Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10. Transplantation 2001; 71: 1214.

Field: Medicine (Health Sciences)

PROSPECTS FOR GENETIC MANIPULATION OF DONOR ORGANS AND TISSUES When considering the application of gene therapy to tissue transplantation, the cornea stands out as having uniquely auspicious properties. The tissue is small (consider the liver by comparison), it can be maintained in culture for several weeks at 32–37°C (instead of a few hours at 0°C), and it is transplanted to an immunologically privileged site. However, the greatest advantage of all is that the key target cell, the corneal endothelial cell, forms a single sheet of cells on the surface of the cornea, and is therefore perfectly accessible to DNA vectors. It is probably not hyperbole to say that the time is right for clinical trials for gene therapy (in the form of genetic manipulation of donor tissue) in corneal transplantation. The two key issues are the vectors to be used, and the genetic manipulations to be evaluated. The other critically important technical factor—the longevity of gene expression—is probably not a major problem for most of the genetic manipulations likely to be considered in a transplant setting, provided that a few weeks’ expression can be guaranteed. The study by Sonja Klebe and co-workers (1) is important as it demonstrates, in a large animal model (sheep), that ex vivo genetic manipulation of corneas can prevent rejection after allotransplantation. These authors used adenovirus vectors carrying the ovine IL-10 gene under the control of a constitutive CMV promoter. Although four of the nine genetically manipulated corneas were rejected in normal time, five showed prolonged survival, and of these two showed indefinite survival, without any other treatment. A key issue is the interaction of the genetic manipulation (in this case local IL-10 production) with conventional antirejection therapy in the form of topical steroids. Drug interactions are not addressed in this report, but would inevitably have to be considered in a clinical setting. Almost certainly, a positive interaction between conventional therapy and gene therapy would be sought in the first instance. Vectors. Klebe et al. chose to use adenovirus vectors. Adenoviruses are well known to be highly efficient gene delivery systems for certain cell types, including corneal endothelial cells (2) and hepatocytes (3). More recently, lentiviruses (4) and polyoma virus pseudocapsids (5) have been used for gene delivery to the corneal endothelium. A major problem with adenovirus (and other virus) vectors is the immune and inflammatory responses to the viral proteins (6,7). Such inflammatory responses are undesirable in any setting, but they could be particularly harmful in transplantation because inflammation might trigger destructive allogeneic responses. It is therefore important that Klebe et al. could find no evidence for inflammatory or immune responses after transplantation in their study. They attribute this to the immune privilege of the setting into which the genetically modified corneas were transplanted, and this quite likely is important. However, there is another possible explanation. Klebe et al. delayed transplantation of the cornea until 48 hr after gene transfer, maintaining the cornea in organ culture at 32°C over this period. How long adenovirus peptides persist in the endothelial cells of an actively metabolising cornea is unknown. However, it is possible that delaying transplantation after adenovirus gene transfer (an advantageous option not open to other forms of tissue transplantation) might reduce problems with antivirus immunity because of the metabolic elimination of viral peptides during the culture period before transplantation. However, if immune privilege is indeed a major factor suppressing inflammation, it is worth noting that it might not be operating effectively in the abnormal setting where gene therapy is likely first to be used in clinical corneal transplantation: the vascularized corneal bed. Nonviral systems such as the dendrimers (8) mentioned by Klebe et al. and integrin-targeted synthetic peptides (9) are effective for gene delivery to corneal endothelial cells. Among other advantages, a particular virtue is absent or minimal immunogenicity, which makes these non-viral vectors attractive candidates for gene delivery in corneal and other forms of transplantation. Genes. The second important issue in this field is what genetic manipulations are likely to be effective. This must be considered in a context where transgene expression is likely to be limited to the few weeks following transplantation. Moreover, the technical limitations of somatic gene therapy as currently practised are such that it can in essence deliver only a “gain of function,” the additional function being that of the protein encoded by the transgene. Suppression of function is difficult, except in the relatively unusual circumstance where dominant-negative effects are possible, i.e., where the structure of the protein product can be engineered into an inactive form which inhibits competitively the function of the native protein. Suppression is also possible with the use of anti-sense genes (the mRNA of which is complementary to the mRNA of the target gene, and forms inactive RNA-RNA duplexes), and the related area of targeted mRNA degradation by ribozymes. However, this approach is not as simple in practice as it sounds in theory. Given the above constraints, the major pathway for gene therapy in transplantation is likely to be via genes coding for regulatory proteins. The potential clinical benefit of this genetic approach is the local delivery of regulatory proteins at concentrations unachievable by systemic administration of recombinant protein. Moreover, such local immunosuppression is obtained without the dangerous consequences of systemic immunosuppression. A general note of caution in this regard is that the uncontrolled expression of soluble regulatory proteins by large donor organs such as the liver (10) can result in high systemic levels of the regulatory protein and consequent uncontrolled systemic immunosuppression. However, this will obviously not be a problem with the cornea, and probably not with other small volume transplants such as pancreatic islets (11). Klebe et al. make a good case for considering IL-10 as a candidate for clinical trials. Another, which has been tested by several groups, is soluble CTLA-4 (CD152) (12). A second general approach to genetic manipulation of donor tissue involves reduction of graft immunogenicity via the suppression of costimulatory ligands or donor MHC antigens. As mentioned above, suppression of function is not readily achieved with current technology. However, powerful suppression of MHC class II expression is possible via dominant-negative mutants of the MHC class II transactivator (CIITA) (13). The final question is that of longevity of gene expression. Klebe et al. demonstrate the presence of IL-10 mRNA after 21 days of in vitro culture. This is of value, but not definitive, as it deals with the virtual world of nucleic acids, rather than the real world of proteins. Whether effective levels of IL-10 protein were present for only a few days after grafting, or for many days beyond day 21, is unknown. Designing DNA expression constructs with long-term stability in cells is certainly one of the major challenges in gene therapy, and one of the major current technical limitations. The eyes have it. Corneal transplantation had a major clinical impact several decades before organ transplantation became established in the 1960s, and it seems likely also to be in the vanguard of assessing if gene therapy will make a substantive contribution to organ transplantation.