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US-20260125653-A1 - MODIFIED PORCINE CELLS AND METHODS OF USING THE SAME

US20260125653A1US 20260125653 A1US20260125653 A1US 20260125653A1US-20260125653-A1

Abstract

This disclosure provides compositions and methods for reducing hyperacute rejection in xenotransplantation using porcine cells including a sequence encoding human ST8Sia6. The disclosure further provides methods of producing porcine cells and porcine tissues encoding human ST8Sia6. The disclosure further provides methods of treatment using said porcine cells and porcine tissues.

Inventors

  • Jon D. Piganelli
  • Saptarshi Roy

Assignees

  • THE TRUSTEES OF INDIANA UNIVERSITY

Dates

Publication Date
20260507
Application Date
20251104

Claims (20)

  1. 1 . A porcine cell comprising a heterologous polynucleotide comprising a sequence encoding human ST8Sia6.
  2. 2 . The porcine cell of claim 1 , wherein the porcine cell is an endothelial cell, a hepatocyte, or a kidney cell.
  3. 3 . The porcine cell of claim 2 , wherein the endothelial cell is an aortic endothelial cell.
  4. 4 . The porcine cell of claim 1 , wherein the human ST8Sia6 comprises (SEQ ID NO: 1), or a sequence with at least 85% identity to SEQ ID NO: 1.
  5. 5 . The porcine cell of claim 1 , wherein the sequence encoding human ST8Sia6 is operably linked to a regulatory element.
  6. 6 . The porcine cell of claim 5 , wherein the regulatory element comprises a promoter or an enhancer.
  7. 7 . A method of generating the porcine cell of claim 1 , the method comprising introducing a heterologous polynucleotide comprising a sequence encoding human ST8Sia6 into a porcine cell.
  8. 8 . A method of generating a porcine tissue composition, the method comprising culturing the porcine cell of claim 1 to generate a porcine tissue composition.
  9. 9 . The method of claim 8 , wherein culturing the porcine cell is performed in a container comprising at least one substrate to promote cell attachment.
  10. 10 . The method of claim 9 , wherein the at least one substrate comprises gelatin.
  11. 11 . A method of treating a subject in need of an organ transplant, the method comprising administering the porcine tissue composition of claim 8 to the subject.
  12. 12 . The method of claim 11 , wherein the methods reduce immune activation by the porcine tissue composition and/or reduces effector CTL killing function in response to the porcine tissue composition, as compared to a porcine tissue composition not comprising the sequence encoding human ST8Sia6.
  13. 13 . The method of claim 11 , wherein the method reduces secretion of IFN-gamma, porphyrin, CD107a, and/or granzyme-B by T cells in response to the porcine tissue composition as compared to a porcine tissue composition not comprising the sequence encoding human ST8Sia6.
  14. 14 . The method of claim 11 , wherein the method reduces the severity or incidence of xenograft rejection compared to a method comprising transplanting a porcine tissue composition not comprising the sequence encoding human ST8Sia6.
  15. 15 . The method of claim 11 , wherein the method is used to mitigate instant blood mediated inflammatory reaction in the subject.
  16. 16 . The method of claim 11 , wherein the subject is in need of a solid organ transplant.
  17. 17 . The method of claim 16 , wherein the subject is in need of a heart transplant or a kidney transplant.
  18. 18 . A pharmaceutical composition comprising the porcine tissue composition of claim 7 and at least one pharmaceutically acceptable carrier.
  19. 19 . A genetically modified pig comprising porcine cells comprising a heterologous polynucleotide comprising a sequence encoding human ST8Sia6.
  20. 20 . A method comprising obtaining a tissue from the genetically modified pig of claim 19 and transplanting the tissue into a subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority benefit from U.S. Application No. 63/716,017, filed Nov. 4, 2024, the entirety of which is incorporated herein by reference. SEQUENCE LISTING A sequence listing (file name: “144578_00469.xml”; size: 2,108 bytes, date generated: Nov. 3, 2025) is hereby incorporated by reference in its entirety. BACKGROUND There exists a need to increase the availability of organs for individuals in need of an organ transplant. Xenogeneic species such as pigs are being considered as potential tissue donors. However, there is an unmet need to address immunological hurdles to xenogeneic organ transplant. SUMMARY In an aspect of this disclosure, porcine cells are provided. In some embodiments, the porcine cells comprise a heterologous polynucleotide comprising a sequence encoding human ST8Sia6. In an aspect of this disclosure, methods of generating a porcine cell are provided. In some embodiments, the methods comprise introducing a heterologous polynucleotide comprising a sequence encoding human ST8Sia6 into a porcine cell. In an aspect of this disclosure, methods of generating a porcine tissue composition are provided. In some embodiments, the methods comprise culturing a porcine cell comprising a heterologous polynucleotide comprising a sequence encoding human ST8Sia6 to generate a porcine tissue composition. In an aspect of this disclosure, methods of treating a subject in need of an organ transplant are provided. In some embodiments, the methods comprise administering a porcine tissue composition comprising porcine cells comprising a heterologous polynucleotide comprising a sequence encoding human ST8Sia6 to the subject. In an aspect of this disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a porcine tissue composition comprising porcine cells comprising a heterologous polynucleotide comprising a sequence encoding human ST8Sia6 and at least one pharmaceutically acceptable carrier. In an aspect of this disclosure, genetically modified pigs are provided. In some embodiments, the genetically modified pigs comprise porcine cells comprising a heterologous polynucleotide comprising a sequence encoding human ST8Sia6. In an aspect of this disclosure, methods are provided. In some embodiments, the methods comprise obtaining a tissue from a genetically modified pig comprising porcine cells comprising a heterologous polynucleotide comprising a sequence encoding human ST8Sia6 and transplanting the tissue into a subject. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIGS. 1A-1F. (FIG. 1A) GFP fluorescent images at different time points after overexpression of ST8Sia6 in B16-F10 cell line. (FIG. 1B, FIG. 1C) Flowcytometric analysis of Peanut Agglutinin (PNA) lectin positive cells number and MFI of ST8Sia6 plasmid or Control GFP transfect B16-F10 cells. (FIG. 1D, FIG. 1E) Flow cytometry gating strategy for analysis of ST8Sia6 transfection efficiency in B16-F10 and B16-Ova cells. (FIG. 1F) Flow cytometry gating strategy to assess the Siglec-E binding efficiency of ST8Sia6 transfected B16-F10 and B16-Ova cells. Significance analyzed by two-tailed unpaired t test between groups, significant different (P<0.05). FIGS. 2A-2F show overexpression of ST8Sia6 in B16-F10 and B16-Ova cell lines, and estimating ligands generation for Siglec-E binding. (FIG. 2A) GFP fluorescent images showed transfection efficiency of ST8Sia6 in B16-F10 and B16-Ova cell line. (FIG. 2B) Assessment of transfection efficiency using flow cytometry in B16-F10 cells. (FIG. 2C) Lysates from B16-F10 and B16-Ova transfected cells were examined for expression of ST8Sia6 by Western blotting. The molecular weight of ST8Sia6 is 43 kDa. (FIG. 2D) Peanut Agglutinin (PNA) lectin binding assay in ST8Sia6 plasmid or Control GFP transfect B16-F10 cell line. (FIG. 2E, FIG. 2F) B16-F10 and B16-Ova murine cancer cell lines were stably transfected with ST8Sia6 plasmid or Control GFP and were probed for ligands with recombinant Siglec-E, expression was measured by flowcytometry using anti-Siglec-E antibody. Siglec-E ligand positive cells percentage, numbers and geometric mean fluorescence intensity (MFI) was quantified across three (n—3) independent experiments, and significance analyzed by two-tailed unpaired t test between groups, significant different (P<0.05). FIGS. 3A-3J show overexpression of ST8Sia6 in B16-F10 and B16-Ova cells suppress OT-1 CD8+ T cells mediated cytotoxic effects. In these experimental conditions, B16-F10 cells transfected with a control GFP plasmid (grey curve) and those transfected with ST8Sia6 plasmid (red curve) were cocultured with OT-1 CD8+ T cells, which are specific effector cells, at various effector to