Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34⁺ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8⁺ and CD4⁺ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8⁺ and CD4⁺ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34⁺ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.     

Enhancement of Myogenic and Muscle Repair Capacities of Human Adipose�Cderived Stem Cells With Forced Expression of MyoD

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments, and mesenchymal stem cells (MSCs) may constitute an attractive cell therapy alternative because they are multipotent and accessible in adult tissues. We have previously shown that human multipotent adipose–derived stem (hMADS) cells were able to restore dystrophin expression in the mdx mouse. The goal of this work was to improve the myogenic potential of hMADS cells and assess the impact on muscle repair. Forced expression of MyoD in vitro strongly induced myogenic differentiation while the adipogenic differentiation was inhibited. Moreover, MyoD-expressing hMADS cells had the capacity to fuse with DMD myoblasts and to restore dystrophin expression. Importantly, transplantation of these modified hMADS cells into injured muscles of immunodepressed Rag2^−/−γC^−/− mice resulted in a substantial increase in the number of hMADS cell–derived fibers. Our approach combined the easy access of MSCs from adipose tissue, the highly efficient lentiviral transduction of these cells, and the specific improvement of myogenic differentiation through the forced expression of MyoD. Altogether our results highlight the capacity of modified hMADS cells to contribute to muscle repair and their potential to deliver a repairing gene to dystrophic muscles.     

Aldehyde Dehydrogenase Activity Identifies a Population of Human Skeletal Muscle Cells With High Myogenic Capacities

Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH⁺ cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDH^br) cells with low side scatter (SSC^lo), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD⁺ (for skeletal muscle ALDH⁺) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD⁺/CD34⁻ and SMALD⁺/CD34⁺ cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD⁺/CD34⁺ cells developed in vitro as a heterogeneous population of CD56⁻ cells able to differentiate in adipoblasts, the SMALD⁺/CD34⁻ fraction developed in vitro as a highly enriched population of CD56⁺ myoblasts able to form myotubes. Moreover, only the SMALD⁺/CD34⁻ population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy.     

In Vivo Myogenic Potential of Human CD133+ Muscle-derived Stem Cells: A Quantitative Study

In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle–derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2^−/− γC^−/− C5^−/− mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle–derived CD133⁺ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133⁺/CD34⁺ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133⁺ cells could be an attractive candidate for cellular therapy.     

Long-term Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using Adeno associated Viral Gene Therapy

Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating metabolites, transgene expression, and whole animal propionate oxidation were examined as outcome parameters after gene therapy. One-hundred percent of the untreated Mut^−/− mice (n = 58) died by day of life (DOL) 72, whereas >95% of the adeno-associated virus–treated Mut^−/− mice (n = 27) have survived for ≥1 year. Despite a gradual loss of transgene expression and elevated circulating metabolites in the treated Mut^−/− mice, the animals are indistinguishable from unaffected control littermates in size and activity levels. These experiments provide the first definitive evidence that gene therapy will have clinical utility in the treatment of MMA and support the development of gene therapy for other organic acidemias.     

Correction of Murine Rag2 Severe Combined Immunodeficiency by Lentiviral Gene Therapy Using a Codon-optimized RAG2 Therapeutic Transgene

Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2^−/− mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2^−/− mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.     

Mesenchymal Stromal Cells Treatment Attenuates Dry Eye in Patients With Chronic Graft-versus-host Disease

Cell therapy is a promising approach for the treatment of refractory ocular disease. This study investigated the efficacy of mesenchymal stromal cells (MSCs) for the treatment of dry eye associated with chronic graft-versus-host disease (cGVHD) and assessed the immunomodulatory effects of MSCs on regulatory CD8⁺CD28⁻ T lymphocytes. A total of 22 patients with refractory dry eye secondary to cGVHD were enrolled. The symptoms of 12 out of 22 patients abated after MSCs transplantation by intravenous injection, improving in the dry eye scores, ocular surface disease index scores and the Schirmer test results. The clinical improvements were accompanied by increasing level of CD8⁺CD28⁻ T cells, but not CD4⁺CD25⁺ T cells, in the 12 patients who were treated effectively. They had significantly higher levels of Th1 cytokines (interleukin (IL)-2 and interferon-γ) and lower levels of Th2 cytokines (IL-10 and IL-4). In addition, CD8⁺ T cells were prone to differentiation into CD8⁺CD28⁻ T cells after co-culture with MSCs in vitro. In conclusion, transfusion of MSCs improved the clinical symptoms in patients (54.55%) with refractory dry eye secondary to cGVHD. MSCs appear to exert their effects by triggering the generation of CD8⁺CD28⁻ T cells, which may regulate the balance between Th1 and Th2.     

Overexpression of Sonic Hedgehog in the Lung Mimics the Effect of Lung Injury and Compensatory Lung Growth on Pulmonary Sca-1 and CD34 Positive Cells

Cells localized in the bronchioalveolar duct junction of the murine lung have been identified as potential bronchioalveolar stem cells. Based on the surface marker expression, two main phenotypes have been proposed: Sca-1⁺, CD34⁺, CD45⁻, Pecam⁻ and Sca-1^low, CD34⁻ CD45⁻, Pecam⁻ cells. An increase in the number of Sca-1⁺, CD34⁺ CD45⁻, Pecam⁻ cells and activation of the sonic hedgehog (Shh) pathway was observed following unilateral pneumonectomy and naphthalene-induced airway injury. Overexpression of Shh in the respiratory tract also resulted in an increase of this cell population. Syngeneic transplantation of β-galactosidase-expressing bone marrow cells demonstrated that the increase of Sca-1⁺, CD34⁺, CD45⁻, Pecam⁻ cells in the lung was a result of local proliferation. Intratracheal administration of purified Shh-stimulated Sca-1⁺, CD45⁻, Pecam⁻ cells coexpressing CD34 to syngeneic mice following pneumonectomy resulted in engraftment of these cells predominantly in the airways for up to 3 months, whereas Sca-1⁻, CD45⁻, Pecam⁻ cells did not engraft. This study suggests that local Sca-1⁺, CD34⁺, CD45⁻, Pecam⁻ cells are stimulated during compensatory lung growth, following airway injury and overexpression of Shh and have some potential to engraft in the airways, without showing clonal properties in vivo.     

Noninvasive Imaging and Radiovirotherapy of Prostate Cancer Using an Oncolytic Measles Virus Expressing the Sodium Iodide Symporter

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 (¹²⁵I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of ¹²³I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 × 10⁶ TCID₅₀) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of ¹³¹I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by ¹³¹I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.     

ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice

Leukocytosis is associated with increased cardiovascular disease risk in humans and develops in hypercholesterolemic atherosclerotic animal models. Leukocytosis is associated with the proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) in mice with deficiencies of the cholesterol efflux–promoting ABC transporters ABCA1 and ABCG1 in BM cells. Here, we have determined the role of endogenous apolipoprotein-mediated cholesterol efflux pathways in these processes. In Apoe^–/– mice fed a chow or Western-type diet, monocytosis and neutrophilia developed in association with the proliferation and expansion of HSPCs in the BM. In contrast, Apoa1^–/– mice showed no monocytosis compared with controls. ApoE was found on the surface of HSPCs, in a proteoglycan-bound pool, where it acted in an ABCA1- and ABCG1-dependent fashion to decrease cell proliferation. Accordingly, competitive BM transplantation experiments showed that ApoE acted cell autonomously to control HSPC proliferation, monocytosis, neutrophilia, and monocyte accumulation in atherosclerotic lesions. Infusion of reconstituted HDL and LXR activator treatment each reduced HSPC proliferation and monocytosis in Apoe^–/– mice. These studies suggest a specific role for proteoglycan-bound ApoE at the surface of HSPCs to promote cholesterol efflux via ABCA1/ABCG1 and decrease cell proliferation, monocytosis, and atherosclerosis. Although endogenous apoA-I was ineffective, pharmacologic approaches to increasing cholesterol efflux suppressed stem cell proliferative responses.     

A Novel Model of SCID-X1 Reconstitution Reveals Predisposition to Retrovirus-induced Lymphoma but No Evidence of ��C Gene Oncogenicity

The emergence of leukemia following gene transfer to restore common cytokine receptor γ chain (γC) function in X-linked severe combined immunodeficiency (SCID-X1) has raised important questions with respect to gene therapy safety. To explore the risk factors involved, we tested the oncogenic potential of human γC in new strains of transgenic mice expressing the gene under the control of the CD2 promoter and locus control region (LCR). These mice demonstrated mildly perturbed T-cell development, with an increased proportion of thymic CD8 cells, but showed no predisposition to tumor development even on highly tumor prone backgrounds or after γ-retrovirus infection. The human CD2-γC transgene rescued T and B-cell development in γC^−/− mice but with an age-related delay, mimicking postnatal reconstitution in SCID-X1 gene therapy subjects. However, we noted that γC^−/− mice are acutely susceptible to murine leukemia virus (MLV) leukemogenesis, and that this trait was not corrected by the γC transgene. We conclude that the SCID-X1 phenotype can be corrected safely by stable ectopic expression of γC and that the transgene is not significantly oncogenic when expressed in this context. However, an underlying predisposition conferred by the SCID-X1 background appears to collaborate with insertional mutagenesis to increase the risk of tumor development.     

Reducing Mitochondrial ROS Improves Disease-related Pathology in a Mouse Model of Ataxia-telangiectasia

The disease ataxia-telangiectasia (A-T) has no cure and few treatment options. It is caused by mutations in the ATM kinase, which functions in the DNA-damage response and redox sensing. In addition to severe cerebellar degeneration, A-T pathology includes cancer predisposition, sterility, immune system dysfunction, and bone marrow abnormalities. These latter phenotypes are recapitulated in the ATM null (ATM^−/−) mouse model of the disease. Since oxidative stress and mitochondrial dysfunction are implicated in A-T, we determined whether reducing mitochondrial reactive oxygen species (ROS) via overexpression of catalase targeted to mitochondria (mCAT) alleviates A-T–related pathology in ATM^−/− mice. We found that mCAT has many beneficial effects in this context, including reduced propensity to develop thymic lymphoma, improved bone marrow hematopoiesis and macrophage differentiation in vitro, and partial rescue of memory T-cell developmental defects. Our results suggest that positive effects observed on cancer development may be linked to mCAT reducing mitochondrial ROS, lactate production, and TORC1 signaling in transforming double-positive cells, whereas beneficial effects in memory T cells appear to be TORC1-independent. Altogether, this study provides proof-of-principle that reducing mitochondrial ROS production per se may be therapeutic for the disease, which may have advantages compared with more general antioxidant strategies.     

Protein-anchoring Strategy for Delivering Acetylcholinesterase to the Neuromuscular Junction

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defects of ColQ cause endplate AChE deficiency and myasthenic syndrome. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq^−/− mice recovered motor functions, synaptic transmission, as well as the morphology of the NMJ. ColQ-tailed AChE was specifically anchored to NMJ and its amount was restored to 89% of the wild type. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq^−/− mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in noninjected limbs. Furthermore, the in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq^−/− mice led to accumulation of AChE in noninjected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring strategy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules.     

Generation of a Hypomorphic Model of Propionic Acidemia Amenable to Gene Therapy Testing

Propionic acidemia (PA) is a recessive genetic disease that results in an inability to metabolize certain amino acids and odd-chain fatty acids. Current treatment involves restricting consumption of these substrates or liver transplantation. Deletion of the Pcca gene in mice mimics the most severe forms of the human disease. Pcca⁻ mice die within 36 hours of birth, making it difficult to test intravenous systemic therapies in them. We generated an adult hypomorphic model of PA in Pcca⁻ mice using a transgene bearing an A138T mutant of the human PCCA protein. Pcca^−/−(A138T) mice have 2% of wild-type PCC activity, survive to adulthood, and have elevations in propionyl-carnitine, methylcitrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy similar to those in patients with PA. This adult model allowed gene therapy testing by intravenous injection with adenovirus serotype 5 (Ad5) and adeno-associated virus 2/8 (AAV8) vectors. Ad5-mediated more rapid increases in PCCA protein and propionyl-CoA carboxylase (PCC) activity in the liver than AAV8 and both vectors reduced propionylcarnitine and methylcitrate levels. Phenotypic correction was transient with first generation Ad whereas AAV8-mediated long-lasting effects. These data suggest that this PA model may be a useful platform for optimizing systemic intravenous therapies for PA.     

Cystinosis]

Recent progress of the study of the pathogenesis, diagnosis, and treatment of a lysosomal transport disorder, cystinosis is reviewed. Cystinosis is an autosomal recessively inherited disease that is caused by the accumulation of cystine in lysosome due to lack of the cystine transport system in lysosome. Renal transplantation has been a successful treatment for the cystinosis patients who are in the end stage renal failure, and this therapy has markedly prolonged the life span of cystinosis patients. Recently, oral cysteamine therapy has been successful in the excretion of accumulated cystine in cystinosis patients, and in improving the symptoms. Studies are now under way to see if early (within a month of life) start of cysteamine therapy would achieve the most satisfactory therapeutic effect and maintain normal renal function.     

Cysteamine,the Molecule Used To Treat Cystinosis,Potentiates the Antimalarial Efficacy of Artemisinin

Malaria continues to be a major threat to global health. Artemisinin combination therapy (ACT) is the recommended treatment for clinical malaria; however, recent reports of parasite resistance to artemisinin in certain areas where malaria is endemic have stressed the need for developing more efficacious ACT. We report that cysteamine (Cys), the aminothiol used to treat nephropathic cystinosis in humans, strongly potentiates the efficacy of artemisinin against the Plasmodium parasite in vivo. Using a mouse model of infection with Plasmodium chabaudi AS, we observe that Cys dosing used to treat cystinosis in humans can strongly potentiate (by 3- to 4-fold) the antimalarial properties of the artemisinin derivatives artesunate and dihydroartemisinin. Addition of Cys to suboptimal doses of artemisinin delays the appearance of blood parasitemia, strongly reduces the extent of parasite replication, and significantly improves survival in a model of lethal P. chabaudi infection. Cys, the natural product of the enzyme pantetheinase, has a history of safe use for the clinical management of cystinosis. Our findings suggest that Cys could be included in novel ACTs to improve efficacy against Plasmodium parasite replication, including artemisinin-resistant isolates. Future work will include clinical evaluation of novel Cys-containing ACTs and elucidation of the mechanism underlying the potentiation effect of Cys.Malaria still represents a huge global health burden, with 500 to 600 million clinical cases resulting in 1 to 2 million deaths annually (www.who.int). The impact is particularly devastating in resource-poor countries of sub-Saharan Africa and Southeast Asia, where malaria is endemic and access to appropriate antimalarial drugs can be limited. Furthermore, as a result of widespread use and misuse of antimalarial drugs, the Plasmodium parasite has developed resistance to commonly used drugs, such as chloroquine, mefloquine, and sulfadoxine-pyrimethamine (23). Artemisinin is a sesquiterpene lactone endoperoxide (extracted from Artemisia annua) with potent antimalarial activity, and artemisinin combination therapy (ACT) is the strategy recommended by the World Health Organization for clinical care of malaria (www.who.int). However, recent reports of delayed parasite clearance times following standard ACT treatment in patients from the Pailin province of Cambodia have suggested emergence of resistance to artemisinin in the Plasmodium parasite (12). These alarming reports have highlighted the urgency for the development of novel and more effective chemotherapeutic strategies, including modification of current ACT composition.We have used a mouse model of blood-stage infection with P. chabaudi AS to identify novel genetic factors affecting host response to malaria. We mapped two major loci, Char4 (chromosome 3) and Char9 (chromosome 10), that control differential responses of innately susceptible A/J (high parasitemia, high mortality) and uniquely resistant AcB55 (low parasitemia, high rate of survival) mice to P. chabaudi infection. Char4-associated resistance was found to result from a loss-of-function mutation (I90N) in the erythrocyte enzyme pyruvate kinase (Pklr) (28). Subsequently, we observed that erythrocytes heterozygous or homozygous for defective variants in human PKLR are resistant to P. falciparum infection ex vivo (reduced parasite replication and increased phagocytosis), validating our initial observations with the mouse (5). On the other hand, we established that Char9-associated susceptibility is caused by a loss of activity in the pantetheinase enzyme encoded by the Vnn1/Vnn3 genes (27). Pantetheinase is an amidohydrolase that hydrolyzes pantetheine (product of coenzyme A degradation) to pantothenic acid (also called pantothenate or vitamin B5) and the small aminothiol cysteamine (Cys) (NH₂-CH₂-CH₂-SH₂) (13). Strikingly, exogenous administration of cysteamine to pantetheinase-deficient malaria-susceptible A/J mice reversed the phenotype by reducing blood parasitemia levels and increasing survival time (26, 27).Cysteamine, in the form of cysteamine bitartrate (Cystagon), is currently used for the clinical treatment of nephropathic cystinosis in humans (21). Cystinosis is a lysosomal storage disease caused by mutations in the lysosomal cystine transporter cystinosin, which result in lysosomal accumulation of cystine and concomitant cytopathic effects (3). Cysteamine depletes cells of cystine in vitro and in vivo and dramatically improves the prognosis for children with cystinosis. Using a mouse model of P. chabaudi infection, we have investigated the potential antimalarial activity of cysteamine at pharmacological doses that mimic those currently used in patients suffering from cystinosis. We have also tested the potential for cysteamine to enhance the antimalarial activity of known drugs, including artemisinin derivatives. Our results show that cysteamine can strongly potentiate and synergize with artemisinin derivatives to reduce blood parasitemia and rescue lethality in P. chabaudi-infected mice.     

AAV Vectors Avoid Inflammatory Signals Necessary to Render Transduced Hepatocyte Targets for Destructive T Cells

Studies in mice indicate that gene transfer to liver with vectors based on adeno-associated viruses (AAVs) is characterized by immunological tolerance to antigenic transgene products. Mechanisms to explain host nonresponsiveness have focused on aberrant T-cell responses. We propose a distinct mechanism for conferring tolerance to AAV-transduced hepatocytes that relates to diminished sensitivity of the target organ to T cell–mediated effects. T cells to β-galactosidase (β-gal) were adoptively transferred into RAG^−/− mice expressing β-gal in hepatocytes due to prior administration of either Ad or AAV vectors. Adoptive transfer was associated with extinction of LacZ expression in Ad-LacZ-transduced RAG^−/− mice and had no effect on liver LacZ expression in AAV-LacZ-transduced RAG^−/− mice. Systemic administration of TLR ligands lipopolysaccharide (LPS) and CpG at the time of adoptive transfer did lead to extinction of LacZ expression. Systemic TLR ligands were associated with upregulation of major histocompatibility complex (MHC) class I and the cell adhesion molecules ICAM and VCAM as was seen with Ad-LacZ alone. These data indicate that AAV transduction lacks the inflammatory signals necessary to render hepatocyte targets for cytotoxic T lymphocytes (CTLs). Underlying liver pathology may confound vector performance and should be considered in the design of clinical trials.