One approach to gene therapy for hepatic diseass is to remove hepatocytes from an affected individual, genetically alter them in vitro, and reimplant them into a receptive locus. Although returning hepatocytes to the liver itself would be advantageous, the feasibility of this approach has never been evaluated due to the inability to distinguish donor from host hepatocytes. To unambiguously identify transplanted hepatocytes after transplantation, and to better quantitate their number and degree of liver function, two transgenic mouse lines were generated in a C57BL/6 background. The first expresses the Escherichia cofi p-galactosidase gene from the relatively liver-specific human a1-antitrypsin (hAAT) promoter and allows transgenic hepatocytes to be readily identified after 5-bromo-4-chloro-3-indolyl .8-D-galactoside staining; the second produces the hAAT protein under control of the same promoter, which enables hepatocyte survival and maintenance of liver function to be quantitated by measuring the serum levels of hAAT. Hepatocytes isolated from transgenic donors were transplanted into nontransgenic C57BL/6 recipients by intrasplenic injection. Surprisingly, a large fraction of these cells were identified within the liver parenchyma but not the spleen at 2 months after transplantation. The high levels of serum hAAT detected in transplant recipients were stable for >6 months, suggesting that established cells will survive indefinitely. These results have important implications for liver organogenesis and hepatic gene therapy.

The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

Hemophilia B is a bleeding disorder caused by mutations in the factor IX gene. The disorder is X-linked recessive with a prevalence of about 1 in 30,000 Caucasian males. Factor IX is naturally synthesized in the liver and secreted into blood. Here we report the construction of recombinant adenoviral vectors containing the canine factor IX cDNA that are capable of transducing hepatocytes in mice at high efficiencies in vivo without partial hepatectomy. The recombinant viral vector was used to treat hemophilia B dogs by direct vector infusion into the portal vasculature of deficient animals. Plasma factor IX concentrations in the treated hemophilia B dogs increased from 0 to 300% of the level present in normal dogs, resulting in complete amelioration of the disease as demonstrated by normal blood coagulation and hemostatic measurements. Although plasma factor IX concentration started to decline after a few days, therapeutic levels of factor IX persisted for 1-2 months in the treated animals. The results validate the principle of in ivo hepatic gene delivery to reconstitute the genetic deficiency in a large animal model and suggest that gene therapy is achievable when long-acting vectors are developed.Hemophilia B is an X-linked disorder resulting in a deficiency of plasma factor IX, which is normally synthesized and secreted from the liver. The disease affects 1 in 30,000 males (1). Various human protein replacement therapies have significantly improved the clinical outcome of affected individuals, yet there still exists a high degree of morbidity and mortality in part due to previous contamination with human immunodeficiency virus and other hepatitis viruses. The current expense of virus-free factor IX usually limits therapy to treatment ofbleeding episodes once they occur. Due to the relatively short half-life of factor IX in the circulation, affected individuals have life-long risks of central nervous system bleeds, chronic arthritis, and other life-threatening hemorrhage. Replacement therapy has improved, but has failed to abrogate, chronic arthropathy, particularly in severely affected patients. A number of cell types in different organs have been targeted for somatic gene therapy of hemophilia B, which include fibroblasts, myoblasts, endothelial cells, keratinocytes, and hepatocytes (2-8). We have pursued the liver as the target organ because hepatocytes represent the natural site offactor IX synthesis and secretion. We have previously been able to transduce hepatocytes in hemophilia B dogs after partial hepatectomy using a retroviral vector containing the factor IX cDNA (9). This resulted in long-term expression of about 0.1% of the normal plasma factor IX concentration. To attempt to produce greater levels of recombinant factor IX in animals, we have pursued replication-deficient recombinant adenovirus (10-12) as a gene delivery vector for the treatment of hemophilia B. METHODSCanine Factor IX Adenoviral Vectors. The isolation of a functional canine factor IX cDNA has been described (9). This cDNA ...

The liver represents an excellent target organ for gene therapy. The current strategy for hepatic gene therapy involves the isolation of primary hepatocytes from a resected liver lobe, transduction of therapeutic genes in vitro followed by autologous hepatocellular transplantation. This ex vivo approach is a rather complex procedure in its entirety; thus, a simple method for direct gene delivery into hepatocytes in vivo has been developed. The procedure involves partial hepatectomy followed by the portal vein infusion of recombinant retroviral vectors. Histological analysis of hepatocytes after in vivo delivery of a recombinant retrovirus bearing the E. coli beta-galactosidase gene showed that 1-2% of the parenchymal cells were transduced. Direct hepatic transfer of human alpha 1-antitrypsin cDNA under the transcriptional direction of the albumin promoter-enhancer led to constitutive expression of the human protein in the sera of recipients at concentrations of 30-1,400 ng/ml for at least 6 months. The experimental animals showed no signs of illness and histologic analysis of the liver revealed no evidence of pathologic abnormalities. The results suggest that the in vivo approach is an attractive alternative for hepatic gene therapy.

The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. We have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes.Here we demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. At least 1 x 109 hepatocytes or 5% of the liver mass can be transplanted by the portal vasculature. In two animals we have transplanted hepatocytes transduced with a retroviral vector containing the human a,-antitrypsin cDNA under transcriptional control of the cytomegalovirus promoter. Both animals had sificant human a1-antitrypsin in the serum for 1 month. Although the serum levels of human a1-antitrypsin eventually fell due to inactivation of the cytomegalovirus promoter, PCR analysis demonstrated that a significant fraction of transduced hepatocytes migrated to the liver and continued to survive in vivo.The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstitution with the use of promoters of cellular genes that are active in the normal liver.Many laboratories have developed methods for the transduction of genes into cultured primary hepatocytes (1-5). To use this method for gene therapy, one must be able to introduce the genetically altered cells back into the animal, preferably to the liver. Using transgenic mouse hepatocytes as a model, we (6) and others (7) have recently demonstrated that freshly isolated mouse hepatocytes can be transplanted into the liver of congenic recipients by direct splenic or portal vein injection. The grafted cells migrated to the liver and functioned indefinitely as hepatocytes after transplantation.The next critical step is to determine whether hepatocytes that undergo in vitro manipulation and retroviral transduction can be transplanted back into the animal and function in a manner similar to the transgenic mouse models. Wilson et al. (8) transplanted heterologous hepatocytes after retroviral transduction with the human low density lipoprotein receptor into low density lipoprotein-deficient rabbits but found limited short-term expression. We have, however, selected the canine model to develop methodologies for autologous transplantation of virally transduced hepatocytes because (i) the size of a young dog parallels that of a human infant and (ii) there are canine hepatic-deficiency disorders that can be used as models for gene therapy. We selected the human a1-antitrypsin (hAAT) cDNA as a marker gene since it encodes a secretable protein that can be easily detected in the serum of recipients with an RIA that utilizes a species-specific hAAT antibody. We show here that at least 1 x 109 hepatocytes can be transplanted into the animals and, after retroviral gene transduction, autologous hepatocytes transplanted by the portal circulation remain in the liver...