TISSUE FOR USE AS ALLOGENEIC OR XENOGENEIC TRANSPLANT AND METHOD FOR ITS PRODUCTION

Abstract

Tissue for use as a transplant, which tissue is allogeneic or xenogeneic and respectively the tissue may express an MHC I molecule that is immunologically incompatible to the transplant recipient and/or may express an MHC II molecule immunologically incompatible to the transplant recipient. The tissue suitable for use as a transplant and the method for its production include a genetic alteration of the tissue that provides for immunologic compatibility of the tissue with a transplant recipient. In the tissue for use as a transplant, which tissue expresses allogeneic or xenogeneic MHC I and/or allogeneic or xenogeneic MHC II molecules, the expression of the allogeneic or xenogeneic MHC I is downregulated by at least 50% to up to 90%, preferably the expression of the allogeneic or xenogeneic MHC I is downregulated by at least 60%.

Claims

1. Tissue for use as a transplant, which tissue expresses allogeneic or xenogeneic MHC I and/or allogeneic or xenogeneic MHC II molecules, wherein the expression of the allogeneic or xenogeneic MHC I is downregulated by at least 50% to up to 90%, wherein the expression of the allogeneic or xenogeneic MHC II is downregulated by at least 50% to up to 90%, wherein the downregulation of the allogeneic or xenogeneic MHC I and/or of the allogeneic or xenogeneic MHC II is in relation to its expression in wild-type tissue, and wherein the tissue additionally expresses at least one complement inhibitory molecule on the tissue.

2. Tissue for use as a transplant according to claim 1, characterized in that the expression of the allogeneic or xenogeneic MHC I is downregulated by 80% up to 90%, and wherein the expression of the allogeneic or xenogeneic MHC II is downregulated by at least 80% to up to 90%.

3. Tissue according to claim 1, wherein the expression of the allogeneic or xenogeneic MHC I and/or the expression of the allogeneic or xenogeneic MHC II is downregulated by the tissue being genetically manipulated to contain at least one nucleic acid construct comprising at least one expression cassette encoding inhibitory RNA interfering with at least one transcript encoding the allogeneic or xenogeneic MHC I molecule and/or encoding the allogeneic or xenogeneic MHC II molecule.

4. Tissue according to claim 1, wherein the expression of the allogeneic or xenogeneic MHC I is downregulated by the tissue being genetically manipulated to contain a nucleic acid construct comprising at least one expression cassette encoding an siRNA which hybridizes to the transcript of the 02-microglobuline gene (β2m), and/or encoding an siRNA which hybridizes to the transcript of at least one, or two, preferably for transcripts of all three heavy chains of HLA-A, HLA-B and HLA-C molecules or SLA-1, SLA-2 and SLA-3 molecules.

5. Tissue according to claim 1, wherein the expression of the allogeneic or xenogeneic MHC II is downregulated by the tissue being genetically manipulated to contain a nucleic acid construct comprising at least one expression cassette encoding an siRNA which hybridizes to transcripts of at least one of the alpha chain and/or beta chain of HLA-DR, HLA-DQ and/or of HLA-DP or SLA-DR and/or SLA-DQ, and/or by encoding an siRNA which is specific for transcripts of the class II transactivator gene.

6. Tissue according to claim 1, wherein complement inhibitory molecule is selected from CD46, CD55 and CD59.

7. Tissue according to claim 1, comprising use for or excluding or reducing immunosuppressants for permanent use in the treatment of HvG disease.

8. Tissue according to claim 1, wherein tissue is a portion of an organ or of an extremity or a complete organ or extremity.

9. Tissue according to claim 1, wherein the expression of the allogeneic or xenogeneic MHC I is downregulated by 80% up to 90% in relation to the expression level of MHC I in the wild-type tissue, and wherein the expression of the allogeneic or xenogeneic MHC II is downregulated by at least 80% to up to 90% in relation to the expression level of MHC I in the wild-type tissue.

10. Tissue according to claim 1, wherein the expression of the allogeneic or xenogeneic MHC I is downregulated by 80% up to 90% in relation to the expression level of MHC I in the recipient of the transplant, and wherein the expression of the allogeneic or xenogeneic MHC II is downregulated by at least 80% to up to 90% in relation to the expression level of MHC I in the recipient of the transplant.

11. Tissue according to claim 1, wherein the tissue consists of or comprises single cells, e.g. islet cells, a portion of an organ or a complete organ, which organ is e.g. a kidney, liver, heart, lung, pancreas, skin, or an extremity.

12. Tissue according to claim 1, being devoid of expression of an additional MHC I molecule and/or MHC II molecule.

13. Tissue according to claim 1, being devoid of expression of HLA-G.

14. Non-human mammalian animal which is genetically manipulated to comprise tissue according to claim 1.

15. Non-human animal according to claim 14, wherein the animal is genetically manipulated to contain, in its germline or by somatic genetic manipulation, nucleic acid constructs which encode inhibitory RNA under the control of a conditional promoter system which can be regulated in a dose-dependent manner by the presence of a synthetic compound, wherein inhibitory RNA under the control of the conditional promoter is arranged to downregulate expression of the MHC I by at least 50% to up to 90%, wherein inhibitory RNA under the control of the conditional promoter is arranged to downregulate expression of MHC II by at least 50% to up to 90%, wherein the downregulation of the MHC I and/or of the MHC II is in relation to its expression in wild-type tissue, and wherein the nucleic acid construct, optionally under the control of the conditional promoter, additionally expresses at least one complement inhibitory molecule.

16. Non-human animal according to claim 15, being devoid of expression of HLA-G.

17. Process for producing a tissue expressing an MHC I and an MHC II for use as a transplant, comprising providing an isolated tissue with a nucleic acid construct that comprises at least one expression cassette encoding at least one siRNA set up to downregulate the expression of the MHC I by at least 50% to up to 90% in relation to its expression in wild-type tissue, and comprises at least one expression cassette encoding at least one siRNA set up to downregulate the expression of the MHC II by at least 50% to up to 90% in relation to its expression in wild-type tissue, and comprising providing the isolated tissue with a nucleic acid construct containing an expression cassette encoding at least one complement inhibitory molecule.

18. Process according to claim 17, wherein the MHC I and MHC II are allogeneic or xenogeneic for a recipient of the transplant.

19. Process according to claim 1, wherein no genetic manipulation of the isolated tissue is performed that would result in expression of HLA-G.

20. Method of treatment of a patient by transplanting a tissue of claim 1 into the patient.

21. Nucleic acid construct for use in the treatment of HvG disease, the nucleic acid construct comprising at least one expression cassette encoding an siRNA set up to downregulate expression of an MHC I of a tissue by at least 50% to up to 90%, at least one expression cassette encoding an siRNA set up to downregulate expression of an MHC II of a tissue by at least 50% to up to 90%, and expression cassettes encoding at least one complement inhibitory molecule.

22. Nucleic acid construct according to claim 21, comprising at least two nucleic acid molecules.

23. Nucleic acid construct according to claim 21, having one nucleic acid molecule.

Description

[0044] The invention is now described by way of examples with reference to the figures, which show in

[0045] FIG. 1 expression levels of CD55 and of CD59 in cells that were lentivirally transduced,

[0046] FIG. 2 PCR analytical results for integration of expression cassettes in tissue cells,

[0047] FIG. 3 transcript levels of f32m and of MHC II in tissue cells expressing inhibitory RNA,

[0048] FIG. 4 FACS results detecting Treg cells expanded in presence of cells with downregulated expression of MHC I and MHC II in comparison to MHC II knockout cells,

[0049] FIG. 5 the percentage of lysed cells of tissue according to the invention which contain expression cassettes for different complement inhibitory molecules.

EXAMPLE 1

Genetic Manipulation of Xenogeneic Pig Islet Cells for use as Transplant

[0050] As an example for a tissue expressing xenogeneic MHC I and MHC II molecules, pig islet cells were used. For genetic manipulation of these xenogeneic cells a lentiviral vector backbone was used, which contained an expression cassette encoding according to the invention an siRNA (SEQ ID NO: 6) specific for the transcript of the Sus scrofa J32-microglobulin gene (SEQ ID NO: 30) and/or an expression cassette encoding an siRNA (SEQ ID NO: 8) specific for the transcript of the class II transactivator gene (CIITA) (SEQ ID NO: 31), preferably both expression cassettes. The inhibitory RNAs were designed and sufficient for a downregulation of MHC class I and MHC class II by 80% but not more than 90%. Following transplantation into mice as transplant recipients it was found that no cellular immunogenic response was elicited in the recipient, and no T-cell-mediated graft rejection occurred.

[0051] It was found that antibodies directed against the xenogeneic MHC I and MHC II molecules can occur, but the formation of anti-MHC I xeno-antibodies and of anti-MHC II xeno-antibodies would not result in antibody-mediated graft rejection when the suppression, i.e. downregulation, of the xenogeneic MHC I and of the xenogeneic MHC II in the tissue was by at least 80% compared to expression of the xenogeneic MHC I and MHC II, respectively, determined for wild-type pig islet cells, e.g. determined for cells treated in parallel but without genetic manipulation. For improved survival of the tissue, it was transduced additionally by a lentiviral vector which contained expression cassettes encoding complement inhibitors CD55 (SEQ ID NO: 17) and CD59 (SEQ ID NO: 19). It was found that a less intense downregulation of expression of the xenogeneic MHC I and MHC II does not result in antibody-mediated graft rejection when the xenogeneic cells expressed higher levels of complement inhibitors.

EXAMPLE 2

Genetic Manipulation of Allogeneic Kidney for use as Transplant

[0052] As an example for a tissue expressing allogeneic MHC I and MHC II molecules, a rat kidney that was genetically manipulated according to the invention was used as an allogeneic transplant in a recipient rat.

[0053] For genetic manipulation of the exemplary tissue, a nucleic acid construct was made which contained expression cassettes for a shRNA encoding SEQ ID NO: 9, which according to the invention is specifically targeting the rat 02-microglobulin mRNA (SEQ ID NO: 26), and an shRNA encoding SEQ ID NO: 10, according to the invention specifically targeting the rat MHC class II transactivator (CIITA) (SEQ ID NO: 27), as well as the coding sequences for the complement inhibitors CD55 (SEQ ID NO: 18) and CD59 (SEQ ID NO: 20). The shRNA targeting the 02-microglobulin mRNA causes silencing of MHC class I in a specific and selective manner without exceeding a level of suppression above 80% compared to untreated, i.e. wild-type, cells and the shRNA targeting the MHC class II transactivator (CIITA) causes silencing of MHC class II in a specific and selective manner without exceeding a level of suppression of 70% compared to untreated cells. The nucleic acid construct further contained an expression cassette for Luciferase, suitable for labelling cells in which the nucleic acid construct is integrated.

[0054] This nucleic acid construct in addition to shRNA targeting MHC transcripts optionally contained the coding sequences for CD55 and CD59. The nucleic acid constructs with or without the additional coding sequences for CD55 and CD59 were packaged in lentiviral particles using packaging cells, were concentrated by centrifugation at 30 000×g for 8 h and resuspended in the preservation solution. The viral particle titer was determined using p24 ELISA and the lentiviral vector particles were stored was at −80° C.

[0055] As an example for an allogeneic isolated vascularized tissue, the explanted rat kidneys of LEW1U.RT1 rats were flushed with preservation solution (composed of Custodiol) containing 80 IE heparin at a temperature of 4° C. immediately after explantation. The flushed rat kidneys were connected to an ex vivo perfusion system, warmed up to 37° C. and perfused for 2-3 h with the vector encoding the shRNA sequences directed against MHC I and MHC II, or with the vector encoding both the shRNA sequences directed against MHC I and MHC II and expression cassettes encoding CD55 and CD59 sequences, using Custodiol supplemented with 8 μg/mL Protamin sulphate and 4mg Urbason. 10.sup.9-10.sup.10 lentiviral particles were used per kidney. During perfusion the temperature was maintained at 37° C. and a flow rate of 10-14 mL/min was used. After perfusion with the vector, the perfusion solution was replaced with Custodiol containing 3-6mL of heparinized peripheral rat blood and the kidneys were perfused for 20-30 min to remove the free residual vector. Afterwards, the kidneys were washed with fresh Custodiol solution and cooled down to 4° C.

[0056] For analyses, kidney tissue was taken from different regions and digested with collagenase VI and dispase (both obtained from Sigma). At 96 h after the addition of the lentiviral vector to the second perfusion solution, endothelial cells of the kidney macrovasculature and micro-vasculature after labelling with anti-CD31 antibody were isolated using paramagnetic beads (Dyna-beads, obtained from Thermo Fisher) for magnetic separation. In parallel, endothelial cells were isolated from a non-transduced kidney which was treated in the same way, except that no viral particle was added. Analyses showed that endothelial cells were isolated at purities above 70%. Endothelial cells were cultivated in gelatine-coated tissue culture plates. Five days after perfusion with the lentiviral vector the MHC silencing effect and the levels of expression of CD55 and CD59 were evaluated by flow cytometric analyses.

[0057] At 24 h after addition of the viral particles to the second perfusion solution, luciferase activity was detected in the culture supernatant. The levels of this bioluminescence increased during the 96 h of cultivation. Remarkably, cells from the kidneys showed to be transduced, indicating integration and activity of the nucleic acid construct in the entire organ endothelium. Luciferase activity was measured after addition of luciferase substrate (Furimazine, Promega).

[0058] FIG. 1 depicts the expression levels of CD55 and CD59 in rat kidney endothelial cells isolated from two different regions designated R1 and R2 of the kidney perfused with the lentiviral vector encoding for CD55 and CD59 in comparison to the native levels expressed in a kidney perfused without vector (non-transduced). The result shows that lentiviral particles provided for stable and permanent integration the nucleic acid construct including the expression cassettes into the genome of the vascularized tissue.

[0059] FIG. 2 depicts the results of real-time PCR using DNA that was isolated from the cultivated rat endothelial cells and primers that were specific for the woodchuck hepatitis virus posttranscriptional element WPRE, which is present in the nucleic acid construct. The result shows the high levels of WPRE, respectively of the nucleic acid construct according to the invention, in the endothelial cells treated by the method of the invention, and background levels only in the endothelial cells from the kidneys that were treated in parallel but without addition of the lentiviral vector

[0060] Isolated cells were cultured in EBM-2 endothelial cell growth medium (obtained from Lonza). Endothelial cells were stimulated with IFN-γ (100ng/mL) for 48 h. It was found that endothelial cells from the microvasculature of the non-transduced parallel treatment were able to up-regulate MHC class I (RT1-A) and MHC class II (RT1-B) transcript levels (p<0.001) upon exposure to IFN-65 , but microvascular endothelial cells from kidneys treated with the nucleic acid construct according to the invention, contained in the lentiviral particle, upon IFN-γ stimulation did not show a significant increase in transcript levels for (32-microglobulin and RT1-B. These results demonstrate that the method of the invention effectively silences expression of MHC class I and of MHC class II in the vascularized tissue and leads to an expression of the transgenes CD55 and CD59. Also, no ischemic damage was found, which could be caused by undersupply with oxygen.

[0061] FIG. 3 depicts the levels detected by RT-PCR for transcripts of (32-microglobulin and for transcripts of MHC class II (RT1-B) after stimulation with IFNy for 48 h in the cultivated endothelial cells. As a negative control, non-transduced cells were used and cells that were transduced with a vector encoding a non-binding non-specific shRNA (shNS). Transcript levels are normalized to levels of transcripts for (3-actin (***p<0.001), also detected by RT-PCR. These results show that the nucleic acid construct encoding shRNA specific for transcripts of (32-microglobulin, respectively for transcripts of CIITA, effectively downregulate expression of (32-microglobulin and of CIITA, and hence downregulate expression of MHC I and MHC II.

[0062] FIG. 4 depicts the analytical result of FACS for Treg cells (CD4+CD25+FoxP3+). The result shows that frequencies of Treg cells that were expanded in the presence of cells containing an expression cassette for shRNA directed against MHC II transcripts for downregulation by 60% (HLA class II silenced (60%)) or by 90% (HLA class II silenced (90%)), are higher than the frequencies of Tregs in the presence of MHC class II knockout cells (HLA-DR knockout, 100% downregulated) and in the moderate downregulation of MHC class II transcripts by 60% even higher than with non-transduced cells. HLA-DR knockout single donor HUVEC cells or HLA class II silenced single donor HUVEC cells that were downregulated for up to 60% or up to 90% were co-cultured in presence of PBMCs in RPMI medium supplemented with 5% AB serum and 100U/mL IL-2 and 20ng TGF-(3. Non-transduced cells were used as controls (Non-Transduced).

[0063] For allogeneic transplantation, the kidneys from explanted kidneys of LEW1U.RT1 donor rats were used as allogeneic transplants in LEW.RT1 rats as recipients. The right kidney was perfused and genetically manipulated as described above and then was transplanted in rat LEW.RT1 recipients in an orthotopic manner. During the operation the animals are maintained on a thermoplate to maintain the body temperature constant. The recipient was ventilated via a respiratory mask. For anaesthesia, Isofluran was administered. After laparotomy the right kidney was resected and the vessels clamped. The genetically modified or unmodified control donor kidneys, respectively, was meanwhile cooled in the ex vivo perfusion system. The kidney was removed from the ex vivo perfusion system, flushed with fresh cold perfusion solution and prepared for implantation. The kidney was then transplanted using standard techniques. After discontinuation of anesthesia and removal of the respiratory mask, the animals were put into boxes provided with drinking water. The animals were regularly controlled for signs of rejection and cytokine levels such as IP-10, MCP-1 or RANTES were monitored. In the first 14 days after transplantation the animals received a mild immunosuppression (10mg/kg body weight Cyclosporin/day). Animals that were transplanted with a genetically modified organ did not show clinical, immunological or morphological signs of immunological organ rejection within an observation period of 90 days, whereas animals that were transplanted with an unmodified kidney rejected the graft at the latest 30 days after transplantation.

[0064] FIG. 5 depicts the percentage of lysed cells which contain expression cassettes for different complement inhibitory molecules. The results show that the overexpression of CD55 and CD59 in MHC-silenced rat endothelial cells (LEW.1W) substantially decreases antibody-mediated cytotoxicity nearly to the level of the negative control. Rat endothelial cells (ECs) were incubated with serum of an immunized rat containing anti-RT1u antibodies and complement. MHC-silenced ECs overexpressing CD55 and CD59 were best protected against complement-dependent antibody-mediated cell lysis. Only MHC-silenced ECs showed an improved protection compared to non-MHC-silenced only CD55 and CD59 transgenes expressing ECs. The data was normalized to the positive control. The negative control used serum from the EC donor (autologous control).

[0065] This example shows that the transplantation of an allogeneic kidney does not induce an immunogenic cellular reaction by the recipient and does not induce a T-cell and B-cell-mediated rejection (HvG), which kidney was genetically manipulated by retroviral transduction to contain expression cassettes for downregulating expression of its MHC I and MHC II by at least 80%, leaving a residual expression of at most 20%, compared to wild-type expression, and to contain expression cassettes for expression of complement inhibitors CD55 an CD59.