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In vivo gene transfer is being considered in the systemic delivery of therapeutic proteins. This report evaluates the use of AAV vectors administered into muscle to deliver erythropoietin (Epo) for the treatment of anemia in a mouse model of beta-thalassemia. Injection of vector expressing Epo from a constitutive promoter resulted in Epo overproduction and improved erythropoiesis. However, severe and lethal polycythemia developed. In order to titrate the expression of Epo to therapeutic and non-toxic levels, vectors were constructed to allow pharmacologic control of Epo transcription. Specifically, expression of Epo was dependent on the presence of a chimeric transcription factor that is activated by the orally available small molecule drug rapamycin. beta-thalassemic mice injected with vectors containing the regulated Epo gene failed to show any effect until they were administered a regimen of rapamycin, which led to the production of Epo and an increase in hematocrit values. Epo expression and its hematologic consequences were directly dependent on the dose of rapamycin and were completely reversed when rapamycin was withdrawn. The increase in hematocrit was associated with partial improvements in the abnormalities of red blood cell morphology. This study confirms the value of pharmacologic regulation of transgene expression in the development of safe and effective gene therapies in which biologically active secreted proteins are produced.

Ornithine transcarbamylase deficiency (OTCD) is an inborn error of urea synthesis that has been considered as a model for liver-directed gene therapy. Current treatment has failed to avert a high mortality or morbidity from hyperammonemic coma. Restoration of enzyme activity in the liver should suffice to normalize metabolism. An E1- and E4-deleted vector based on adenovirus type 5 and containing human OTC cDNA was infused into the right hepatic artery in adults with partial OTCD. Six cohorts of three or four subjects received 1/2 log-increasing doses of vector from 2 x 10(9) to 6 x 10(11) particles/kg. This paper describes the experience in all but the last subject, who experienced lethal complications. Adverse effects included a flu-like episode and a transient rise in temperature, hepatic transaminases, thrombocytopenia, and hypophosphatemia. Humoral responses to the vector were seen in all research subjects and a proliferative cellular response to the vector developed in apparently naive subjects. In situ hybridization studies showed transgene expression in hepatocytes of 7 of 17 subjects. Three of 11 subjects with symptoms related to OTCD showed modest increases in urea cycle metabolic activity that were not statistically significant. The low levels of gene transfer detected in this trial suggest that at the doses tested, significant metabolic correction did not occur.

Hepatic artery infusion of adenoviral vectors has been shown to increase transduction of certain hepatocellular malignancies in preclinical studies. In addition, clinical trials have begun evaluating the efficacy of gene transfer of cytotoxic genes to metastatic colorectal tumors through hepatic artery infusion. Here we evaluate the extent of gene expression and therapeutic effect following various routes of administration of recombinant adenovirus in a rat model of metastatic colorectal carcinoma. We administered adenovirus (AdCMVlacZ) to rats with established colorectal metastases through infusion into the hepatic artery, intravenous infusion, or direct injection into a tumor. Intravenous administration resulted in transduction of hepatocytes, but not tumor cells. Hepatic arterial administration failed to substantially increase transduction of tumor cells. In addition, ligation of the hepatic artery following infusion of adenovirus or the addition of lipiodol infusion had no effect on the transduction of tumor cells. We administered AdCMVp53 by direct injection into tumors, intravenous administration, or hepatic artery infusion to evaluate the delivery of a therapeutic gene. Direct injection of AdCMVp53 into established hepatic colorectal metastases resulted in a therapeutic response in comparison with both hepatic arterial and intravenous infusion of vector. These preclinical studies fail to support a strategy of infusion through the hepatic artery of recombinant adenovirus targeting tumor cells in the treatment of colorectal cancer liver metastases.

The innate immune response to intraportally infused adenoviral vector was evaluated in rhesus monkeys. A first-generation adenovirus-expressing lacZ (Ad-lacZ) was administered at a dose just below that which causes severe morbidity. The response to vector was evaluated for the initial 24 h following infusion. Clinical findings during this time were primarily limited to petechiae, consistent with the development of thrombocytopenia and biochemical evidence of disseminated intravascular coagulation. Serum transaminases were elevated and a lymphopenia developed. Tracking of fluorescent-labeled vector demonstrated distribution to macrophages and dendritic cells of the spleen and Kupffer cells of the liver. A systemic release of the cytokine IL-6 occurred soon after vector infusion. Analysis of splenic cells revealed acute activation of macrophages and dendritic cells followed by massive apoptosis. Bone marrow cultures demonstrated normal erythroid and primitive progenitors with a significant decrease in myeloid progenitors. Similar findings, except the abnormality in bone marrow cultures, were observed in monkeys who received an identical dose of Ad-lacZ in which vector genes were inactivated with psoralen and UV irradiation. These data suggest that inadvertent targeting of antigen-presenting cells following intraportal infusion of vector leads to a systemic cytokine syndrome which may be triggered by the viral capsid proteins.

The use of recombinant adeno-associated virus (rAAV) as gene therapy vector for treating liver metabolic diseases is limited by its low transduction efficiency. We describe a strategy for achieving stable and efficient genetic reconstitution in liver after direct administration of rAAV and selective expansion of transduced cells. We have exploited the biology of apoptosis to develop a generic approach for selectively repopulating liver with vector-transduced hepatocytes. Low-level, stable transduction of hepatocytes was achieved by direct injection of rAAV into mouse liver. Expansion of these vector-transduced cells was achieved by incorporating into the construct a minigene expressing Bcl-2 followed by induction of apoptosis in non-vector-containing hepatocytes by systemic administration of a Fas antibody (Ab). Western and Southern blot analysis demonstrated amplification of bcl-2 gene product and viral copy number, respectively, in vector-treated mouse liver when placed under selection. In addition, the percentage of vector transduced cells increased from 2 to 20% after three administrations of Fas Ab, based on immunohistochemical studies.

Adeno-associated viral vectors were used to deliver the gene for very-low-density lipoprotein (VLDL) receptor (VLDLR) to liver of a murine model of familial hypercholesterolemia (FH). Infusion of adeno-associated virus-VLDLR into the portal circulation of FH mice resulted in a 40% reduction in serum cholesterol and triglyceride that was stable for the duration of the study (30 weeks). Fractionation of serum lipids revealed a reduction of both VLDL and low-density lipoprotein. Expression of transgene-derived VLDLR was confirmed in livers of recipient animals by Western blot analysis and immunohistochemistry; vector DNA was present at 1 copy/cell. Vector-treated animals had significantly less lipid accumulation in liver and reduced atherosclerosis in the aorta.

We have previously reported a factor VIII knockout (FVIII KO) mouse model for hemophilia A. Here we demonstrate the presence of nonfunctional heavy chain factor VIII protein in the mouse, making it an excellent model for cross-reacting material (CRM)-positive hemophilia A patients, who express normal levels of a dysfunctional FVIII protein. We attempted to correct these mice phenotypically by transduction of wild-type mouse factor VIII cDNA delivered in an E1/E3-deleted adenoviral vector by tail vein injection. All treated mice displayed initial high-level FVIII expression that diminished after 1 month. Ten of 12 mice administered between 6 x 10(9) and 1 x 10(11) particles/mouse along with anti-CD4 antibody showed long-term FVIII activity (0.03-0.05 IU/ml, equivalent to 3-5% of normal FVIII) that corrected the phenotype. Wild-type murine FVIII was a neo-antigen to the KO mice, generating both cytotoxic and humoral immune responses. Immune suppression with anti-CD4 antibody abrogated these immune responses. These data demonstrate that despite the presence of endogenous FVIII protein the immune system still recognizes a species-specific transgene protein as a neo-antigen, eliciting a cytotoxic T cell response. This phenomenon may exist in the treatment of other genetic disorders by gene therapy.

The early successes of adenoviral vector-mediated gene therapies in the lung have been hampered by the immune response directed against viral proteins and transgene product. Intratracheal administration of adenovirus vector in immune-competent mice transduces bronchioepithelial cells of lung extremely efficiently; however, transgene expression is eliminated within 2 weeks. Extinction of transgene expression has been attributed to infiltrating cytotoxic T lymphocytes (CTLs). Fas-Fas ligand (Fas-FasL) interactions play a critical role in the effector function of the CTL killing. We have previously demonstrated that this interacting pair of molecules plays a critical role in elimination of transgene expression in mouse liver. In this study we investigated the role of Fas-FasL interactions in CTL effector functions in vivo in mouse lung. Analyses of these molecules in lung showed constitutive expression of Fas in bronchiooepithelial cells. On the other hand, FasL was inducible in the bronchioepithelial cells after adenovirus vector treatment. The in vivo role of the Fas-FasL interactions was determined by adoptive transfer of splenocytes from normal immune-competent mice to Fas-deficient mice (B6-lpr); likewise, the function of FasL on CTLs was analyzed by the ability of splenocytes from FasL-deficient mice (B6-gld) to eliminate transgene expression in Rag1-deficient mice. These studies demonstrate that despite expression of Fas and FasL in bronchioepithelial cells and CTLs, these interacting molecules do not play a critical role in elimination of transgene expression in the lung.

A murine model for hereditary tyrosinemia Type I (HTI) was evaluated for in vivo gene therapy with adeno-associated viral (AAV) vectors expressing the enzyme fumarylacetoacetate hydrolase. Transduction of a limited number of hepatocytes was accomplished following infusion of vector into the portal circulation. Corrected hepatocytes were expanded in vivo by withdrawing a drug which prevents the accumulation of toxic metabolites. The liver was eventually repopulated with hepatocytes harboring a functional and apparently integrated AAV provirus. Recipient animals regained normal liver function and architecture and the underlying metabolic derangements were normalized. After 9 months, vector-treated animals showed benign hepatomas, whereas in untreated animals areas of marked dysplasia were present within hepatomas.

Vectors based on adeno-associated viruses (AAV) type 2 show promise for treating chronic diseases because transgene expression appears to be stable. This study evaluated the impact of humoral immunity to the capsid proteins on vector uptake by hepatocytes following an intravascular approach. Route of vector administration in mice had a qualitative effect on antivector B cell responses. Administration of vector into the tail vein resulted in T-cell-dependent (TD) B cell responses that were completely inhibited with depleting CD4 antibody. Delivery of vector into the portal circulation via the spleen yielded B cell response that were partially T cell independent (TI) rendering strategies based on T cell inhibition ineffective in allowing vector readministration. The TI B cell response was short lived in comparison to the TD response. Rhesus monkeys produced a B cell memory response to intraportal vector which appeared to be T cell dependent based on Ig isotypes. Gene therapy strategies that require AAV vector readministration should consider vector biodistribution and its impact on B cell activation.

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (

CD40 is thought to play a central role in T cell-dependent humoral responses through two distinct mechanisms. CD4+ T helper cells are activated via CD40-dependent Ag presentation in which CD80/CD86 provides costimulation through CD28. In addition, engagement of CD40 on B cells provides a direct pathway for activation of humoral responses. We used a model of adenovirus-mediated gene transfer of β-galactosidase (lacZ) into murine lung to evaluate the specific CD40-dependent pathways required for humoral immunity at mucosal surfaces of the lung. Animals deficient in CD40L failed to develop T and B cell responses to vector. Activation of Th2 cells, which normally requires CD40-dependent stimulation of APCs, was selectively reconstituted in CD40 ligand-deficient mice by systemic administration of an Ab that is agonistic to CD28. Surprisingly, this resulted in the development of a functional humoral response to vector as evidenced by formation of germinal centers and production of antiadenovirus IgG1 and IgA that neutralized and prevented effective readministration of vector. The CD28-dependent B cell response required CD4+ T cells and was mediated via IL-4. These studies indicate that CD40 signals to the B cells are not necessary for CD4+ Th2 cell-dependent humoral responses to be generated.

Recombinant adeno-associated virus (rAAV) is a promising vector for therapy of retinal degenerative diseases. We evaluated the efficiency, cellular specificity, and safety of retinal cell transduction in nonhuman primates after subretinal delivery of an rAAV carrying a cDNA encoding green fluorescent protein (EGFP), rAAV.CMV. EGFP . The treatment results in efficient and stable EGFP expression lasting >1 year. Transgene expression in the neural retina is limited exclusively to rod photoreceptors. There is neither electroretinographic nor histologic evidence of photoreceptor toxicity. Despite significant serum antibody responses to the vector, subretinal readministration results in additional transduction events. The findings further characterize the retinal cell tropism of rAAV. They also support the development of studies aimed ultimately at treating inherited retinal degeneration by using rAAV-mediated gene therapy.

Factors relevant to the successful application of adeno-associated virus (AAV) vectors for liver-directed gene therapy were evaluated. Vectors with different promoters driving expression of human α-1-antitrypsin (α-1AT) were injected into the portal circulation of immunodeficient mice. α-1AT expression was stable but dependent on the promoter. Southern analysis of liver DNA revealed approximately 0.1 to 2.0 provirus copies/diploid genome in presumed head-to-tail concatamers. In situ hybridization and immunohistochemical analysis revealed expression in approximately 5% of hepatocytes clustered in the pericentral region. These results support the use of AAV as a vector for diseases treatable by targeting of hepatocytes.