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1.
Blood ; 129(24): 3184-3195, 2017 06 15.
Article in English | MEDLINE | ID: mdl-28468798

ABSTRACT

Adeno-associated virus (AAV) is a replication-deficient parvovirus that is extensively used as a gene therapy vector. CD8+ T-cell responses against the AAV capsid protein can, however, affect therapeutic efficacy. Little is known about the in vivo mechanism that leads to the crosspriming of CD8+ T cells against the input viral capsid antigen. In this study, we report that the Toll-like receptor 9 (TLR9)-MyD88 pattern-recognition receptor pathway is uniquely capable of initiating this response. By contrast, the absence of TLR2, STING, or the addition of TLR4 agonist has no effect. Surprisingly, both conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are required for the crosspriming of capsid-specific CD8+ T cells, whereas other antigen-presenting cells are not involved. TLR9 signaling is specifically essential in pDCs but not in cDCs, indicating that sensing of the viral genome by pDCs activates cDCs in trans to cross-present capsid antigen during CD8+ T-cell activation. Cross-presentation and crosspriming depend not only on TLR9, but also on interferon type I signaling, and both mechanisms can be inhibited by administering specific molecules to prevent induction of capsid-specific CD8+ T cells. Thus, these outcomes directly point to therapeutic interventions and demonstrate that innate immune blockade can eliminate unwanted immune responses in gene therapy.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Capsid Proteins/immunology , Dendritic Cells/immunology , Dependovirus/immunology , Lymphocyte Activation , Plasma Cells/immunology , Animals , Capsid Proteins/genetics , Dependovirus/genetics , Genetic Therapy , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/immunology
2.
Mol Ther ; 24(6): 1042-1049, 2016 06.
Article in English | MEDLINE | ID: mdl-27019999

ABSTRACT

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.


Subject(s)
Capsid Proteins/genetics , Dependovirus/genetics , Genetic Vectors/administration & dosage , Hepatocytes/ultrastructure , Animals , Cells, Cultured , Dependovirus/metabolism , Hepatocytes/metabolism , Humans , Mice , Organ Specificity , Protein Engineering , Transduction, Genetic
3.
Mol Ther Methods Clin Dev ; 2: 15029, 2015.
Article in English | MEDLINE | ID: mdl-26445723

ABSTRACT

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

5.
N Engl J Med ; 371(21): 1994-2004, 2014 Nov 20.
Article in English | MEDLINE | ID: mdl-25409372

ABSTRACT

BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).


Subject(s)
Factor IX/genetics , Genetic Therapy , Genetic Vectors/administration & dosage , Hemophilia B/therapy , Adult , Alanine Transaminase/blood , Dependovirus/genetics , Factor IX/metabolism , Follow-Up Studies , Gene Expression , Genetic Therapy/adverse effects , Hemophilia B/blood , Hemophilia B/genetics , Humans , Infusions, Intravenous , Male , Middle Aged , Transgenes , Young Adult
6.
Front Immunol ; 5: 350, 2014.
Article in English | MEDLINE | ID: mdl-25101090

ABSTRACT

Adeno-associated virus (AAV) vectors are one of the most efficient in vivo gene delivery platforms. Over the past decade, clinical trials of AAV vector-mediated gene transfer led to some of the most exciting results in the field of gene therapy and, recently, to the market approval of an AAV-based drug in Europe. With clinical development, however, it became obvious that the host immune system represents an important obstacle to successful gene transfer with AAV vectors. In this review article, we will discuss the issue of cytotoxic T cell responses directed against the AAV capsid encountered on human studies. While over the past several years the field has acquired a tremendous amount of information on the interactions of AAV vectors with the immune system, a lot of questions are still unanswered. Novel concepts are emerging, such as the relationship between the total capsid dose and the T cell-mediated clearance of transduced cells, the potential role of innate immunity in vector immunogenicity highlighted in preclinical studies, and the cross talk between regulatory and effector T cells in the determination of the outcome of gene transfer. There is still a lot to learn about immune responses in AAV gene transfer, for example, it is not well understood what are the determinants of the kinetics of activation of T cells in response to vector administration, why not all subjects develop detrimental T cell responses following gene transfer, and whether the intervention strategies currently in use to block T cell-mediated clearance of transduced cells will be safe and effective for all gene therapy indications. Results from novel preclinical models and clinical studies will help to address these points and to reach the important goal of developing safe and effective gene therapy protocols to treat human diseases.

7.
Front Immunol ; 5: 28, 2014.
Article in English | MEDLINE | ID: mdl-24570676

ABSTRACT

Transitioning to human trials from pre-clinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL) responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity and explored methods for bypassing these responses. Many efforts toward measuring innate immunity have utilized Toll-like receptor deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T-cells as well as cytotoxicity toward AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T-cell proliferation, but actual transgene level reduction and parameters of cytotoxicity toward transduced target cells have only been shown in one model. The model utilized adoptive transfer of capsid-specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells as well as modulating the response pharmacologically has also been explored.

8.
Mol Ther ; 21(9): 1727-37, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23857231

ABSTRACT

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.


Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Capsid Proteins/immunology , Dependovirus/immunology , Epitopes, T-Lymphocyte/immunology , Genetic Vectors , Immunoglobulin G/immunology , Animals , Antigens, Viral/genetics , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Capsid/immunology , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cells, Cultured , Dependovirus/genetics , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/metabolism , Genetic Therapy , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/immunology , Humans , Immunoglobulin G/genetics , Male , Mice , Mice, Inbred C57BL , Spleen/immunology , T-Lymphocytes, Regulatory/immunology
9.
Sci Transl Med ; 5(194): 194ra92, 2013 Jul 17.
Article in English | MEDLINE | ID: mdl-23863832

ABSTRACT

Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.


Subject(s)
Capsid/immunology , Dependovirus/immunology , Gene Transfer Techniques , Immunity, Humoral/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Humans , Macaca mulatta/immunology , Male , Mice , Mice, Inbred C57BL , Mutation/genetics , Neutralization Tests
10.
PLoS One ; 8(5): e61396, 2013.
Article in English | MEDLINE | ID: mdl-23667438

ABSTRACT

Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.


Subject(s)
Choroideremia/genetics , Choroideremia/therapy , Dependovirus/genetics , Genetic Therapy/methods , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line , Female , Genetic Therapy/adverse effects , Humans , Male , Mice , Plasmids/genetics , Precision Medicine , Protein Transport/genetics , Safety , rab GTP-Binding Proteins/metabolism
11.
Blood ; 121(12): 2224-33, 2013 Mar 21.
Article in English | MEDLINE | ID: mdl-23325831

ABSTRACT

Recent clinical trials have shown that evasion of CD8(+) T-cell responses against viral capsid is critical for successful liver-directed gene therapy with adeno-associated viral (AAV) vectors for hemophilia. Preclinical models to test whether use of alternate serotypes or capsid variants could avoid this deleterious response have been lacking. Here, the ability of CD8(+) T cells ("cap-CD8," specific for a capsid epitope presented by human B*0702 or murine H2-L(d) molecules) to target AAV-infected hepatocytes was investigated. In a murine model based on adoptive transfer of ex vivo expanded cap-CD8, AAV2-transduced livers showed CD8(+) T-cell infiltrates, transaminitis, significant reduction in factor IX transgene expression, and loss of transduced hepatocytes. AAV8 gene transfer resulted in prolonged susceptibility to cap-CD8, consistent with recent clinical findings. In contrast, using an AAV2(Y-F) mutant capsid, which is known to be less degraded by proteasomes, preserved transgene expression and largely avoided hepatotoxicity. In vitro assays confirmed reduced major histocompatibility complex class I presentation of this capsid and killing of human or murine hepatocytes compared with AAV2. In conclusion, AAV capsids can be engineered to substantially reduce the risk of destruction by cytotoxic T lymphocytes, whereas use of alternative serotypes per se does not circumvent this obstacle.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Capsid Proteins/immunology , Dependovirus/physiology , Genetic Therapy/methods , Genetic Vectors/physiology , Hepatocytes/immunology , Adoptive Transfer/methods , Animals , CD8-Positive T-Lymphocytes/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cells, Cultured , Dependovirus/genetics , Dependovirus/immunology , Dependovirus/metabolism , Genetic Engineering , Genetic Vectors/genetics , Hepatocytes/metabolism , Humans , Male , Mice , Mice, Inbred BALB C , T-Cell Antigen Receptor Specificity/genetics , Transduction, Genetic
12.
N Engl J Med ; 365(25): 2357-65, 2011 Dec 22.
Article in English | MEDLINE | ID: mdl-22149959

ABSTRACT

BACKGROUND: Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS: We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS: AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS: Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).


Subject(s)
Dependovirus , Factor IX/genetics , Genetic Therapy , Genetic Vectors , Hemophilia B/therapy , Adult , Dependovirus/genetics , Factor IX/therapeutic use , Genetic Therapy/adverse effects , Genetic Vectors/immunology , Humans , Infusions, Intravenous , Middle Aged , Transgenes/immunology
13.
Mol Ther ; 18(7): 1318-29, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20424599

ABSTRACT

Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector-mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4(+)FoxP3(+)IL-10(+) T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle.


Subject(s)
Dependovirus/genetics , Factor IX/genetics , Genetic Vectors/adverse effects , Genetic Vectors/genetics , Hemophilia B/therapy , Muscle, Skeletal/metabolism , Animals , CD4-Positive T-Lymphocytes/metabolism , Cell Line , Dogs , Factor IX/metabolism , Flow Cytometry , Hemophilia B/metabolism , Humans , Immunoglobulin G/metabolism , Interferon-gamma/metabolism , Interleukin-10/metabolism , Muscle, Skeletal/pathology
14.
Blood ; 114(10): 2077-86, 2009 Sep 03.
Article in English | MEDLINE | ID: mdl-19506302

ABSTRACT

In a clinical trial for adeno-associated virus serotype 1 (AAV-1)-mediated gene transfer to muscle for lipoprotein lipase (LPL) deficiency, 1 subject from the high-dose cohort experienced a transient increase in the muscle enzyme creatine phosphokinase (CPK) 4 weeks after gene transfer. Simultaneously, after an initial downward trend consistent with expression of LPL, plasma triglyceride levels returned to baseline. We characterized B- and T-cell responses to the vector and the transgene product in the subjects enrolled in this study. IFN-gamma enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining assays performed on peripheral blood mononuclear cells (PBMCs) from the subject who experienced the CPK elevation showed the activation of capsid-specific CD4(+) and CD8(+) T cells. Four of 8 subjects had detectable T-cell responses to capsid with dose-dependent kinetics of appearance. Subjects with detectable T-cell responses to capsid also had higher anti-AAV-1 IgG3 antibody titer. No subject developed B- or T-cell responses to the LPL transgene product. These findings suggest that T-cell responses directed to the AAV-1 capsid are dose-dependent. Whether they also limit the duration of expression of the transgene at higher doses is unclear, and will require additional analyses at later time points.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Capsid/immunology , Dependovirus/immunology , Genetic Therapy , Hyperlipoproteinemia Type I/immunology , Lipoprotein Lipase/immunology , Lymphocyte Activation/immunology , Muscle, Skeletal/immunology , Transgenes/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Capsid/metabolism , Creatine Kinase/biosynthesis , Creatine Kinase/immunology , Dependovirus/genetics , Dose-Response Relationship, Immunologic , Female , Humans , Hyperlipoproteinemia Type I/enzymology , Hyperlipoproteinemia Type I/genetics , Hyperlipoproteinemia Type I/therapy , Immunoenzyme Techniques , Immunoglobulin G/blood , Immunoglobulin G/immunology , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Lipoprotein Lipase/biosynthesis , Lipoprotein Lipase/genetics , Lymphocyte Activation/genetics , Male , Muscle, Skeletal/enzymology , Transduction, Genetic , Transgenes/genetics , Triglycerides/blood
15.
J Clin Invest ; 119(6): 1688-95, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19436115

ABSTRACT

Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid-derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.


Subject(s)
Antigen Presentation/immunology , Capsid Proteins/immunology , Cytotoxicity, Immunologic/immunology , Dependovirus/genetics , Genetic Vectors/genetics , Hepatocytes/immunology , Hepatocytes/metabolism , Capsid Proteins/metabolism , Cell Line , Hepatocytes/cytology , Histocompatibility Antigens/immunology , Humans , Protein Multimerization , Receptors, Antigen, T-Cell/immunology , Solubility , Substrate Specificity , T-Lymphocytes, Cytotoxic/immunology
16.
Blood ; 110(7): 2334-41, 2007 Oct 01.
Article in English | MEDLINE | ID: mdl-17609423

ABSTRACT

Adeno-associated virus (AAV)-mediated gene transfer of factor IX (F.IX) to the liver results in long-term expression of transgene in experimental animals, but only short-term expression in humans. Loss of F.IX expression is likely due to a cytotoxic immune response to the AAV capsid, which results in clearance of transduced hepatocytes. We used a nonhuman primate model to assess the safety of AAV gene transfer coupled with an anti-T-cell regimen designed to block this immune response. Administration of a 3-drug regimen consisting of mycophenolate mofetil (MMF), sirolimus, and the anti-IL-2 receptor antibody daclizumab consistently resulted in formation of inhibitory antibodies to human F.IX following hepatic artery administration of an AAV-hF.IX vector, whereas a 2-drug regimen consisting only of MMF and sirolimus did not. Administration of daclizumab was accompanied by a dramatic drop in the population of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). We conclude that choice of immunosuppression (IS) regimen can modulate immune responses to the transgene product upon hepatic gene transfer in subjects not fully tolerant; and that induction of transgene tolerance may depend on a population of antigen-specific Tregs.


Subject(s)
Adenoviridae/genetics , Immune Tolerance/immunology , Liver/immunology , Liver/metabolism , Macaca mulatta/genetics , Macaca mulatta/metabolism , Transgenes/genetics , Adenoviridae/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , B-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Daclizumab , Factor IX/genetics , Factor IX/metabolism , Gene Expression , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors/genetics , Humans , Immunoglobulin G/pharmacology , Immunosuppressive Agents/pharmacology , Male , Models, Animal , Mycophenolic Acid/analogs & derivatives , Mycophenolic Acid/pharmacology , Receptors, Interleukin-2/immunology , Sirolimus/pharmacology
17.
Eur J Cell Biol ; 86(11-12): 817-26, 2007 Dec.
Article in English | MEDLINE | ID: mdl-16928407

ABSTRACT

The skin is an attractive target for antigen-specific vaccination. Particle bombardment of the epidermis with plasmid DNA using the gene gun results in antigen expression in keratinocytes of the epidermis leading to antigen presentation in the draining lymph nodes by migratory dendritic cells (DC). In order to better understand the role of the skin in stimulating antigen-specific CD8+cytotoxic T cells (CTL), we compared gene gun immunization with intracutaneous injections of antigen-transduced DC. A single intracutaneous injection of antigen-transduced DC was able to induce in vivo expansion of CD8+CTL specific for the model antigen chicken ovalbumin while four simultaneous shots with the gene gun were not effective. Antigen-transduced DC were much more efficient than particle bombardment of the epidermis in stimulating adoptively transferred TCR-transgenic CD8+CTL in the draining lymph nodes. Employing the novel technique of in vivo bioluminescence imaging, we demonstrated efficient gene transfer to the skin following gene gun bombardment and confirmed that a similar amount of antigen reached the lymph node when compared with injection of antigen-transduced DC. Our results suggest that direct transfection of the skin does not optimally reach and activate appropriate antigen-presenting DC. We believe that this reflects the immunological function of the epidermis which must balance immunity and tolerance to foreign antigens. Further investigations will have to address the role of Langerhans cells for the activation of cellular immunity in the skin.


Subject(s)
Antigens/administration & dosage , Biolistics/methods , Dendritic Cells/immunology , Immunization/methods , Skin/immunology , T-Lymphocytes, Cytotoxic/immunology , Transduction, Genetic , Adoptive Transfer , Animals , Cell Proliferation , Dendritic Cells/cytology , Injections, Intradermal , Luciferases/metabolism , Luminescent Proteins/metabolism , Lymph Nodes/immunology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Ovalbumin , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Cytotoxic/cytology , Vaccines, DNA/immunology , Whole Body Imaging
18.
J Gene Med ; 8(11): 1300-6, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16952196

ABSTRACT

BACKGROUND: Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. Here we explore the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). METHODS: DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-alpha as well as IL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RESULTS: RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-alpha upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. CONCLUSIONS: RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches.


Subject(s)
Adenoviridae/genetics , Adenoviridae/immunology , Dendritic Cells/immunology , Interferon-alpha/biosynthesis , Toll-Like Receptor 9/physiology , Transduction, Genetic , Animals , Cell Differentiation , Dendritic Cells/cytology , In Vitro Techniques , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligodeoxyribonucleotides/genetics , Toll-Like Receptor 9/deficiency , Toll-Like Receptor 9/genetics
19.
Am J Pathol ; 169(2): 665-72, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16877364

ABSTRACT

Currently, novel mouse models of melanoma are being generated that recapitulate the histopathology and molecular pathogenesis observed in human disease. Impaired cell-cycle control, which is a hallmark of both familial and sporadic melanoma, promotes slowly growing carcinogen-induced melanomas in the skin of mice carrying a mutated cyclin-dependent kinase 4 (CDK4(R24C)). Deregulated receptor tyrosine kinase signaling, which is another important feature of human melanoma, leads to spontaneous development of metastatic melanoma after a long latency period in mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF mice). Here we report that treatment with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate induced metastatic melanomas in all HGF/SF mice on the C57BL/6 background, which histologically resemble human melanoma. Importantly, mutant CDK4 dramatically increased the number and the growth kinetics of carcinogen-induced primary melanomas in the skin and promoted the growth of spontaneous metastases in lymph nodes and lungs in all HGF/SF mice within the first 3 months of life. Apart from very few skin papillomas, we did not observe tumors of other histology in carcinogen-treated HGF/SF x CDK4(R24C) mice. This new experimental mouse model can now be exploited to study further the biology of melanoma and evaluate new treatment modalities.


Subject(s)
Cyclin-Dependent Kinase 4/genetics , Hepatocyte Growth Factor/genetics , Melanoma, Experimental/chemically induced , Mutation/genetics , Neoplasm Invasiveness/pathology , Neoplasm Metastasis/pathology , Skin Neoplasms/pathology , Animals , Animals, Newborn , Carcinogens , Disease Models, Animal , Gene Expression , Humans , Lung/pathology , Lymph Nodes/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutant Proteins/genetics
20.
Cancer Res ; 66(10): 5427-35, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16707471

ABSTRACT

Malignant melanoma is an attractive model disease for the development of antigen-specific immunotherapy because many antigens recognized by tumor-specific T cells have been identified. In C57BL/6 mice, genetic immunization with recombinant adenovirus encoding xenogeneic human tyrosinase-related protein 2 (Ad-hTRP2) induces protective but not therapeutic cellular immunity against growth of transplanted B16 melanoma cells. Here, we additionally applied CpG DNA and synthetic double-stranded RNA, which activate the innate immune system via Toll-like receptors (TLR). Both adenoviral vaccination and peritumoral injections of TLR ligands were required for rejection of established B16 melanoma in the skin. To more closely mimic the clinical situation in patients with melanoma, we evaluated this combined immunotherapeutic strategy in genetically modified mice, which overexpress hepatocyte growth factor (HGF) and carry an oncogenic mutation in the cyclin-dependent kinase 4 (CDK4)(R24C). HGF x CDK4(R24C) mice rapidly develop multiple invasive melanomas in the skin following neonatal carcinogen treatment, which spontaneously metastasize to lymph nodes and lungs. Vaccination with Ad-hTRP2 followed by injections of TLR ligands resulted in delayed growth of autochthonous primary melanomas in the skin and reduction in the number of spontaneous lung metastases but did not induce tumor regression. Carcinogen-treated HGF x CDK4(R24C) mice bearing multiple autochthonous melanomas did not reject transplanted B16 melanoma despite treatment with Ad-hTRP2 and TLR ligands, suggesting the development of tumor immunotolerance. Further investigations in our novel genetic melanoma model may help to better understand the role of the immune system in the pathogenesis and treatment of this life-threatening disease.


Subject(s)
Cancer Vaccines/pharmacology , Intramolecular Oxidoreductases/immunology , Melanoma, Experimental/therapy , Skin Neoplasms/therapy , Toll-Like Receptors/immunology , Vaccines, DNA/pharmacology , Adenoviridae/genetics , Adenoviridae/immunology , Animals , Cancer Vaccines/immunology , CpG Islands/genetics , CpG Islands/immunology , Cyclin-Dependent Kinase 4/genetics , Epitopes , Female , Hepatocyte Growth Factor/genetics , Humans , Intramolecular Oxidoreductases/genetics , Lung Neoplasms/secondary , Male , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/secondary , Mice , Mice, Inbred C57BL , Mice, Transgenic , Poly I-C/pharmacology , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Vaccines, DNA/immunology
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