Antibodies (Abs) directed at the Galα1,3Galβ1,4GlcNAc-R (αGal) carbohydrate epitope initiate xenograft rejection. Previously, we have shown that bone marrow transplantation (BMT) with lentivirus-mediated gene transfer of porcine α1,3 galactosyltransferase (GalT) is able to induce tolerance to αGal-expressing heart grafts following a lethal dose of irradiation. Here we show the first demonstration of permanent survival of αGal+ hearts following transplantation with autologous, lentivirus-transduced BM using a nonmyeloablative regimen. Autologous BM from GalT knockout (GalT–/–) mice was transduced with a lentiviral vector expressing porcine GalT and transplanted into sublethally irradiated (3 Gy) GalT–/– mice. Chimerism in the peripheral blood cells (PBCs) remained low but was higher in the BM, especially within the stromal cell population. Mice reconstituted with GalT did not produce anti-αGal Abs over time. We immunized these mice with αGal-expressing cells and assessed humoral immune responses. Anti-αGal xenoantibodies were not produced in mice reconstituted with GalT, but normal Ab responses to other xenoantigens were detected. Mice reconstituted with GalT accepted αGal+ heart grafts over 100 days. Transduction with lentiviral vectors results in chimerism at levels sufficient to induce long-term tolerance under nonmyeloablative conditions.

Subjects:

Gene Therapy, Immunobiology and Immunotherapy, Immunotherapy, Neoplasia

Topics:

antibodies, antibodies, heterophile, bone marrow transplantation, carbohydrates, chimerism, epitopes, gene therapy, gene transfer techniques, gut-associated lymphoid tissue, heart

The number of cases of organ transplantation has been increasing, however, the shortage of donor organs is a major problem because the number of donors is limited. Xenotransplantation using nonhuman species as organ donors has received much attention as a possible solution to this problem. Pigs are regarded as the most likely species to serve as donors for clinical xenotransplantation.1  All mammals except humans, apes, and old-world monkeys express α1,3 galactosyltransferase (GalT), hence Galα1,3Galβ1, 4GlcNac-R (αGal) is expressed on most tissues including vascular endothelium.2-4  However, as humans have natural antibodies (Abs) against αGal, it is known that xenotransplantation of pig organs into humans induces hyperacute rejection, acute vascular rejection/delayed xenograft rejection, and even chronic rejection against αGal.5-7  In pig-to-primate discordant combination, 80% to 90% of antipig xenogeneic Abs are anti-αGal Abs.8  Therefore, overcoming this response is presently the biggest problem facing xenotransplantation.

To solve this problem, various approaches have been proposed.9-13  Mixed hematopoietic chimerism can induce tolerance in allogeneic and xenogeneic models of transplantation.14,15  Recently, it has been shown that tolerance to αGal can be induced in GalT knockout (GalT–/–) mice that produce anti-αGal Abs. Yang et al16  applied mixed-cell chimerism to achieve permanent acceptance of transplanted organs by injecting bone marrow (BM) from wild-type (WT) donor mice into GalT–/– mice conditioned with irradiation and T-cell depletion. Bracy et al17  have reported use of gene therapy to induce chimerism and tolerance following transplantation of autologous BM cells expressing a retrovirally transduced gene encoding GalT. We have reported that donor hearts were permanently accepted in GalT–/– mice conditioned with lethal irradiation and injected with autologous BM cells transduced with a lentiviral vector expressing GalT.18  Lethal irradiation conditioning, however, is not reasonable for use in clinical applications. We therefore have focused on establishing the conditions under which tolerance can be achieved using nonlethal irradiation and autologous gene therapy as a combined protocol.

Unlike other retroviruses, lentivirus vectors are able to transduce nondividing cells including hematopoietic stem cells.19  These genes are permanently transmitted and expressed as they are integrated into the genome. Here, we show the first application of gene therapy using lentiviral vectors and nonmyeloablative conditioning to overcome anti-αGal antibody-mediated rejection of WT hearts in GalT–/– mice.

Mice

GalT–/– mice homozygous for the targeted disruption in the GalT do not express the αGal epitope and produce anti-αGal–reactive Abs in their serum. GalT–/– mice were backcrossed 10 times using C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) and were obtained from Dr A. d’Apice (St Vincent’s Hospital in Melbourne, Australia). The mice used for these experiments were 3 to 5 months of age at the time of bone marrow transplantation (BMT). All animals received humane care in compliance with the Principles of Laboratory Animal Care, formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, prepared by the National Institutes of Health.20 

Construction of the lentiviral vector

The vector provirus has less than 850 bp of the human immunodeficiency virus genome, lacks long terminal repeat (LTR) enhancers and promoters (self-inactivating [SIN]), but contains the central polypurine tract and central terminal site sequences to increase the rate and extent of proviral integration. Sequences from Simian virus (SV) 40 were added to augment polyadenylation from the 3′ LTR to enhance gene expression and minimize transcriptional read-through to downstream cellular sequences at the site of proviral integration. The vector contains 80 bp from the gag region and lacks a 5′ splice donor site, making the vector independent of rev. The vector contains an MCU3 promoter that is the U3 region from the MND retroviral vector with substitution of the TATA site with that of the human CMV promoter. Titers were measured by flow cytometry following transduction of HIT293T cells. The woodchuck hepatitis virus posttranscriptional regulatory element reported to increase expression of transgenes delivered by viral vectors was included in the construct.21  The porcine α1,3 Galt gene or a control construct expressing the porcine Galt gene in the antisense orientation (REV) was used to transduce BM from GalT–/– mice. The vector was pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G), as described.22 

Transduction, bone marrow transplantation, and irradiation

Animals were matched for age and anti-αGal Ab levels prior to BMT. BM donors were killed, and bone marrow cells were flushed from the femora and humeri using phosphate-buffered saline. Transduction of BM from autologous GalT–/– mice was done over a 48-hour period using virus at a concentration of 1 × 107 IU/mL in X-VIVO media (Cambrex Bio Science, Walkersville, MD) supplemented with 50 ng/mL IL3, IL6, and SCF (Stem Cell Technologies, Vancouver, BC, Canada).23  Twenty-four to 36 hours after transduction, bone marrow recipients were prepared by treatment with 3 Gy of whole-body irradiation using a 137Cs irradiator. Sublethally irradiated GalT–/– mice were reconstituted with 8 × 106 to 30 × 106 transduced BM cells within 12 to 24 hours after irradiation by tail vein injection. Blood samples from the tail vein were taken prior to transplantation and at time intervals ranging from 1 week to 15 weeks after transplantation to monitor chimerism and Ab levels.

Immunization

To induce a humoral immune response, mice were immunized by intraperitoneal injection of 1 × 106 rabbit red blood cells (RRBCs). Mice were reimmunized by intraperitoneal injection of 1 × 107 pig kidney (MPK) cells. Immunized control mice for enzyme-linked immunospot (ELISPOT) assay were injected intraperitoneally with 1 × 106 RRBCs 2 weeks before the experiment.