Compositions and methods to improve graft survival

Abstract

The present disclosure provides, in part, combination therapies and methods of eliciting immuno-tolerance using PDL1 variants and immune system modulators.

Description

COMPOSITIONS AND METHODS TO IMPROVE GRAFT SURVIVALRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 645,791, filed May 10, 2024, which is hereby incorporated by reference herein in its entirety.REFERENCE TO AN ELECTRONIC 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 AAVAN1 15WOSEQLIST.XML, which was created and last modified on May 8, 2025, which is 24,121 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates in some respects to constructs and methods for increasing solid organ transplant survival.BACKGROUND

[0004] 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. The number of patients with heart failure is expected to increase by nearly 46% by 2030 to approximately 8 million. Nearly 50% of these patients are expected to die within 5 years of diagnosis. For those with end stage heart failure, heart transplantation is the gold standard treatment. Long-term survival following transplant is limited due to graft dysfunction, rejection, vasculopathy, and chronic systemic immunosuppression. There is a strong unmet need to apply major advances in gene therapy to the field of transplantation to decrease transplant rejection, decrease use of immunosuppressants, and expand the donor pool. In view of the foregoing, it would be desirable to provide new targets and methods to minimize organ rejection.SUMMARY

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

[0006] Some embodiments herein are directed to methods of increasing solid organ transplant survival or methods of preparing a subject for a transplant. In some embodiments, the methods comprise: obtaining a solid donor organ, administering truncated PD-L1 (PDL1- TM) and / or secreted PD-L1 (PDL1-SS), and administering one or more immune system modulators to the solid donor organ. In some embodiments the solid donor organ comprises liver, pancreas, spleen, kidney, heart, or adrenal glands. In some embodiments, the PD-L1 is PDL1-TM and / or PDL1-SS. In some embodiments, administering PDL1-TM and / or PDL1-SS comprises administering a nucleic acid encoding PDL1-TM and / or PDL1-SS. In some embodiments, administering PDL1-TM and / or PDL1-SS comprises administering an adeno- associated virus (AAV) encapsidating an expression cassette for expression of the PDL1-TM and / or PDL1-SS.

[0007] In some embodiments, the AAV is a modified AAV. In some embodiments, the modified AAV is derived from an AAV serotype 9 vector. In some embodiments, the AAV vector is derived from an AAV serotype rh74 vector. In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector or an AAV serotype rh74 vector comprising a capsid insertion. In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector comprising a capsid insertion having SEQ ID NO: 13. In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector comprising SEQ ID NO: 14. In some embodiments, the AAV vector comprises AAV-SLB101 having SEQ ID NO: 14.

[0008] 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: 14, 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%-80%, 80%-100%, 80%-95%, 80%-90%, 90%- 100%, 90%-95%, or 95%-100% identity to an AAV serotype 9 vector having SEQ ID NO: 14. In some embodiments, the modified AAV serotype vector has about 70%, 75%, 80%, 85%,90%, 95%, or 100% identity to the parental AAV serotype 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%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to the parental AAV serotype (e.g., AAV rh74).

[0009] In some embodiments, the nucleotide encoding PD-L1 comprises SEQ ID NO: 15. In some embodiments, the expression cassette comprises 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: 5 and / or SEQ ID NO: 5. In some 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: 7. 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: 8. In some embodiments, the expression cassette comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises SEQ ID NO: 9. Some embodiments disclosed herein relate to an 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 comprises PD-L1 and / or variants thereof. In some embodiments, the PD-L1 comprises a peptide comprising SEQ ID NO: 1 or SEQ ID NO: 3. Some embodiments disclosed herein relate to an 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 and a 3’ ITR. In some embodiments, the 5’ ITR comprises SEQ ID NO: 5, the CMV enhancer comprises SEQ ID NO: 7, the CMV promoter comprises SEQ ID NO: 8, the Kozak sequencecomprises SEQ ID NO: 9, the PD-L1 coding sequence comprises SEQ ID NO: 15 or SEQ ID NO: 16 and the 3’ ITR comprises SEQ ID NO: 6.

[0012] 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: 5, a CMV enhancer comprising SEQ ID NO: 7, a CMV promoter comprising SEQ ID NO: 8, a Kozak sequence comprising SEQ ID NO: 8, a PD-L1 coding sequence comprising SEQ ID NO: 15 or SEQ ID NO: 16, and a 3’ ITR comprising SEQ ID NO: 6, 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%-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: 5, a CMV enhancer comprising SEQ ID NO: 7, a CMV promoter comprising SEQ ID NO: 8, a Kozak sequence comprising SEQ ID NO: 9, a PD-L1 coding sequence comprising SEQ ID NO: 15 or SEQ ID NO: 16, and a 3’ ITR comprising SEQ ID NO: 6.

[0013] In some embodiments, administering PDL1-TM and / or PDL1-SS 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 PDL1-TM and / or PDL1-SS 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. In some embodiments, administering PDL1-TM and / or PDL1-SS to the donor organ and one or more immune modifiers to the donor organ or recipient increases survival of the solid donor organ by greater than 100 days following transplantation of the donor organ. In some embodiments, administering PDL1-TM and / or PDL1-SS to the donor organ and one or more immune modifiers to the donor organ or recipient 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 CMVenhancer, a CMV promoter, a Kozak sequence, a PD-L1 coding sequence and a 3’ ITR. In some embodiments, the 5’ ITR comprises SEQ ID NO: 5, the CMV enhancer comprises SEQ ID NO:7, the CMV promoter comprises SEQ ID NO: 8, the Kozak sequence comprises SEQ ID NO: 9, the PD-L1 coding sequence comprises SEQ ID NO: 15 or SEQ ID NO: 16 and the 3’ ITR comprises SEQ ID NO: 6. In some embodiments, the 5’ ITR comprises SEQ ID NO: 5, the CMV enhancer comprises SEQ ID NO: 7, the CMV promoter comprises SEQ ID NO: 8, the Kozak sequence comprises SEQ ID NO: 9, the PD-L1 coding sequence comprises SEQ ID NO: 15 and the 3’ ITR comprises SEQ ID NO: 16. 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: 5, a CMV enhancer comprising SEQ ID NO: 7, a CMV promoter comprising SEQ ID NO: 8, a Kozak sequence comprising SEQ ID NO: 9, a PD-L1 coding sequence comprising SEQ ID NO: 15 and a 3’ ITR comprising SEQ ID NO: 6, 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%-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: 5, a CMV enhancer comprising SEQ ID NO: 7, a CMV promoter comprising SEQ ID NO: 8, a Kozak sequence comprising SEQ ID NO: 9, a PD-L1 coding sequence comprising SEQ ID NO: 15 and a 3’ ITR comprising SEQ ID NO: 6.

[0015] Some aspects of the present disclosure provide a nucleic acid construct comprising a promotor operably connected to a nucleic acid sequence encoding a PDL1 variant or portion thereof including the extracellular portion, wherein the PDL1 variant or portion thereof including the extracellular portion is capable of binding to its receptor. In some embodiments, the construct comprises a secretory signal operably connected to the promoter and functionally linked to the nucleic acid encoding the PDL1 variant or portion thereof. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding PDL1-TM and / or PDL1-SS. In some embodiments, the construct comprises a viral vector, forexample an AAV vector. In some embodiments, the composition comprises a pharmaceutical composition comprising the nucleic acid construct or expression cassette described herein.

[0016] Some aspects of the present disclosure provide a method for expressing a PDL1 variant or portion thereof in a transplantable article. In some embodiments, the method comprises introducing the nucleic acid construct or the pharmaceutical composition described herein into a transplantable article. In some embodiments, the method comprises ex vivo or in vitro perfusion of the nucleic acid construct or the pharmaceutical composition into the article. In some embodiments, the method further comprises transplanting the article into a subject. In some embodiments, the article comprises an organ, a cell population, skin, or a tissue or a combination thereof.

[0017] Some aspects of the present disclosure provide a recombinant AAV (rAAV) vector including an expression cassette, wherein the expression cassette includes a nucleic acid encoding, from 5’ to 3’ : a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDL1-TM or PDL1-SS encoding sequence, and a 3’ ITR.

[0018] Some aspects of the present disclosure provide a method for transfecting a transplantable article with an rAAV vector including an expression cassette, wherein the expression cassette includes a nucleic acid encoding, from 5’ to 3’: a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDL1-TM or PDL1-SS encoding sequence, and a 3’ ITR; the method includes adding the rAAV to a medium to produce a transplant medium, and incubating the transplantable article in the transplant medium. In some embodiments, incubating the transplantable article in the transplant medium produces a conditioned medium. In some embodiments, the method further includes comprising collecting the conditioned medium, and analyzing the conditioned medium for expression of PDLI protein derived from the PDL1-TM or PDL1-SS encoding sequence. In some embodiments, the method further includes transplanting the transplantable article into a subject. In some embodiments, the method further includes administering a therapeutically effective amount of one or more additional therapeutic agents to the subject. In some embodiments, the one or more additional therapeutic agent is administered prior to, during and / or after transplantation of the article to the subject.

[0019] Some aspects of the present disclosure provide a transplantable article produced by the method of transfecting a transpl antable article with an rAAV vector includingan expression cassette, wherein the expression cassette includes a nucleic acid encoding, from 5’ to 3’: a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDL1-TM or PDL1-SS encoding sequence, and a 3’ ITR; the method includes adding the rAAV to a medium to produce a transplant medium, and incubating the transplantable article in the transplant medium. In some embodiments, transplantable article includes one or more articles including heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, and adrenal glands. In some embodiments, the transplantable article includes a rAAV, wherein the rAAV expresses PDLI protein derived from the PDL1-TM or PDL1-SS encoding sequence. In some embodiments, the article for transplantation comprises an article, a cell population, skin, or a tissue. In some embodiments, the transplantable article includes one or more articles including heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, and adrenal glands. In some embodiments, the transplantable article is from a mammalian donor. In some embodiments, the mammalian donor is a human donor. In some embodiments, mammalian donor is a pig donor.

[0020] Some aspects of the present disclosure provide a method for preventing or reducing rejection of a transplanted article in a subject. In some embodiments, the method comprises introducing the nucleic acid construct or a pharmaceutical composition described herein into a transplantable article prior to transplantation of the article into the subject. In some embodiments, the method further comprises transplanting the article into a subject.

[0021] Some aspects of the present disclosure provide a method for introducing a construct into a transplantable article, the method comprising ex vivo perfusion of the construct into the article prior to transplantation.

[0022] Some aspects of the present disclosure provide a method for preventing or reducing rejection of a transplanted article in a subject. In some embodiments, the method comprises introducing a construct or a pharmaceutical composition described herein into a transplantable article prior to transplantation into a subject. In some embodiments, the method further comprises transplanting the article into a subject.

[0023] Some aspects of the present disclosure provide a nucleic acid construct comprising a promotor operably connected to a nucleic acid sequence encoding a PDLI variant or portion thereof including the extracellular portion, wherein the PLD1 variant or portion thereof including the extracellular portion is capable of binding to its receptor. In someembodiments, the nucleic acid construct comprises a secretory signal operably connected to the promoter and functionally linked to the nucleic acid encoding the protein or portion thereof. In some embodiments, the PDL1 variant comprises PDL1-TM and / or PDL1-SS. In some embodiments, the nucleic acid construct comprises a viral vector, for example an AAV vector. In some embodiments, the composition comprises a pharmaceutical composition comprising the nucleic acid construct described herein.

[0024] Some aspects of the present disclosure provide a method for expressing a PDL1 variant or portion thereof in a transplantable article. In some embodiments, the method comprises introducing the nucleic acid construct or the pharmaceutical composition described herein into a transplantable article. In some embodiments, the method comprises ex vivo or in vitro perfusion of the nucleic acid construct or the pharmaceutical composition into the article. In some embodiments, the method further comprises transplanting the article into a subject. In some embodiments, the article comprises an organ, a cell population, skin, and or tissue or combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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 limit the scope of the disclosure herein.

[0026] FIG. 1 is an illustration depicting embodiments of PD-L1 variant mediated T-cell deactivation.

[0027] FIG. 2 is a chart setting forth non-limiting embodiments of PD-L1 expression constructs suitable for use in the formulations and methods disclosed herein.

[0028] FIG. 3 is an illustrative representation of a heterotopic heart transplant model.

[0029] FIG. 4 is a series of images (left) and a graph (right) depicting PDL1-TM and PDL1-SS expression on cardiomyocytes and in the serum of donor animal prior to organ transplant.

[0030] FIG. 5 is an illustrative representation of a heterotopic heart transplant model.

[0031] FIG. 6 depicts graft survival in a heterotopic heart transplant model with donor mouse administered PDL1-TM or PDL1-SS prior to organ transplant and recipient mouse administered abatacept postoperatively.

[0032] FIG. 7 is a pair of bar graphs depicting quantification of allograft function at 4 and 8 weeks after transplant.

[0033] FIG. 8 shows histology of allograft heart tissue treated with PDL1 variants.

[0034] FIG. 9 shows CD3 and CD20 infiltration of allograft heart tissue treated with PDLl variants.

[0035] FIG. 10 shows histology of allograft heart tissue treated with PDL1 variants.

[0036] FIG. 11 shows histology of allograft heart tissue treated with PDL1 variants.DETAILED DESCRIPTION

[0037] 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 per year, 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, and cardiac allograft vasculopathy (CAV). 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. Accordingly, some embodiments herein are directed to methods and constructs for increasing solid donor organ transplant survival.DEFINITIONS:

[0038] 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 iscommonly 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. The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art considering the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any order or configuration to such structures or steps. All methods described herein may be performable in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0039] 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, regulates or suppresses the immune system. Immune system modulators and their ligands include, without limitation, CTLA4-antibody fusion proteins (CTLA4-Ig) such as abatacept and belatacept, ipilimumab, tremelimumab, corticosteroids, biologies, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, and IMDH inhibitors.

[0040] In some embodiments, an immune system modulator as described herein is capable of binding or blocking binding to a receptor. Ligand receptor interactions, or binding may result in a molecular response. Similarly, blocking ligand receptor interactions or binding may result in a molecular response. The molecular response may include a change in immune function, cytokine production, proliferation, cellular migration or signaling.

[0041] 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 AAVvector (or “rAAV vector”), which refers to AAV comprising a polynucleotide sequence not of AAV origin (e.g., a transgene). The term “AAV” includes an AAV serotype 9 vector comprising a capsid insertion having SEQ ID NO: 13, or an AAV serotype 9 vector comprising SEQ ID NO: 14.

[0042] 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.

[0043] 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.

[0044] 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, 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, 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.

[0045] 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.

[0046] 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.

[0047] 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-m ethyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy- aminomethyl- 2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 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.

[0048] 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 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.

[0049] 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.

[0050] 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. , 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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. Promoters useful in the practice of the present disclosure include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissue-preferred, tissue-specific promoters and cell- type specific. The promoter may be an animal, bacterial, fungal, viral or synthetic promoter. Suitable promoters are known and described in the art. In mammalian cells, typical promoters include, without limitation, promoters for Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 virus, chicken beta actin, 3- phosphoglycerate kinase promoter, aMHC-CMV, C5-12, CK8 promoters, TNNT2, JeT,MHCK7, Des, and hDes promoters, translational elongation factor EF-la promoter or ubiquitin promoter, and other suitable promoters for the target article.

[0057] 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.

[0058] In some embodiments, the constructs described herein may comprise a secretory signal. A secretory signal, sometimes called a signal peptide, signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide, is or encodes a short peptide (usually 16-30 amino acids long) present at the N- terminus, C-terminus or internally of most newly synthesized proteins that are destined toward the secretory pathway. Often, a secretory signal functions to prompt a cell to translocate the protein to the cellular membrane and / or extracellular space. Signal peptides adhere to a generic three-domain structure, comprising a basic N-domain, a hydrophobic H-domain, and a slightly polar C-domain.

[0059] 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 be present between a promoter sequence and a coding sequence, with those two sequences still being considered “operably linked”.

[0060] The terms “vector” and “construct” may be used interchangeably herein and refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating 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.” The constructs may be heterogeneous exogenous constructs containing sequences from two or more different sources. Suitable constructs or vectors include constructs that are able to express the protein of interest. A preferred vector is an adeno-associated vector (AAV). Suitable methods of making viral particles are known in the art to be able to transform cells in order to express the protein of interest as described herein.

[0061] 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.”

[0062] 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.

[0063] 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.

[0064] The term “effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a condition or response 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 condition or response experienced by a subject.

[0065] 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.

[0066] 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 described herein.

[0067] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0068] 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 leastone” 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.

[0069] 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 claimed subject 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.

[0070] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 90%” includes “90%. ” In some embodiments, at least 95% homologous or identical includes 96%, 97%, 98%, 99%, and 100% homologous or identical to the reference sequence. In addition, when a sequence is disclosed as “comprising” a nucleotide or amino acid sequence, such a reference shall also include, unless otherwise indicated, that the sequence “comprises,” “consists of’ or “consists essentially of’ the recited sequence.

[0071] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. It will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of thedocuments cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.Non-Limiting Embodiments:

[0072] Disclosed herein are constructs encoding PDL1 variants, for example, truncated PD-L1 (PDL1-TM) and / or secreted PD-L1 (PDL1-SS), and methods of using the constructs comprising PDL1 variants to transduce organs and reduce transplant rejection including in combination therapy with immune modulation agents, for example CTLA-4 Igs, such as belatacept and abatacept.

[0073] Programmed death-ligand 1 (PD-L1 or PDL1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) or programmed cell death protein 1, also known as PD-1 and CD279. Engagement of PDL1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR- mediated activation of IL-2 production and T cell proliferation, this down-regulates the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PDL1 is a type I transmembrane glycoprotein encoded by the CD274 gene on chromosome 9 in humans. Transcription of this gene can produce multiple PDL1 splice variants, including PD-L1 IncRNA splice isoforms, truncated PDL1 (PDL1-TM) and soluble PDL1 (PDL1-SS). In some embodiments, the constructs disclosed herein PDL1-TM) and / or PDL1-SS.

[0074] Some aspects of the present disclosure provide methods for introducing one or more of the constructs described herein into a transplantable article. In some embodiments, the methods comprise introducing any of the constructs described herein or a pharmaceutical composition described herein into a transplantable article. In some embodiments, introducing the constructs described herein or a pharmaceutical composition described herein into a transplantable article includes ex vivo or in vitro perfusion of the construct or the pharmaceutical composition into the article.

[0075] In some embodiments, an article may consist of an organ, a cell population, and a tissue. The article for transplantation may comprise an organ. The organ may include, without limitation heart, heart valves, lung, kidney, liver, pancreas, skin, spleen, middle ear, connective tissue, intestine, colon, eye, stomach, ovary, testes, bladder, uterus and adrenalglands. The cell population may comprise a stem cells, bone marrow and immune cells. Tissues may comprise bones tendons, ligaments, skin, heart valves blood vessels, pancreas islets, nerves, veins, and limbs.

[0076] In some embodiments, the method further comprises transplanting the article into a subject. Organ transplantation is a medical procedure in which an organ is removed from one body and placed in the body of a recipient, to replace a damaged or missing organ. The donor and recipient may be at the same location, or organs may be transported from a donor site to another location. An allograft is a transplant of an organ or tissue between two genetically non-identical members of the same species. Due to the genetic difference between the organ and the recipient, the recipient's immune system may identify the organ as foreign and attempt to destroy it, causing transplant rejection. In addition, in cases of stem cell, bone marrow or other hematopoietic transplants the immune cells of the transplant attack the host cells. This is called Graft-versus-host disease (GvHD). “Graft” refers to transplanted, or donated tissue, and “host” refers to the tissues of the recipient. Transplantation recipients often receive prophylactic treatment to suppress the immune system after the transplant. These treatments continue after transplantation.

[0077] Some aspects of the present disclosure relate to an ex vivo method for prevention or reducing rejection of a transplanted article in a subject. In some embodiments, the methods comprise introducing a construct into a transplantable article prior to transplantation into a subject. The constructs may encode a promotor operably connected to a nucleic acid sequence encoding a PDL1 variant or extracellular portion of a PDL1 variant. The PDL1 variant or extracellular portion of a PDL1 variant may be capable of binding to its receptor and may activate the receptor similarly to the native PD1.

[0078] In some embodiments, the methods disclosed herein comprise obtaining a solid donor organ. In some embodiments, the solid donor organ comprises liver, pancreas, spleen, kidney, heart, and adrenal glands. PDL1-TM and / or PDL1-SS is administered to the solid donor organ and one or more immune system modulators are administered to the recipient. In some embodiments, the PDL1-TM and / or PDL1-SS is human PDL1-TM and / or PDL1-SS. In some embodiments, the PDL1-TM and / or PDL1-SS is mouse PDL1-TM and / or PDL1-SS. In some embodiments, the PDL1-TM and / or PDL1-SS is swine or pig PDL1-TM and / or PDL1-SS. In some embodiments, the PDL1-TM and / or PDL1-SS is non-human primate(NHP), for example, chimp, PDL1-TM and / or PDL1-SS. In some embodiments, the PDL1 - TM and / or PDL1-SS is administered by ex vivo perfusion. In some embodiments, administering PDL1-TM and / or PDL1-SS comprises administering a nucleotide encoding PDL1-TM and / or PDL1-SS. In some embodiments, administering PDL1-TM and / or PDL1-SS comprises administering an AAV encapsidating an expression cassette for expression of the PDL1-TM and / or PDL1-SS.

[0079] In some embodiments, PDL1-TM is administered. In some embodiments, the PDL1-TM comprises SEQ ID NO: 1. In some embodiments, the PDL1-TM 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: 1, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the PDL1-TM 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: 1. In some embodiments, PDL1-TM is administered. In some embodiments, the PDL1- TM comprises SEQ ID NO: 2. In some embodiments, the PDL1-TM 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: 2, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the PDL1-TM 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: 2.

[0080] In some embodiments, PDL1-SS is administered. In some embodiments, the PDL1-SS comprises SEQ ID NO: 3. In some embodiments, the PDL1-SS 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: 3, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the PDL1-SS 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: 3. In some embodiments, PDL1-SS is administered. In some embodiments, the PDL1-SS comprises SEQ ID NO: 4. In some embodiments, the PDL1-SS 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: 4, or a percentage identity that is in a range defined by any twoof the preceding values. For example, in some embodiments, the PDL1 -SS 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: 4.

[0081] Table 1 depicts some embodiments of PDL1-TM and PDL1-SS amino acid sequences suitable for use in some embodiments disclosed herein. In some embodiments, the PDL1-TM or PDL1-SS comprises a PDL1-TM or PDL1-SS sequence in table 1. In some embodiments In some embodiments, the PDL1-TM or PDL1-SS has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a PDL1- TM or PDL1-SS 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 PDL1-TM or PDL1-SS 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 a PDL1-TM or PDL1-SS in Table 1.Table 1

[0082] In some embodiments, one or more immune system modulators are administered before, during and / or after PDL1-TM and / or PDL1-SS administration.

[0083] In some embodiments, the one or more immune system modulators comprises CTLA4-Ig. In some embodiments, the immune system modulators comprise abatacept and belatacept, ipilimumab, tremelimumab, corticosteroids, biologies, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, and IMDH inhibitors. In some embodiments, one or more immune system modulators are administered to recipient before, during and / or after PDL1-TM and / or PDL1-SS administration. In some embodiments, the one or more immune system modulators comprises CTLA4-Ig. In some embodiments, the one or more immune system modulators comprises abatacept and / or belatacept. In some embodiments, CTLA4-Ig comprises belatacept or abatacept.

[0084] In some embodiments, administering PD-L1 variant comprises administering an expression cassette encoding or expressing the PD-L1 variant. In some embodiments, the expression cassette comprises one or more ITRs, enhancers, promoters, selfcleaving peptides, protein coding sequences, Kozak sequences, linkers, or any combination thereof.

[0085] 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: 5 or 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 ITRs comprise 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 or 6. 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: 5 or 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 ITRs 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: 5 or 6.

[0086] 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: 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 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: 7.

[0087] 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: 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 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: 8.

[0088] In some embodiments, the expression cassette comprises one or more Kozak sequences. In some embodiments, the one or more Kozak 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: 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 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: 9.

[0089] In some embodiments, the expression cassette comprises one more selfcleaving peptides coding sequence. In some embodiments, the one or more self-cleaving peptides coding sequence 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 self-cleaving peptides coding sequence 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 self-cleaving peptide coding 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: 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 self-cleaving peptide coding sequences have 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.

[0090] 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: 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 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: 12.

[0091] In some embodiments, the expression cassette comprises from left to right: a 5' ITR, an enhancer, a promoter, a PDL1 variant 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 PDL1 variant coding sequence 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: 5; 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: 7; 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:8; 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: 9; a PDL1-TM or PDL1-SS 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: 1 or SEQ ID NO: 3, respectively; 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: 6; or having a percentage identity that is defined by any 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: 5; 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: 7; 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: 8; 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: 9; a PDL1-TM or PDL1-SS 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: 1 or SEQ ID NO: 3, respectively; 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: 6.

[0092] FIG. 2 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. In some embodiments, an expression construct that is an expression construct in FIG. 2 is administered.

[0093] In some embodiments, the PDL1-TM and / or PDL1-SS administered to the donor article and the one or more immune system modulators administered to the recipient belong to the same species. In some embodiments, the PDL1-TM and / or PDL1-SS and the one or more immune system modulators belong to different species. In some embodiments, the PDL1-TM and / or PDL1-SS is human, mouse, pig, swine, NHP, chimp, or chicken, PDL1-TM and / or PDL1-SS. In some embodiments, the PDLl-TM and / or PDL1-SS has about 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to human, mouse, pig, swine, NHP, chimp, or chicken, PDL1-TM and / or PDL1-SS, 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 PDL1-TM and / or PDL1-SS 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, PDL1-TM and / or PDL1-SS. 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 human, mouse, pig, swine, NHP, chimp, or chicken, PDL1-TM and / or PDL1-SS, 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 PDL1-TM and / or PDL1-SS 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, PDL1-TM and / or PDL1-SS.

[0094] In some embodiments, administering PDL1-TM and / or PDL1-SS and one or more immune system modulators increases survival of the solid donor organ or article after transplant. In some embodiments, administering PDL1-TM and / or PDL1-SS and one or more immune system modulators increases survival of the solid donor organ or article 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 PDL1-TM and / or PDL1-SS and one or more immune system modulators increases survival of the solid donor organ or article 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 PDL1-TM and / or PDL1-SS and one or more immune system modulators increases survival of the solid donor organ or article by more than 24 months. In some embodiments, administering PDL1-TM and / or PDL1-SS and one or more immune system modulators increases survival of the solid donor organ or article 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 PDL1-TM and / or PDL1-SS and one or more immune system modulators increases survival of the solid donor organ or article between about l%-100%, l%-95%, l%-90%, l%-75%, 1%- 50%, l%-25%, l%-20%, l%-10%, l%-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%.

[0095] In some embodiments, administration of the PDL1-TM and / or PDL1-SS comprises administration of a modified AAV encapsidating an expression cassette for expression of the PDL1-TM and / or PDL1-SS and / or the one or more immune system modulators. In some embodiments, comprises an AAV serotype 9 vector comprising a capsid insertion having SEQ ID NO: 13, or an AAV serotype 9 vector comprising SEQ ID NO: 14.

[0096] In some embodiments, the construct may be introduced to the article by ex vivo perfusion. Ex vivo perfusion is also called normothermic perfusion and comprises a machine which keeps organs at body temperature by continuously pumping or perfusing blood or a bloodless solution of nutrients, proteins and oxygen, through them. Ex vivo perfusion may reduce ischemic injury time and allow for graft evaluation. In some embodiments, the construct described herein may be included in the solution being perfused through the article.

[0097] In some embodiments, the rAAV or construct is introduced to the transplantable article via transfection. In some embodiments, the method for transfecting a transplantable article with the rAAV or construct includes adding the rAAV or construct to a medium to produce a transplant medium, and incubating the transplantable article in the transplant medium. In some embodiments, incubating the transplantable article in the transplant medium produces a conditioned medium. In some embodiments, the method includes collecting the conditioned medium, and analyzing the conditioned medium for expression of PDLI protein derived from the PDL1-TM or PDL1-SS encoding sequence.

[0098] In some embodiments, the method further comprises analyzing the article or cells of the article for the presence or expression of the construct or the protein encoded by the construct in the article. Assessment of the construct or protein may be by any means known in the art. Results of the assessment may be used to inform dosage, function or effect of the construct or may be useful in determining the levels or requirements for additional immunosuppressive treatments if the article is used as a transplant organ.Pharmaceutical Compositions

[0099] Some aspects of the present disclosure relate to a pharmaceutical composition comprising a construct described herein and a pharmaceutically acceptablecarrier, diluent and / or excipient. As used herein, the term “carrier” refers to a pharmaceutically acceptable solid or liquid filler, diluent, or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and / or viscosityincreasing agents, tonicity agents, wetting agents, or other biocompatible materials. A tabulation of ingredients listed by the above categories, may be found in the U.S. Pharmacopeia National Formulary, 1857-1859, (1990); which is hereby expressly incorporated by reference in its entirety for all purposes.

[0100] Some non-limiting examples of the materials which may serve as pharmaceutically acceptable carriers are buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, preservatives, amino acids, salts, base buffers, detergents, and, optionally, and stabilizers, or any combination thereof, can also be present in the compositions, according to the desires of the formulator.

[0101] Non-limiting examples of pharmaceutically acceptable antioxidants include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol and the like; and metal-chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

[0102] In some embodiments, the compositions disclosed herein may also comprise other suitable agents such as, but not limited to, a stabilizing delivery vehicle, carrier, support or complex-forming species. The coordinate administration methods and combinatorial formulations of the present disclosure may optionally incorporate effective carriers, processing agents, or delivery vehicles, to provide improved formulations for delivery of the constructs or vectors described herein.

[0103] The compositions of the present disclosure may additionally include a biologically acceptable buffer to maintain a desired pH, including a pH of from 6.0-8.5,preferably from 7.5-8.5 and more preferable about 8.0. Such buffers preferably used are typically phosphates, carboxylates, and bicarbonates, amino acids, and tris. More preferred buffering agents are sodium phosphate, potassium phosphate, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, potassium bicarbonate, tris, histidine, proline, arginine. The buffer may comprise about 0.0001-5% (w / v) of the vaccine formulation, more preferably about 0.001-1% (w / v). Other excipients, if desired, may be included as part of the final pharmaceutical formulation.

[0104] Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservative.

[0105] In some embodiments, the compositions and methods provided herein may be utilized in xenotransplantation. Xenotransplantation, or heterologous transplant, is the transplantation, implantation or infusion of living cells, tissues, or organs from one species to another, for example from a nonhuman animal source to a human recipient. Such cells, tissues or organs are called xenografts or xenotransplants. Nonhuman organs, cells or tissue may be genetically modified through ex vivo viral, or non-viral transduction with vectors permitting the expression of PDL1 genes and variants as described herein. Such approach may suppress the immune mechanism response for organ rejection and allow for more successful xenotranspl antati on .Numbered Arrangements:

[0106] Some embodiments provided herein are described by way of the following provided numbered arrangements and also provided as possible combinations or overlapping embodiments:

[0107] 1 A method of increasing solid article transplant survival, the method comprising: obtaining a solid donor article or article, administering a PDL1 variant, to the solid donor article, wherein said PDL1 variant comprises a truncated (PDL1-TM) and / or secreted (PDL1-SS) PD-L1; and administering one or more immune system modulators to a solid donor article recipient.

[0108] 2 The method of arrangement 1, wherein the method is for preparing a subject for a transplant or for increasing solid article transplant survival.

[0109] 3 The method of any one of arrangements 1 or 2, wherein the solid donor article comprises a liver, pancreas, spleen, kidney, heart, or adrenal glands.

[0110] 4. The method of any one of arrangements 1-3, wherein administeringPDL1-TM and / or PDL1-SS comprises administering a nucleic acid encoding PDL1-TM and / or PDL1-SS.

[0111] 5 The method of any one of arrangements 1 to 4, wherein administering thePDL1 variant comprises administering an adeno-associated virus (AAV) encapsidating an expression cassette for expression of the PDL1-TM and / or PDL1-SS.

[0112] 6 The method of any one of the preceding arrangements, wherein administering the one or more immune system modulators comprises administering one or more immunosuppressant agents.

[0113] 7. The method of any one of arrangements 1 to 6, wherein administering the one or more immune system modulators comprises administering one or more anti-CTLA-4 antibodies.

[0114] 8 The method of any one of the preceding arrangements, wherein the one or more immune system modulators comprises CTLA-4 Ig.

[0115] 9 The method of arrangement 8, wherein the one or more immune system modulators comprise abatacept and / or belatacept.

[0116] 10. The method of arrangement 8, wherein the one or more immune system modulators comprise abatacept.

[0117] 11 The method of arrangement 8, wherein the one or more immune system modulators comprise belatacept.

[0118] 12. The method of any one of arrangements 5-11, wherein the AAV is a recombinant AAV (rAAV).

[0119] 13. The method of arrangements 12, wherein the rAAV vector is derived from an AAV serotype 9 vector.

[0120] 14. The method of any one of the preceding arrangements, wherein the nucleic acid encoding PDL1-TM comprises a sequence comprising or consisting of SEQ ID NO: 15 or at least 90% percent identity thereto.

[0121] 15. The method of any one of the preceding arrangements, wherein the nucleic acid encoding PDL1-SS comprises a sequence comprising or consisting of SEQ ID NO: 16 or at least 90% percent identity thereto.

[0122] 16. The method of any one of the preceding arrangements, wherein administering the PDL1-TM and / or PDL1-SS comprises administering an expression cassette for expression of PDL1-TM and / or PDL1-SS.

[0123] 17. The method of any one of the preceding arrangements, wherein the expression cassette comprises a nucleic acid sequence encoding one or more compounds including ITRs, enhancers, promoters, self-cleaving peptides, protein coding sequences, and / or Kozak sequences.

[0124] 18. The method of arrangement 17, wherein the expression cassette comprises a nucleic acid sequence encoding one or more ITRs.

[0125] 19. The method of arrangement 18, wherein the one or more ITRs a sequence comprising or consisting of SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0126] 20. The method of arrangement 17, wherein the expression cassette comprises a nucleic acid sequence encoding an enhancer.

[0127] 21. The method of arrangement 20, wherein the enhancer comprises a CMV enhancer.

[0128] 22. The method of arrangement 21, wherein the CMV enhancer comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 7.

[0129] 23. The method of arrangement 17, wherein the expression cassette comprises a nucleic acid sequence encoding a promoter.

[0130] 24. The method of arrangement 23, wherein the promoter comprises a CMV promoter.

[0131] 25. The method of arrangement 24, wherein the CMV promoter comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 8.

[0132] 26. The method of arrangement 16, wherein the PDL1-TM comprises a protein sequence comprising or consisting of SEQ ID NO: 1.

[0133] 27. The method of arrangement 16, wherein the PDL1-SS comprises a protein sequence comprising or consisting of SEQ ID NO: 2.

[0134] 28. The method of arrangement 17, wherein the expression cassette comprises a Kozak sequence.

[0135] 29. The method of arrangement 28, wherein the Kozak sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 9.

[0136] 30. The method of arrangement 17, wherein the expression cassette comprises a nucleic acid encoding, from 5’ to 3’: a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDL1-TM or PDL1-SS encoding sequence, a T2A self-cleaving peptide, a P2A self-cleaving peptide, and a 3’ ITR.

[0137] 31. The method of arrangement 30, wherein the 5’ ITR comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 5, the CMV enhancer comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 7, the CMV promoter comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 8, the Kozak sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 9, the PD-L1 encoding sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 15 or SEQ ID NO: 16, and the 3’ ITR comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

[0138] 32. The method of any one of the preceding arrangements, wherein administering PDL1-TM and / or PDL1-SS and one or more immune system modifiers increases survival of the solid donor article by up to about 500%.

[0139] 33. The method of any one of the preceding arrangements, wherein administering PDL1-TM and / or PDL1-SS and one or more immune system modifiers increases survival of the solid donor article by up to about 100 days following transplantation of the donor article.

[0140] 34. The method of any one of the preceding arrangements, wherein administering PDL1-TM or PDL1-SS and one or more immune system modifiers increases survival of the solid donor article by greater than 100 days following transplantation of the donor article.

[0141] 35. The method of any one of the preceding arrangements, wherein administering PDL1-TM and / or PDL1-SS and one or more immune system modifiers increases survival of the solid donor article by up to about 12 months.

[0142] 36. An expression cassette comprising from 5’ to 3’: a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDL1-TM and / or PDL1-SS encoding sequence, and a 3 ’ ITR.

[0143] 37. The expression cassette of arrangement 36, wherein the 5’ ITR comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 5, the CMV enhancer comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 7, the CMV promoter comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 8, the Kozak sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 9, the PD-L1 encoding sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 15 or SEQ ID NO: 16, and the 3’ ITR comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

[0144] 38. A recombinant adeno-associated virus (rAAV) comprising the expression cassette of any one of arrangements 36-37.

[0145] 39. Use of the rAAV of arrangement 38, in the manufacture of a medicament for increasing solid article transplant survival.

[0146] 40. A recombinant AAV vector comprising an expression cassette, wherein the expression cassette comprises a nucleic acid encoding, from 5’ to 3’ : a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDL1-TM or PDL1-SS encoding sequence, and a 3 ’ ITR.

[0147] 41. A method for transfecting a transplantable article with the rAAV of arrangement 40, the method comprising adding the rAAV to a medium to produce a transplant medium, and incubating the transplantable article in the transplant medium.

[0148] 42. The method of arrangement 41, wherein incubating the transplantable article in the transplant medium produces a conditioned medium.

[0149] 43. The method of arrangement 41, further comprising collecting the conditioned medium, and analyzing the conditioned medium for expression of PDLI protein derived from the PDL1-TM or PDL1-SS encoding sequence.

[0150] 44. The method of arrangement 43, further comprising transplanting the transplantable article into a subject.

[0151] 45. The method of arrangement 44, further comprising administering a therapeutically effective amount of one or more additional therapeutic agents to the subject.

[0152] 46. The method of arrangement 45, wherein the one or more additional therapeutic agent is administered prior to, during and / or after transplantation of the article to the subject.

[0153] 47. The transplantable article produced by the method of arrangement 41, wherein the transplantable article comprises one or more articles including heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, and adrenal glands.

[0154] 48. The transplantable article of arrangement 47, wherein the transplantable article comprises a rAAV, wherein the rAAV expresses PDLI protein derived from the PDL1- TM or PDL1-SS encoding sequence.

[0155] 49. The transplantable article of arrangement 47 or 48, wherein the article for transplantation comprises an article, a cell population, skin, or a tissue.

[0156] 50. The transplantable article of arrangement 49, comprising one or more articles including heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, and adrenal glands.

[0157] 51. The transplantable article of any one of arrangements 47-50, wherein the transplantable article is from a mammalian donor.

[0158] 52. The transplantable article of arrangement 51, wherein the mammalian donor is a human donor.

[0159] 53. The transplantable article of arrangement 51, wherein the mammalian donor is a pig donor.

[0160] 54. The method of arrangement 51, comprising ex vivo perfusion of the construct into the transplantable article prior to transplantation.

[0161] 55. The method of arrangement 54, wherein the transplantable article comprises a heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, adrenal glands, or a combination thereof.

[0162] 56. An ex vivo method for preventing or reducing rejection of a transplanted article in a subject, the method comprising introducing a construct into a transplantable article prior to transplantation into a subject, wherein the construct encodes a promotor operably connected to a nucleic acid sequence encoding a PDLI variant or extracellular portion of aPDL1 variant, wherein the PDL1 variant or extracellular portion of the PDL1 variant is capable of binding to its receptor.

[0163] 57. The method of arrangement 56, further comprising transplanting the article into a subject.

[0164] 58. The method of arrangement 56 or 57, wherein the article comprises article, cell population, skin, or a tissue.

[0165] 59. The method of arrangement 58, wherein the article comprises a heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, adrenal glands, or a combination thereof.EXAMPLES

[0166] There is strong unmet need to apply major advances in gene therapy to the field of transplantation to decrease transplant rejection, decrease use of immunosuppressants, and expand the donor pool. The development of successful gene therapy strategies relies upon four components: 1) a delivery approach, 2) a vector (usually viral based), 3) a well-defined disease or injury state and disease models, and 4) molecular targets appropriately therapeutic for the disease or indication. Previously, the lack of an efficient delivery approach for targeted delivery of transgenes to donor hearts was a barrier to gene therapy for cardiac transplantation. Recently an ex vivo warm blood perfusion system (TransMedics OCS™) has been FDA- approved to prolong donor heart transport times and decrease ischemic injury. This system can also be opportunistically used for viral vector delivery to genetically modify transplanted organs prior to implantation.

[0167] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the disclosure or of the appended claims.

[0168] To test the potential of vector mediated delivery of this approach, PDL1-SS expressing AAV vectors were generated and utilized to transduce mouse hearts in vivo in a heterotropic Heart Transplant Model.

[0169] FIG. 3 is an illustrative representation of some embodiments of a murine heterotopic heart transplant model.

[0170] Wild-type BALBc mice were injected with IxlO14vg / kg of vector and their heart was subsequently transplanted into a mismatched wild type B6 recipient in the abdominalposition two weeks post-injection. The graft was examined daily via abdominal palpation and echocardiographic assessment. The endpoint was defined as complete cessation of graft function as confirmed by both exam and echocardiography.

[0171] Heterotopic heart transplantation will be performed as follows: The donor mouse will be anesthetized and prepared. After sternotomy, the heart is explanted in sterile fashion. The heart is arrested with cold buffer delivered into the aortic root. The heart is then implanted in an HLA mismatched recipient. The ascending aorta of the donor is attached in an end-to-side fashion to the abdominal aorta of the recipient, and the donor pulmonary artery is attached in an end-to-side fashion to the recipient's inferior vena cava. The graft is de-aired. The abdomen is closed, and the recipient mouse is recovered.

[0172] Assessment of cardiac rejection will be primarily performed by three methods: 1) Daily physical exam. Graft function can be easily assessed with simple palpation on the animal's abdomen and graded on a 4-point standard International Society for Heart and Lung Transplantation scale; 2) Echocardiography weekly; 3) Subsets of animals (n=S per time point) will be euthanized for blood and tissue weekly. Peripheral blood mononuclear cells (PBMCs) will be isolated and stored for immune profding. Hearts will be harvested and processed for sectioning and H&E staining. Mice will be continuously examined for intimal hyperplasia or other signs of cardiac allograft occlusive vasculopathy / rejection.

[0173] PDL1 variant expression in donor heart tissue and serum was assessed after administration of AAV-SLB101-PDL1 variant vectors to donor mice.

[0174] FIG. 4 is a series of images (left) and a graph (right) depicting some embodiments of PDL1-TM and PDL1-SS expression on cardiomyocytes and in the serum of the donor animal after administration of AAV-SLB101-PDL1-TM and AAV-SB101-PDL1- SS vectors prior to organ transplant.

[0175] Robust PDL1 expression throughout the myocardium among membrane bound donors (IHC on left) and in the plasma of donors (ELISA on right) was validated at the two week timepoint after injection prior to transplantation. Surprisingly, significant quantities of PDL1 were detected in the serum of mice treated with PDL1-SS and PDL1-TM, which -TM were approximately 4- to 5-fold higher than previously reported PDL1 serum levels in PDL1- SS and PDL1-TM transgenic mice, suggesting that administration of PDL1-SS and / or PDL1- TM in an AAV-SLB101 vector may improve allograft survival over previously reportedimprovements in allograft survival following heterotopic heart transplant from a PDL1 -SS and PDL1-TM transgenic donor mouse.

[0176] FIG. 5 depicts co-stimulation blockade with PDL1 administration. 1X1014vg / kg of AAV-SLB101-PDL1 variant vector was administered to wild-type donor mice pre- operatively and their heart was subsequently transplanted into a mismatched wild type recipient in the abdominal position 2 weeks post-administration. Three doses of 250 pg / mouse of abatacept was administered postoperatively to recipient mice. Four groups of transplants were performed: 1) a control co-stimulation blockade-only group, 2) a co-stimulation blockade + sham vector group, 3) a PDL1-TM treated group, and 4) a PDL1-SS treated group.

[0177] FIG. 6 depicts graft survival in a heterotopic heart transplant model where the donor mouse was administered PDL1-TM or PDL1-SS via AAV-SLB101 prior to organ transplant and recipient mouse was administered abatacept by IP at PODO, POD2 and POD4. As can be seen in FIG. 6, a significant graft survival benefit was observed, particularly among those mice recipients of PDL1-TM containing allograft. Recipients of control grafts with just co-stimulation blockade along demonstrated median survival of 5 — 60 days with a final 100 day survival of 30-40%, while the PDL1-TM recipients demonstrated nearly 100% tolerance at 100 days. As discussed above with respect to FIG. 4, the PDL1-SS allograft recipients with abatacept co-stimulation demonstrated greater than 60% tolerance at 80 days, approximately a 2-fold increase in tolerance as compared to previously reported improvements in tolerance following heterotopic heart transplant from a PDL1-SS transgenic mouse. Additionally, PDL1- TM allograft recipients with abatacept co-stimulation demonstrated 100% tolerance at 80 days and nearly 90% tolerance at 100 days before being taken down.

[0178] Survival data was corroborated by functional assessment.

[0179] FIG. 7 is a pair of bar graphs depicting some embodiments of quantification of allograft function at 4 and 8 weeks after transplant of hearts treated with AAV-SLB101- PDL1 variant vectors and recipients with CTLA-4 Ig co-stimulation as well as controls administered either CTLA-4 Ig alone and sham (empty) vector with CTLA-4 Ig costimulation. Allograft function was scored on a scale of 1-4. A score of 1 indicates minimal single-wall function such as septal or RV wall. A score of 2 indicates moderate single wall function or weak / partial LV function, very weak global function. A score of 3 indicates moderate globalfunction including all LV and RV. A score of 4 indicates strong / robust global LV and RV function.

[0180] As can be seen in FIG. 7, at 1 month, all groups’ grafts functioned well as expected after administration of co-stimulation blockade with a score of 3-4 / 4. However, by 2 months, 4 grafts each in both control groups had failed completely. Among those that were still alive, there was still a significant difference in graft function between the PDL1 -containing grafts and control grafts.

[0181] Echocardiograms indicate minimal motion (single-wall at best) in the control mice two-months post-transplant while experimental transplanted grafts still demonstrated robust contractility and function.

[0182] FIG. 8 shows some embodiments of the histology of allograft heart tissue treated with AAV-SLB101-PDL1 variants and recipients with CTLA-4 Ig co-stimulation, as well as control. As can be seen in FIG. 8, histology confirmed a stark difference in rejection. On the left, these representative sections demonstrate classic 3R ISHLT rejection with diffuse lymphocytic infiltration, edema, hemorrhage, and a high degree of myocardial necrosis. In contrast, the experimental mice demonstrated significantly improved myocardial viability, integrity, and a more localized foci of lymphocytic infiltration without edema, hemorrhage, or necrosis.

[0183] FIG. 9 shows some embodiments of CD3, and CD20 infiltration of allograft heart tissue treated with AAV-SLB101-PDL1 variants vectors and recipients with CTLA-4 Ig co-stimulation, as well as control. As can be seen in FIG. 9, subclassification of the immune cell profile demonstrates a T cell predominance among control groups in contrast to a reduced T cell predominance in experimental groups and a higher B cell composition.

[0184] FIG. 10-11 shows some embodiments of the histology of allograft heart tissue treated with AAV-SLB101-PDL1 variants vectors and recipients with CTLA-4 Ig costimulation, as well as control. As can be seen in FIG. 10, the foci of lymphocytic infiltration in experimental groups were consistently endocardial in location unlike control groups. As can be seen in FIG. 11, the endocardial lymphocytic aggregates demonstrates co-existence of both T and B cell populations which is uncharacteristic of acute cellular rejection. In the clinical setting, this can represent a Quilty lesion, a benign foci of lymphocytes seen in heart transplant patients that is typically not treated.

[0185] As seen in the results disclosed herein, some of the PDL1-TM and PDL1 - SS treated transplanted (including AAV-SLB101-PDL1 variants vector treated) hearts survived over 80 days before being sacrificed to assess cardiac tissue. These experiments demonstrate the potential for PDL1-TM and PDL1-SS to prevent solid organ rejection in recipients.

[0186] 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.

[0187] 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 in detail, 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.

[0188] It should be understood, however, that this detailed description, while indicating preferred embodiments, is given by way of illustration only, since various changesand modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art.

[0189] 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 TS:

1. A method comprising: obtaining a solid donor article or article, administering a PDL1 variant, to the solid donor article, wherein said PDL1 variant comprises a truncated (PDL1-TM) and / or secreted (PDL1-SS) PD-L1; and administering one or more immune system modulators to a solid donor article recipient.

2. The method of claim 1, wherein the method is for preparing a subject for a transplant, or for increasing solid article transplant survival.

3. The method of claim 1, wherein the solid donor article comprises a liver, pancreas, spleen, kidney, heart, or adrenal glands.

4. The method of claim 1, wherein administering PDL1-TM and / or PDL1-SS comprises administering a nucleic acid encoding PDL1-TM and / or PDL1-SS.

5. The method of claim 1, wherein administering the PDL1 variant comprises administering an adeno-associated virus (AAV) encapsidating an expression cassette for expression of the PDL1-TM and / or PDL1-SS.

6. The method of claim 1, wherein administering the one or more immune system modulators comprises administering one or more immunosuppressant agents.

7. The method of claim 1, wherein administering the one or more immune system modulators comprises administering one or more T-cell co-stimulatory molecules.

8. The method of claim 1, wherein the one or more immune system modulators comprises CTLA-4 Ig.

9. The method of claim 1, wherein the one or more immune system modulators comprise abatacept and / or belatacept.

10. The method of claim 9, wherein the one or more immune system modulators comprise abatacept.

11. The method of claim 9, wherein the one or more immune system modulators comprise belatacept.

12. The method of claim 5, wherein the AAV is a recombinant AAV (rAAV).

13. The method of claims 12, wherein the rAAV vector is derived from an AAV serotype 9 vector.

14. The method of claim 1, wherein the nucleic acid encoding PDL1 -TM comprises a sequence comprising or consisting of SEQ ID NO: 15 or at least 90% percent identity thereto.

15. The method of claim 1, wherein the nucleic acid encoding PDL1-SS comprises a sequence comprising or consisting of SEQ ID NO: 16 or at least 90% percent identity thereto.

16. The method of claim 1, wherein administering the PDL1-TM and / or PDL1-SS comprises administering an expression cassette for expression of PDL1-TM and / or PDL1-SS.

17. The method of claim 1, wherein the expression cassette comprises a nucleic acid sequence encoding one or more compounds including ITRs, enhancers, promoters, selfcleaving peptides, protein coding sequences, and / or Kozak sequences.

18. The method of claim 17, wherein the expression cassette comprises a nucleic acid sequence encoding one or more ITRs.

19. The method of claim 18, wherein the one or more ITRs a sequence comprising or consisting of SEQ ID NO: 5 and / or SEQ ID NO: 6.

20. The method of claim 17, wherein the expression cassette comprises a nucleic acid sequence encoding an enhancer.

21. The method of claim 20, wherein the enhancer comprises a CMV enhancer.

22. The method of claim 21, wherein the CMV enhancer comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 7.

23. The method of claim 17, wherein the expression cassette comprises a nucleic acid sequence encoding a promoter.

24. The method of claim 23, wherein the promoter comprises a CMV promoter.

25. The method of claim 24, wherein the CMV promoter comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 8.

26. The method of claim 16, wherein the PDL1-TM comprises a protein sequence comprising or consisting of SEQ ID NO: 1.

27. The method of claim 16, wherein the PDL1-SS comprises a protein sequence comprising or consisting of SEQ ID NO: 2.

28. The method of claim 17, wherein the expression cassette comprises a Kozak sequence.

29. The method of claim 28, wherein the Kozak sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 9.

30. The method of claim 17, wherein the expression cassette comprises a nucleic acid encoding, from 5’ to 3’ : a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDL1-TM or PDL1-SS encoding sequence, a T2A self-cleaving peptide, a P2A self-cleaving peptide, and a 3’ ITR.

31. The method of claim 30, wherein the 5’ ITR comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 5, the CMV enhancer comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 7, the CMV promoter comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 8, the Kozak sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 9, the PD-L1 encoding sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 15 or SEQ ID NO: 16, and the 3’ ITR comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

32. The method of claim 1, wherein administering PDL1-TM and / or PDL1-SS and one or more immune system modifiers increases survival of the solid donor article by up to about 500%.

33. The method of claim 1, wherein administering PDL1-TM and / or PDL1-SS and one or more immune system modifiers increases survival of the solid donor article by up to about 100 days following transplantation of the donor article.

34. The method of claim 1, wherein administering PDL1-TM or PDL1-SS and one or more immune system modifiers increases survival of the solid donor article by greater than 100 days following transplantation of the donor article.

35. The method of claim 1, wherein administering PDL1-TM and / or PDL1-SS and one or more immune system modifiers increases survival of the solid donor article by up to about 12 months.

36. An expression cassette comprising from 5’ to 3’ : a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDLl-TM and / or PDL1-SS encoding sequence, and a 3’ ITR.

37. The expression cassette of claim 36, wherein the 5’ ITR comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 5, the CMV enhancer comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 7, the CMV promoter comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 8, the Kozak sequencecomprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 9, the PD-L1 encoding sequence comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 15 or SEQ ID NO: 16, and the 3’ ITR comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

38. A recombinant adeno-associated virus (rAAV) comprising the expression cassette of claim 36.

39. Use of the rAAV of claim 38, in the manufacture of a medicament for increasing solid article transplant survival.

40. A recombinant AAV vector comprising an expression cassette, wherein the expression cassette comprises a nucleic acid encoding, from 5’ to 3’ : a 5’ ITR, a CMV enhancer, a CMV promoter, a Kozak sequence, a PDL1-TM or PDL1-SS encoding sequence, and a 3 ’ ITR.

41. A method for transfecting a transplantable article with the rAAV of claim 40, the method comprising adding the rAAV to a medium to produce a transplant medium, and incubating the transplantable article in the transplant medium.

42. The method of claim 41, wherein incubating the transplantable article in the transplant medium produces a conditioned medium.

43. The method of claim 41, further comprising collecting the conditioned medium, and analyzing the conditioned medium for expression of PDLI protein derived from the PDL1- TM or PDL1-SS encoding sequence.

44. The method of claim 43, further comprising transplanting the transplantable article into a subj ect.

45. The method of claim 44, further comprising administering a therapeutically effective amount of one or more additional therapeutic agents to the subject.

46. The method of claim 45, wherein the one or more additional therapeutic agent is administered prior to, during and / or after transplantation of the article to the subject.

47. The transplantable article produced by the method of claim 41, wherein the transplantable article comprises one or more articles including heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, and adrenal glands.

48. The transpl antable article of claim 47, wherein the transplantable article comprises a rAAV, wherein the rAAV expresses PDLI protein derived from the PDL1-TM or PDL1-SS encoding sequence.

49. The transplantable article of claim 47, wherein the article for transplantation comprises an article, a cell population, skin, or a tissue.

50. The transplantable article of claim 49, comprising one or more articles including heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, and adrenal glands.

51. The transplantable article of claim 47, wherein the transpl antable article is from a mammalian donor.

52. The transplantable article of claim 51, wherein the mammalian donor is a human donor.

53. The transplantable article of claim 51, wherein the mammalian donor is a pig donor.

54. The method of claim 51, comprising ex vivo perfusion of the construct into the transplantable article prior to transplantation.

55. The method of claim 54, wherein the transplantable article comprises a heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, adrenal glands, or a combination thereof.

56. An ex vivo method for preventing or reducing rejection of a transplanted article in a subject, the method comprising introducing a construct into a transplantable article prior to transplantation into a subject, wherein the construct encodes a promotor operably connected to a nucleic acid sequence encoding a PDLI variant or extracellular portion of a PDLI variant, wherein the PDLI variant or extracellular portion of the PDLI variant is capable of binding to its receptor.

57. The method of claim 56, further comprising transplanting the article into a subject.

58. The method of claim 56, wherein the article comprises article, cell population, skin, or a tissue.

59. The method of claim 58, wherein the article comprises a heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testes, bladder, uterus, adrenal glands, or a combination thereof.

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