transplant survival. In some embodiments, an expression construct that is an expression construct in FIG. 8A or FIG. 8B is administered. In some embodiments, the expression constructs comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to an expression construct in FIG. 8A or FIG. 8B, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression construct comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%,80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an expression construct in FIG. 8 A or FIG. 8B.

[0126] FIG. 11 is an illustrative representation of some embodiments of expression constructs for increasing solid organ transplant survival. In some embodiments, an expression construct that is an expression construct in FIG. 11 is administered. In some embodiments, the expression constructs comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to an expression construct in FIG. 11, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the expression construct comprises a nucleotide having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-95%, or 95%-l 00% identity to an expression construct in FIG. 11.

[0127] FIG. 12 is an illustrative representation of the modularity of some immune system modulators according to some embodiments disclosed herein. In some embodiments, a vector that is a vector in FIG. 12 is administered. In some embodiments, the vector comprises a nucleotide having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to a vector in FIG. 12, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the vector comprises a nucleotide having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%- 100% identity to a vector in FIG. 11.

[0128] FIG. 13 is a series of graphs depicting some embodiments of identity and similarity between some embodiments of PD-L1 and CMTM6 belonging to different species. In some embodiments, the full-length PD-L1 and the one or more immune system modulators belong to the same species. In some embodiments, the full-length PD-L1 and the one or more immune system modulators belong to different species. In some embodiments, the full-length PD-L1 is human, mouse, pig, swine, NHP, chimp, or chicken, PD-L1. In some embodiments, the full-length PD-L1 has about 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to full-length human, mouse, pig, swine, NHP, chimp, or chicken, PD-L1, or has a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the In some embodiments, the full- length PD-L1 has between about 50%-100%, 50%-90%, 50%-75%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-75%, 75%-100%, 75%-90%, 80%-100%, 80%-90%, or 90%-100% identity to human, mouse, pig, swine, NHP, chimp, or chicken, full-length PD-L1. In some embodiments, the one or more immune system modulators belong to different species. In some embodiments, the one or more immune system modulators is a human, mouse, pig, swine, NHP, chimp, or chicken, immune system modulator. In some embodiments, the one or more immune system modulators has about 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to full-length human, mouse, pig, swine, NHP, chimp, or chicken, PD-L1, or has a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the In some embodiments, the full-length PD-L1 has between about 50%-100%, 50%-90%, 50%-75%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-75%, 75%-100%, 75%-90%, 80%-100%, 80%-90%, or 90%-100% identity to human, mouse, pig, swine, NHP, chimp, or chicken, full- length PD-L1.

[0129] In some embodiments, administering full-length PD-L1 and one or more immune system modulators increases survival of the solid donor organ. In some embodiments, administering full-length PD-L1 and one or more immune system modulators increases survival of the solid donor organ by up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months, or by a range defined by any two of the preceding values. For example, in some embodiments, administering full-length PD-L1 and one or more immune system modulators increases survival of the solid donor organ between about 1-24, 1- 18, 1-12, 1-6, 1-3, 1-2, 2-24, 2-18, 2-12, 2-6, 6-24, 6-18, 6-12, 12-24, 12-18, or 18-24 months. In some embodiments, administering full-length PD-L1 and one or more immune system modulators increases survival of the solid donor organ by more than 24 months. In some embodiments, administering full-length PD-L1 and one or more immune system modulators increases survival of the solid donor organ by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or by a percentage that is in range defined by any two of the preceding values. For example, in some embodiments, administering full-length PD-L1 and one or more immune system modulators increases survival of the solid donor organ between about l%-100%, l%-95%, l%-90%, l%-75%, l%-50%, l%-25%, l%-20%, l%-10%, 1%- 5%, 5%-100%, 5%-95%, 5%-90%, 5%-75%, 5%-50%, 5%-25%, 5%-20%, 5%-10%, 10%- 100%, 10%-95%, 10%-90%, 10%-75%, 10%-50%, 10%-25%, 10%-20%, 25%-100%, 25%-95%, 25%-90%, 25%-75%, 25%-50%, 50%-100%, 50%-95%, 50%-90%, 50%-75%, or 75%- 100%.

[0130] In some embodiments, administration of the full-length PD-L1 and / or one or more immune system modulators comprises administration of AAV encapsidating an expression cassette for expression of the full-length PD-L1 and / or the one or more immune system modulators. In some embodiments, the AAV is an AAV serotype 1 (AAV-1), AAV serotype 2 (AAV-2), AAV serotype 3 (AAV-3), AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV-7), AAV serotype 8 (AAV-8), AAV serotype 9 (AAV-9), serotype rhlO AAV, serotype rh74 AAV, or a pseudotyped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9).

[0131] Some embodiments provided herein are described by way of the following provided numbered arrangements and also provided as possible combinations or overlapping embodiments:

[0132] 1. A method of increasing solid organ transplant survival, the method comprising: obtaining a solid donor organ, administering PD-L1 and one or more immune system modulator to the solid donor organ.

[0133] 2. The method of arrangement 1, wherein the solid donor organ is selected from the group comprising: liver, pancreas, spleen, kidney, heart, and adrenal glands.

[0134] 3. The method of arrangement 1 or 2, wherein the PD-L1 is full-length PD-Ll.

[0135] 4. The method of any one of arrangements 1-3, wherein administering PD-L1 comprises administering a nucleotide encoding PD-L1.

[0136] 5. The method of any one of arrangements 1 to 4, wherein administering the one or more immune system modulators comprises administering an adeno-associated virus (AAV) encapsidating an expression cassette for expression of the PD-L1.

[0137] 6. The method of any one of the preceding arrangements, wherein administering the one or more immune system modulators comprises administering a nucleotide encoding the one or more immune system modulators.

[0138] 7. The method of any one of arrangements 1 to 5, wherein administering the one or more immune system modulators comprises administering an adeno-associated virus(AAV) encapsidating an expression cassette for expression of the one or more immune system modulators.

[0139] 8. The method of any one of the preceding arrangements, wherein the one or more immune system modulators is selected from the group comprising: CTLA-4, Qa-ld, HLA-E, HLA-G, CMTM6, and indolamine-2, 3 -dioxygenase (IDO).

[0140] 9. The method of arrangement 8, wherein the one or more immune system modulator is CTLA-4.

[0141] 10. The method of arrangement 8, wherein the one or more immune system modulator is Qa-ld.

[0142] 11. The method of arrangement 8, wherein the one or more immune system modulator is IDO.

[0143] 12. The method of arrangement 8, wherein the one or more immune system modulator is CMTM6.

[0144] 13. The method of any one of the preceding arrangements, wherein theAAV is an AAV serotype 1 (AAV-1), AAV serotype 2 (AAV-2), AAV serotype 3 (AAV-3), AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV-7), AAV serotype 8 (AAV-8), AAV serotype 9 (AAV-9), serotype rhlO AAV, serotype rh74 AAV, or a pseudotyped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9).In some embodiments, the AAV comprises SLB-101.

[0145] 14. The method of any one of arrangements 1-13, wherein the AAV vector comprises an AAV serotype 9, or is derived from an AAV serotype 9 vector.

[0146] 15. The method of any one of arrangements 1-13, wherein the AAV vector comprises an AAV serotype rh74, or is derived from an AAV serotype rh74 vector.

[0147] 16. The method of any one of the preceding arrangements, wherein the nucleotide encoding PD-L1 comprises SEQ ID NO: 5.

[0148] 17. The method of any one of the preceding arrangements, wherein the nucleotide encoding CTLA-4 comprises SEQ ID NO: 8 or SEQ ID NO: 9.

[0149] 18. The method of any one of the preceding arrangements, wherein the nucleotide encoding CMTM6 comprises SEQ ID NO: 10.

[0150] 19. The method of any one of the preceding arrangements, wherein the nucleotide encoding IDO comprises SEQ ID NO: 11.

[0151] 20. The method of any one of the preceding arrangements, wherein the nucleotide encoding Qa-ld comprises SEQ ID NO: 13.

[0152] 21. The method of any one of the preceding arrangements, wherein administering the full-length PD-L1 and one or more immune system modulators comprises administering an expression cassette for expression of full-length PD-L1 and one or more immune system modulators.

[0153] 22. The method of any one of the preceding arrangements, wherein the expression cassette comprises one or more genetic elements selected from the group comprising one or more: ITRs, enhancers, promoters, self-cleaving peptides, protein coding sequences, or Kozak sequences.

[0154] 23. The method of arrangement 22, wherein the expression cassette comprises one or more ITRs.

[0155] 24. The method of arrangement 22, wherein the one or more ITRs compriseSEQ ID NO: 1 and / or SEQ ID NO: 15.

[0156] 25. The method of arrangement 22, wherein the expression cassette comprises an enhancer.

[0157] 26. The method of arrangement 25, wherein the enhancer comprises a CMV enhancer.

[0158] 27. The method of arrangement 26, wherein the CMV enhancer comprisesSEQ ID NO: 2.

[0159] 28. The method of arrangement 22, wherein the expression cassette comprises a promoter.

[0160] 29. The method of arrangement 28, wherein the promoter comprises a CMV promoter.

[0161] 30. The method of arrangement 29, wherein the CMV promoter comprisesSEQ ID NO: 3.

[0162] 31. The method of arrangement 22, wherein the expression cassette comprises one or more self-cleaving peptides.

[0163] 32. The method of arrangement 31, wherein the one or more self-cleaving peptides comprises a 2A self-cleaving peptide.

[0164] 33. The method of arrangement 32, wherein the 2A self-cleaving peptide comprises a T2A or P2A self-cleaving peptide.

[0165] 34. The method of arrangement 33, wherein the T2A self-cleaving peptide comprises SEQ ID NO: 6.

[0166] 35. The method of arrangement 33, wherein the P2A self-cleaving peptide comprises SEQ ID NO: 12.

[0167] 36. The method of arrangement 22, wherein the expression cassette comprises one or more protein coding sequences.

[0168] 37. The method of arrangement 36, wherein the one or more protein coding sequences is selected from the group comprising: PD-L1, CTLA-4, Qa-ld, HLA-E, HLA-G, CMTM6, and indolamine-2, 3 -dioxygenase (IDO).

[0169] 38. The method of arrangement 37, wherein the PD-L1 comprises a peptide comprising SEQ ID NO: 23.

[0170] 39. The method of arrangement 37, wherein the CTLA-4 comprises a peptide comprising SEQ ID NO: 24 or SEQ ID NO: 25.

[0171] 40. The method of arrangement 37, wherein the Qa-ld comprises a peptide comprising SEQ ID NO: 26 or SEQ ID NO: 27.

[0172] 41. The method of arrangement 37, wherein the IDO comprises a peptide comprising SEQ ID NO: 28.

[0173] 42. The method of arrangement 37, wherein the CMTM6 comprises a peptide comprising SEQ ID NO: 29.

[0174] 43. The method of arrangement 22, wherein the expression cassette comprises a Kozak sequence.

[0175] 44. The method of arrangement 43, wherein the Kozak sequence comprisesSEQ ID NO: 4.

[0176] 45. The method of arrangement 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2A selfcleaving peptide, a Qa-ld coding sequence, and a 3' ITR.

[0177] 46. The method of arrangement 45, wherein the 5' ITR comprises SEQ IDNO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3 ' ITR comprises SEQ ID NO: 15.

[0178] 47. The method of arrangement 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CMTM6 coding sequence, a P2A selfcleaving peptide, a Qa-ld coding sequence, and a 3' ITR.

[0179] 48. The method of arrangement 47, wherein the 5' ITR comprises SEQ IDNO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15.

[0180] 49. The method of arrangement 47, wherein the 5' ITR comprises SEQ IDNO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM-6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15.

[0181] 50. The method of arrangement 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2A selfcleaving peptide, a Qa-ld coding sequence, and a 3' ITR.

[0182] 51. The method of arrangement 50, wherein the 5' ITR comprises SEQ IDNO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ IDNO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 9, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3 ' ITR comprises SEQ ID NO: 15.

[0183] 52. The method of arrangement 50, wherein the 5' ITR comprises SEQ IDNO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3 ' ITR comprises SEQ ID NO: 15.

[0184] 53. The method of arrangement 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a linker, an IDO coding sequence, and a 3' ITR.

[0185] 54. The method of arrangement 53, wherein the 5' ITR comprises SEQ IDNO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the linker comprises SEQ ID NO: 7, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15.

[0186] 55. The method of arrangement 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, an IDO coding sequence, and a 3' ITR.

[0187] 56. The method of arrangement 55, wherein the 5' ITR comprises SEQ IDNO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15.

[0188] 57. The method of any one of the preceding arrangements, wherein administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 500%.

[0189] 58. The method of any one of the preceding arrangements, wherein administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 100 days following transplantation of the donor organ.

[0190] 59. The method of any one of the preceding arrangements, wherein administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by greater than 100 days following transplantation of the donor organ.

[0191] 60. The method of any one of the preceding arrangements, wherein administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 12 months.

[0192] 61. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, aPD-Ll coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2A self-cleaving peptide, a Qa-ld coding sequence, and a 3 ' ITR.

[0193] 62. The expression cassette of arrangement 61, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15.

[0194] 63. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, aPD-Ll coding sequence, a T2A self-cleaving peptide, a CMTM6 coding sequence, a P2A self-cleaving peptide, a Qa-ld coding sequence, and a 3' ITR.

[0195] 64. The expression cassette of arrangement 63, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3TTR comprises SEQ ID NO: 15.

[0196] 65. The expression cassette of arrangement 63, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM-6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15.

[0197] 66. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, aPD-Ll coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2A self-cleaving peptide, a Qa-ld coding sequence, and a 3 ' ITR.

[0198] 67. The expression cassette of arrangement 66, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 9, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15.

[0199] 68. The expression cassette of arrangement 66, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15.

[0200] 69. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a linker, an IDO coding sequence, and a 3' ITR.

[0201] 70. The expression cassette of arrangement 69, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 codingsequence comprises SEQ ID NO: 5, the linker comprises SEQ ID NO: 7, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15.

[0202] 71. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, aPD-Ll coding sequence, a T2A self-cleaving peptide, an IDO coding sequence, and a 3 ' ITR.

[0203] 72. The expression cassette of arrangement 71, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15.

[0204] 73. An adeno-associated virus (AAV) comprising the expression cassette of any one of arrangements 62-72.

[0205] 74. Use of the expression cassette or AAV vector of any one of arrangements 61-73, in the manufacture of a medicament for increasing solid organ transplant survival.EXAMPLESExample 1 : Evaluation of novel combinatory transgene constructs

[0206] To assess the impact of putative immune suppressive genes on tissue engraftment, an established model of tumor cell line rejection, mediated by expression of a strong immunodominant antigen (membrane expressed OVA). This model consists of an E0771 cell line transduced with a puromycin selection marker used to select for cells with membrane OVA expression. Using this model, 100% rejection of established tumor within 3 weeks of implantation in immune competent mice, with fully established tumor occurring in SCID mice has been observed. Using procedures defined below, different immune suppressive genes were engineered as combinatory constructs into E0771-LX301-mOVA cells (using a hygromycin expressing lentivirus). These cells were then assessed for gene expression by standard procedures, including flow cytometry, western blot and QRT-PCR. After verification, 500,000 cells were injected into the flanks and mammary fat pads of immune competent mice to assess engraftment over time by bi-weekly caliper measurement.

[0207] FIG. 10 is an illustrative representation of a method for increasing solid organ transplant survival according to some embodiments disclosed herein.

[0208] FIGs. 2B-8B are illustrations depicting some embodiments of an expression construct for increasing solid organ transplant survival.

[0209] FIG. 11 is an illustrative representation of some embodiments of vectors for increasing solid organ transplant survival.

[0210] FIG. 12 is an illustrative representation of the modularity of some immune system modulators according to some embodiments disclosed herein.Procedure for engineering constructs into lentiviral vectors.Protocol for Gateway Cloning into a Viral Destination VectorMaterials:

[0211] Non-limiting examples of materials used in the examples presented herein include: viral destination vector(s) (e.g., lentiviral, adenoviral, or adeno-associated viral vector(s)) with Gateway™ cassette — pLenti-CMV-Hygro-DEST (Lentiviral plasmid with CMV promoter and Hygromycin resistance); entry clone containing the gene of interest (pENTRlA — vectors clones through restriction enzyme ligation); LR Clonase™ II enzyme mix; TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0); sterile water; agarose gel electrophoresis equipment and reagents; competent E. coli cells for transformation; LB agar plates with appropriate antibiotic selection; LB broth with appropriate antibiotic selection; and plasmid isolation kit(s). It shall be appreciated that other reagents or materials not specifically listed herein can be used as viable alternatives.Procedure:Prepare Entry Clone:

[0212] If the gene of interest was not already in an appropriate entry clone (e.g., a pDONR entry clone), a recombination reaction was performed according to the manufacturer’ s protocol to clone the gene into an entry vector (e.g., a pDONR vector). The presence and orientation of the gene of interest was confirmed in the entry clone by restriction digest analysis or DNA sequencing.Cloning Reaction:

[0213] The viral destination vector and the entry clone were thawed on ice. The cloning reaction was set up as follows: 100 ng of a destination vector (e.g., pLenti-CMV-Hygro) wasmixed with 100 ng of the entry clone DNA (SOLID 1-7) or different clones (e.g., pENTRlA- PDL1) in a total volume of 4 pl (see Table 12). 1 pl of enzyme mix (for example, LR Clonase™ II) was added to the DNA mixture. The reaction mixture was then incubated at 25°C for 1-2 hours or overnight.Table 12: Combinatorial Gene ConstructsTransformation of E, coli:

[0214] Competent E. coli cells were thawed on ice. 2 pl of the cloning reaction mixture was then added to 50 pl of competent E. coli cells in a microcentrifuge tube. The mixture was mixed gently by tapping the tube and incubating on ice for 30 minutes. The cells were then subjected to heat shock at 42°C for 45 seconds, and the tube was immediately transferred back to ice for 2 minutes. 250 pl of SOC medium or LB broth without antibiotics was added to the cells and incubate at 37°C with shaking for 1 hour. 50-100 pl of the transformed cells were plated onto LB agar plates containing the appropriate antibiotic selection. The plates were incubated overnight at 37°C.Screening of Positive Clones:

[0215] 3 -5 colonies were picked and inoculated into 3-5 ml of LB broth with the appropriate antibiotic selection. The cultures were then incubated overnight at 37°C with shaking. Plasmid DNA was isolated from the overnight cultures using a plasmid isolation kit. The presence of the gene of interest in the viral destination vector was then assessed by restriction digest analysis or DNA sequencing.Large-scale Plasmid Preparation:

[0216] A larger culture (e.g., 100 ml) of positive clone(s) was inoculated in LB broth with appropriate antibiotic selection. The culture(s) were incubated overnight at 37°C with shaking. Plasmid DNA was isolated from the overnight cultures using a plasmid isolation kit. The purified plasmid DNA was quantified by measuring the absorbance at 260 nm.Protocol for Lentiviral Production and Cellular TransductionMaterials:

[0217] Non-limiting examples of materials used in the examples presented herein include: lentiviral vector containing the gene of interest; packaging plasmids (e.g., psPAX2 and pMD2.G); tumor cells (e.g., cancer cell lines); cell culture medium (appropriate for the tumor cell line); polybrene (optional); phosphate-buffered saline (PBS); sterile syringes and needles; mice (appropriate strain for tumor studies); anesthetics (e.g., isoflurane); and surgical tools (e.g., scissors, forceps). It shall be appreciated that other reagents or materials not specifically listed herein can be used as viable alternatives.Procedure:Lentivirus Production:

[0218] Cells (e.g., HEK293T cells) were transfected with the lentiviral vector, packaging plasmids (e.g., psPAX2 and pMD2.G), and transfection reagent according to the manufacturer’s protocol. The lentivirus-containing supernatant was collected 48-72 hours post-transfection and filtered through a 0.45 pm filter to remove cell debris. The lentivirus was then concentrated using ultracentrifugation or a commercial virus concentration kit. Following concentration, the lentiviral pellet was resuspended in an appropriate volume of PBS or cell culture medium and aliquoted. Aliquots were stored at -80°C until further use.Tumor Cell Transduction:

[0219] Tumor cells were cultured in appropriate cell culture medium in a T25 flask until they reached 70-80% confluency. The medium was replaced with fresh medium containing lentivirus at an appropriate multiplicity of infection (MOI). Polybrene (8 pg / mL) was added as needed to enhance transduction efficiency. The cells were then incubated with lentivirus for 24-48 hours at 37°C in a CO2 incubator. The lentivirus-containing medium was then replaced with fresh complete medium and incubated for an additional 24 hours.Transduction efficiency was confirmed by observing expression of the gene of interest using fluorescence microscopy or other appropriate assays.Injection of tumor cells into micePreparation of Mice:

[0220] Appropriate procedures were used for handling and anesthesia according to institutional guidelines. A sterile environment was prepared for the injection procedure. The injection site on the mice was shaved as necessary to ensure proper visualization and access. Injection of Transduced Tumor Cells into Mice:

[0221] The number of transduced tumor cells was counted and a suspension was prepared at the desired concentration in PBS. Mice were anesthetized using an appropriate method (e.g., isoflurane inhalation). Analgesics were administered as per institutional guidelines to minimize pain and discomfort. The transduced tumor cells were injected subcutaneously or orthotopically (500,000 E0771 cells) at the desired site using a sterile syringe and needle. Mice were monitored for tumor growth and general health regularly postinjection.Post-injection Monitoring:

[0222] Mice were monitored daily for signs of distress, tumor growth, or any adverse effects. Tumor size was measured regularly using calipers or other appropriate methods. Any changes in behavior or physical condition were recorded.

[0223] FIG. 9 is a graph showing the effect of administration of full-length PD-L1 and one or more immune system modulators on tumor volume. As can be seen from FIG. 9, administration of full-length PD-L1 and one or more immune system modulators result in increased tumor volume, thus indicating that these molecules may be useful in increasing solid organ transplant survival. FIG. 14 is a graph showing the effect of administration of full-length PD-L1 and one or more immune system modulators on tumor volume over an extended period of time.MHC-Mismatched Murine Heterotopic Heart Transplant Model:

[0224] Gene constructs Solid-2 and Solid-5 were packaged into an AAV capsid (SLB-101) and tested in an MHC-mismatched murine heterotopic heart transplant model. BALB / c mice were each injected with one of the viral vectors two weeks prior to transplant. Donor hearts were transplanted into the abdominal position of C57BL / 6 recipients with andwithout concomitant CTLA-4 Ig. Grafts were assessed for function by abdominal palpation and echocardiography. Fulminant graft rejection, defined as complete cessation of allograft function on exam and by echocardiography, was the endpoint of the study.

[0225] FIG. 15 is a graph showing the effect of administration of full-length PD- L1 and one or more immune system modulators on the probability of graft survival in an MHC- mismatched heterotopic heart transplant model.

[0226] Three constructs were tested in the murine heart transplant model: one transplanted control (an unmodified PD-L1 construct without any immune modulator(s)), and two therapeutic constructs (construct Solid-2 and construct Solid-5). In the absence of concomitant immunosuppression, transplanted control and therapeutic grafts were fully rejected within a median of 7-9 days (FIG. 15). With the addition of concomitant CTLA-4 Ig, all treated allografts demonstrated higher graft survival at 100 days compared to grafts with CTLA-4 Ig alone.

[0227] Results from these in vivo model experiments indicate that overexpression of PDL1 in combination with other key immunomodulatory pathways can prevent or delay acute cellular rejection locally without abrogation of systemic immune function.

[0228] Any titles or subheadings used herein are for organizational purposes and should not be used to limit the scope of embodiments disclosed herein. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose, including the disclosures specifically referenced herein. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein.

[0229] Although embodiments described herein have been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the disclosure and obvious modifications and equivalents thereof. In addition, while several variations of the embodiments have been shown and described indetail, other modifications, which are within the scope of this disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. It should be understood that various features and aspects of the disclosed embodiments can be combined with, or substituted for, one another in order to form varying modes or embodiments. Thus, it is intended that the scope of the present disclosure should not be limited by the particular disclosed embodiments described above.

[0230] It should be understood, however, that this detailed description, while indicating preferred embodiments, is given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art.

[0231] The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive manner. Rather, the terminology is simply being utilized in conjunction with a detailed description of embodiments of the systems, methods, and related components. Furthermore, embodiments may comprise several novel features, no single one of which is solely responsible for its desirable attributes or is believed to be essential to practicing the embodiments herein described.

Claims

WHAT IS CLAIMED IS:

1. A method of increasing solid organ transplant survival, the method comprising: obtaining a solid donor organ, administering PD-L1 and one or more immune system modulator to the solid donor organ.

2. The method of claim 1 , wherein the solid donor organ is selected from the group comprising: liver, pancreas, spleen, kidney, heart, and adrenal glands.

3. The method of claim 1, wherein the PD-L1 is full-length PD-L1.

4. The method of claim 1, wherein administering PD-L1 comprises administering a nucleotide encoding PD-L1.

5. The method of claim 1, wherein administering the one or more immune system modulators comprises administering an adeno-associated virus (AAV) encapsidating an expression cassette for expression of the PD-L1.

6. The method of claim 1, wherein administering the one or more immune system modulators comprises administering a nucleotide encoding the one or more immune system modulators.

7. The method of claim 1, wherein administering the one or more immune system modulators comprises administering an adeno-associated virus (AAV) encapsidating an expression cassette for expression of the one or more immune system modulators.

8. The method of claim 1, wherein the one or more immune system modulators is selected from the group comprising: CTLA-4, Qa-ld, HLA-E, HLA-G, CMTM6, and indolamine-2, 3 -dioxygenase (IDO).

9. The method of claim 8, wherein the one or more immune system modulator is CTLA-4.

10. The method of claim 8, wherein the one or more immune system modulator is Qa-ld.

11. The method of claim 8, wherein the one or more immune system modulator is IDO.

12. The method of claim 8, wherein the one or more immune system modulator is CMTM6.

13. The method of claim 1, wherein the AAV is an AAV serotype 1 (AAV-1), AAV serotype 2 (AAV-2), AAV serotype 3 (AAV-3), AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV-7), AAV serotype 8 (AAV-8), AAV serotype 9 (AAV-9), serotype rhlO AAV, serotype rh74 AAV, or a pseudotyped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9), an AAV serotype 9 vector comprising a capsid insertion having SEQ ID NO: 46, or an AAV serotype 9 vector comprising SEQ ID NO: 47.

14. The method of claim 1, wherein the AAV vector comprises an AAV serotype 9, or is derived from an AAV serotype 9 vector.

15. The method of claim 1, wherein the AAV vector comprises an AAV serotype rh74, or is derived from an AAV serotype rh74 vector.

16. The method of claim 1, wherein the nucleotide encoding PD-L1 comprises SEQ ID NO: 5.

17. The method of claim 1, wherein the nucleotide encoding CTLA-4 comprises SEQ ID NO: 8 or SEQ ID NO: 9.

18. The method of claim 1, wherein the nucleotide encoding CMTM6 comprises SEQ ID NO: 10.

19. The method of claim 1, wherein the nucleotide encoding IDO comprises SEQ ID NO: 11.

20. The method of claim 1, wherein the nucleotide encoding Qa-ld comprises SEQ ID NO: 13.

21. The method of claim 1, wherein administering the full-length PD-L1 and one or more immune system modulators comprises administering an expression cassette for expression of full-length PD-L1 and one or more immune system modulators.

22. The method of claim 1, wherein the expression cassette comprises one or more genetic elements is selected from the group comprising one or more: ITRs, enhancers, promoters, self-cleaving peptides, protein coding sequences, or Kozak sequences.

23. The method of claim 22, wherein the expression cassette comprises one or more ITRs.

24. The method of claim 22, wherein the one or more ITRs comprise SEQ ID NO: 1 and / or SEQ ID NO: 15.

25. The method of claim 22, wherein the expression cassette comprises an enhancer.

26. The method of claim 25, wherein the enhancer comprises a CMV enhancer.

27. The method of claim 26, wherein the CMV enhancer comprises SEQ ID NO:2.

28. The method of claim 22, wherein the expression cassette comprises a promoter.

29. The method of claim 28, wherein the promoter comprises a CMV promoter.

30. The method of claim 29, wherein the CMV promoter comprises SEQ ID NO:3.

31. The method of claim 22, wherein the expression cassette comprises one or more self-cleaving peptides.

32. The method of claim 31, wherein the one or more self-cleaving peptides comprises a 2A self-cleaving peptide.

33. The method of claim 32, wherein the 2A self-cleaving peptide comprises a T2A or P2A self-cleaving peptide.

34. The method of claim 33, wherein the T2A self-cleaving peptide comprises SEQ ID NO: 6.

35. The method of claim 33, wherein the P2A self-cleaving peptide comprises SEQ ID NO: 12.

36. The method of claim 22, wherein the expression cassette comprises one or more protein coding sequences.

37. The method of claim 36, wherein the one or more protein coding sequences is selected from the group comprising: PD-L1, CTLA-4, Qa-ld, HLA-E, HLA-G, CMTM6, and indolamine-2, 3 -dioxygenase (IDO).

38. The method of claim 37, wherein the PD-L1 comprises a peptide comprising SEQ ID NO: 23.

39. The method of claim 37, wherein the CTLA-4 comprises a peptide comprising SEQ ID NO: 24 or SEQ ID NO: 25.

40. The method of claim 37, wherein the Qa-ld comprises a peptide comprising SEQ ID NO: 26 or SEQ ID NO: 27.

41. The method of claim 37, wherein the IDO comprises a peptide comprising SEQID NO: 28.

42. The method of claim 37, wherein the CMTM6 comprises a peptide comprising SEQ ID NO: 29.

43. The method of claim 22, wherein the expression cassette comprises a Kozak sequence.

44. The method of claim 43, wherein the Kozak sequence comprises SEQ ID NO: 4.

45. The method of claim 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2A self-cleaving peptide, a Qa- Id coding sequence, and a 3' ITR.

46. The method of claim 45, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15.

47. The method of claim 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CMTM6 coding sequence, a P2A self-cleaving peptide, a Qa- ld coding sequence, and a 3' ITR.

48. The method of claim 47, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15.

49. The method of claim 47, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, theT2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM-6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15.

50. The method of claim 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2A self-cleaving peptide, a Qa- ld coding sequence, and a 3' ITR.

51. The method ofclaim 50, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 9, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15.

52. The method of claim 50, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15.

53. The method of claim 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a linker, an IDO coding sequence, and a 3 'ITR.

54. The method of claim 53, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the linker comprises SEQ ID NO: 7, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15.

55. The method of claim 22, wherein the expression cassette comprises, from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, an IDO coding sequence, and a 3' ITR.

56. The method of claim 55, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15.

57. The method of claim 1, wherein administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 500%.

58. The method of claim 1, wherein administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 100 days following transplantation of the donor organ.

59. The method of claim 1, wherein administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by greater than 100 days following transplantation of the donor organ.

60. The method of claim 1, wherein administering PD-L1 and one or more immune modifiers increases survival of the solid donor organ by up to about 12 months.

61. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2 A self-cleaving peptide, a Qa- 1 d coding sequence, and a 3 ' ITR.

62. The expression cassette of claim 61, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3 ' ITR comprises SEQ ID NO: 15.

63. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CMTM6 coding sequence, a P2A self-cleaving peptide, a Qa-ld coding sequence, and a 3' ITR.

64. The expression cassette of claim 63, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO:3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3' ITR comprises SEQ ID NO: 15.

65. The expression cassette of claim 63, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CMTM-6 coding sequence comprises SEQ ID NO: 10, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3' ITR comprises SEQ ID NO: 15.

66. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, a CTLA-4 coding sequence, a P2 A self-cleaving peptide, a Qa- 1 d coding sequence, and a 3 ' ITR.

67. The expression cassette of claim 66, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 9, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 13, and the 3 ' ITR comprises SEQ ID NO: 15.

68. The expression cassette of claim 66, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the CTLA-4 coding sequence comprises SEQ ID NO: 8, the P2A self-cleaving peptide comprises SEQ ID NO: 12, the Qa-ld coding sequence comprises SEQ ID NO: 14, and the 3 ' ITR comprises SEQ ID NO: 15.

69. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a linker, an IDO coding sequence, and a 3' ITR.

70. The expression cassette of claim 69, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the linker comprises SEQ ID NO: 7, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15.

71. An expression cassette comprising from 5' to 3': a 5' ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence, a T2A self-cleaving peptide, an IDO coding sequence, and a 3 ' ITR.

72. The expression cassette of claim 71, wherein the 5' ITR comprises SEQ ID NO: 1, the CMV enhancer comprises SEQ ID NO: 2, the CMV promoter comprises SEQ ID NO: 3, the Kozak sequence comprises SEQ ID NO: 4, the PD-L1 coding sequence comprises SEQ ID NO: 5, the T2A self-cleaving peptide comprises SEQ ID NO: 6, the IDO coding sequence comprises SEQ ID NO: 11, and the 3' ITR comprises SEQ ID NO: 15.

73. An adeno-associated virus (AAV) vector comprising the expression cassette of any one of claims 61-72.

74. Use of the expression cassette of any one of claims 61-72 or the AAV vector of claim 73, in the manufacture of a medicament for increasing solid organ transplant survival.

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