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2.
J Struct Biol ; 186(2): 308-17, 2014 May.
Article in English | MEDLINE | ID: mdl-24704217

ABSTRACT

The Adeno-associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ∼40-70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded ß-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral twofold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ∼61%, ∼81%, and ∼63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system.


Subject(s)
Capsid/ultrastructure , Dependovirus/chemistry , Gene Transfer Techniques , Genetic Vectors/chemistry , Models, Molecular , Amino Acid Sequence , Crystallography, X-Ray , Genetic Vectors/genetics , Genetic Vectors/ultrastructure , Humans , Molecular Sequence Data , Protein Conformation
3.
Mol Ther ; 22(1): 28-41, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23778424

ABSTRACT

Following gene transfer of adeno-associated virus 2/8 (AAV2/8) to the muscle, C57BL/6 mice show long-term expression of a nuclear-targeted LacZ (nLacZ) transgene with minimal immune activation. Here, we show that pre-exposure to AAV2/8 can also induce tolerance to the more immunogenic AAV2/rh32.33 vector, preventing otherwise robust T-cell activation and allowing stable transgene expression. Depletion and adoptive transfer studies showed that a suppressive factor was not sufficient to account for AAV2/8-induced tolerance, whereas further characterization of the T-cell population showed upregulation of the exhaustion markers PD1, 2B4, and LAG3. Furthermore, systemic administration of Toll-like receptor (TLR) ligands at the time of AAV2/rh32.33-administration broke AAV2/8-induced tolerance, restoring T-cell activation and ß-gal clearance. As such, AAV2/8 transduction appears to lack the inflammatory signals necessary to prime a functional cytotoxic T-cell response. Inadequate T-cell priming could be explained upstream by AAV2/8's poor transduction and activation of antigen-presenting cells (APCs). Immunohistochemical analysis indicates that AAV2/8 transduction also fails to upregulate major histocompatibility complex class I (MHCI) expression on the surface of myocytes, rendering transduced cells poor targets for T-cell-mediated destruction. Overall, AAV2/8-induced tolerance in the muscle is multifactorial, spanning from poor APC transduction and activation to the subsequent priming of functionally exhausted T-cells, while simultaneously avoiding upregulation of MHCI on potential targets.


Subject(s)
Antigen-Presenting Cells/immunology , Dependovirus/immunology , Genetic Vectors/immunology , Histocompatibility Antigens Class I/immunology , Immune Tolerance , Muscle, Skeletal/immunology , T-Lymphocyte Subsets/immunology , Transduction, Genetic , Animals , Antigen-Presenting Cells/metabolism , Dependovirus/genetics , Gene Expression , Genetic Vectors/genetics , Humans , Inflammation/immunology , Inflammation/metabolism , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Regulatory/immunology , Toll-Like Receptors/metabolism , Transgenes/genetics , Transgenes/immunology
4.
J Virol ; 87(17): 9473-85, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23720715

ABSTRACT

Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8(+) T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33.


Subject(s)
Dependovirus/immunology , Macaca mulatta/virology , T-Lymphocytes/immunology , T-Lymphocytes/virology , Amino Acid Sequence , Animals , Capsid/immunology , Capsid Proteins/chemistry , Capsid Proteins/genetics , Capsid Proteins/immunology , Dependovirus/classification , Dependovirus/genetics , Epitope Mapping , Hybridization, Genetic , Lymphocyte Activation , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid , Serotyping
5.
J Clin Invest ; 123(3): 1216-28, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23391720

ABSTRACT

Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and Th17 inflammation by regulating innate immune cell recruitment through an IL-10-dependent pathway. The protective effects of FOXP3 could be reversed by depletion of IL-10 or administration of recombinant IL-17A or IL-23. Delivery of Foxp3 mRNA to sites of inflammation may offer a novel, safe therapeutic tool for the treatment of allergic asthma and other diseases driven by an imbalance in helper T cell responses.


Subject(s)
Asthma/prevention & control , Forkhead Transcription Factors/genetics , Interleukin-10/metabolism , RNA, Messenger/genetics , Airway Remodeling , Airway Resistance , Animals , Asthma/immunology , Asthma/metabolism , Cell Line , Cytidine/analogs & derivatives , Cytidine/chemistry , Female , Forkhead Transcription Factors/biosynthesis , Gene Expression , Genetic Therapy , Humans , Immunity, Innate , Inflammation Mediators/pharmacology , Inflammation Mediators/physiology , Interleukin-17/pharmacology , Interleukin-17/physiology , Interleukin-23/pharmacology , Interleukin-23/physiology , Mice , Mice, Inbred BALB C , Mice, Knockout , Pyroglyphidae/immunology , RNA, Messenger/chemistry , Th17 Cells/immunology , Th17 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism , Thiouridine/analogs & derivatives , Thiouridine/chemistry , Transfection
6.
Eur Respir J ; 39(6): 1385-90, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22088968

ABSTRACT

Cystic fibrosis (CF) lung disease severity is largely independent on the CF transmembrane conductance regulator (CFTR) genotype, indicating the contribution of genetic modifiers. The chemokine receptors CXCR1 and CXCR2 have been found to play essential roles in the pathogenesis of CF lung disease. Here, we determine whether genetic variation of CXCR1 and CXCR2 influences CF lung disease severity. Genomic DNA of CF patients in Germany (n = 442) was analysed for common variations in CXCR1 and CXCR2 using a single-nucleotide polymorphism (SNP) tagging approach. Associations of CXCR1 and CXCR2 SNPs and haplotypes with CF lung disease severity, CXCR1 and CXCR2 expression, and neutrophil effector functions were assessed. Four SNPs in CXCR1 and three in CXCR2 strongly correlated with age-adjusted lung function in CF patients. SNPs comprising haplotypes CXCR1_Ha and CXCR2_Ha were in high linkage disequilibrium and patients heterozygous for the CXCR1-2 haplotype cluster (CXCR1-2_Ha) had lower lung function compared with patients with homozygous wild-type alleles (forced expiratory volume in 1 s ≤ 70% predicted, OR 7.24; p = 2.30 × 10(-5)). CF patients carrying CXCR1-2_Ha showed decreased CXCR1 combined with increased CXCR2 mRNA and protein expression, and displayed disturbed antibacterial effector functions. CXCR1 and CXCR2 genotypes modulate lung function and antibacterial host defence in CF lung disease.


Subject(s)
Cystic Fibrosis/immunology , Haplotypes/genetics , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8B/genetics , Adolescent , Adult , Child , Cystic Fibrosis/microbiology , Female , Genetic Variation , Germany , Humans , Linkage Disequilibrium/genetics , Lung/immunology , Lung/physiology , Male , Neutrophils/immunology , Neutrophils/microbiology , Pneumonia, Bacterial/genetics , Pneumonia, Bacterial/immunology , Polymorphism, Single Nucleotide , Receptors, Interleukin-8A/biosynthesis , Receptors, Interleukin-8A/immunology , Receptors, Interleukin-8B/biosynthesis , Receptors, Interleukin-8B/immunology , Severity of Illness Index , Young Adult
7.
Nat Biotechnol ; 29(2): 154-7, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21217696

ABSTRACT

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.


Subject(s)
Erythropoietin/biosynthesis , Gene Transfer Techniques , Proteolipids/biosynthesis , RNA, Messenger/administration & dosage , Animals , Erythropoietin/genetics , Histocytochemistry , Kaplan-Meier Estimate , Lung/metabolism , Mice , Mice, Transgenic , Proteolipids/genetics , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics
8.
Mol Ther ; 19(1): 16-27, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21119617

ABSTRACT

Original reports of adeno-associated virus (AAV) vector-mediated gene transfer to the muscle resulted in high-level ß-galactosidase (ß-gal) expression and the promise of a viral vector that was largely nonimmunogenic. Subsequent attempts to utilize these vectors for genetic vaccination, however, demonstrated that it was possible to activate cellular and humoral immunity to AAV-encoded antigens. These findings fueled years of investigation into factors impacting the immunogenicity of recombinant AAV-mediated gene delivery, including route of administration, dose, host species, capsid serotype, and transgene product. In cases where AAV vectors could avoid transgene-directed immunity, it became clear that mechanisms of tolerance were at work, varying between ignorance, anergy/deletion, or active suppression. Here, we follow the field of AAV gene therapy from inception, as investigators have worked to understand the delicate balance between AAV-mediated tolerance and the activation of immunity. This review discusses our current appreciation of AAV vector immunology, with a specific focus on the transgene-specific T cell response.


Subject(s)
Capsid Proteins/immunology , Dependovirus/immunology , Genetic Therapy/methods , Genetic Vectors/immunology , T-Lymphocytes/immunology , Transgenes/immunology , Animals , Capsid Proteins/administration & dosage , Capsid Proteins/genetics , Dependovirus/genetics , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Humans , Immune Tolerance/immunology , Lymphocyte Activation/immunology , Transgenes/genetics
9.
J Gene Med ; 11(12): 1095-102, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19777488

ABSTRACT

BACKGROUND: Adeno-associated virus (AAV) is an ideal gene therapy vector and is non-immunogenic in many small animal models. The stable gene expression commonly seen in murine models does not necessarily translate to nonhuman primates and higher-order species, highlighting the need for a better understanding of immune activation to these vectors. One capsid variant, AAVrh32.33, demonstrates a unique phenotype in murine muscle, reminiscent of what is often seen in higher-order species. AAVrh32.33 generates a strong CD8+ T-cell response to both capsid and encoded transgene antigens in a manner independent of transgene product or major histocompatability complex haplotype, making it an ideal candidate for studying immune activation to AAV in the mouse. METHODS: To map the H-2b and H-2d dominant epitopes of the AAVrh32.33 capsid, C57BL/6 or Balb/C mice received an intramuscular injection of 1 x 10(11) genome copies of AAV2/rh32.33.CB.nLacZ. Three weeks later, splenocytes were harvested and stimulated in vitro with pooled or individual peptides from the AAVrh32.33 capsid peptide library and analysed by an interferon (IFN)-gamma enzyme-linked immunosorbent spot assay or intracellular cytokine staining. RESULTS: The immunodominant epitopes within the AAVrh32.33 capsid responsible for driving CD8+ T-cell responses to the capsid protein in C57BL/6 (SSYELPYVM) and Balb/C (KIPASGGNAL) mice were defined. CONCLUSIONS: Identification of dominant capsid epitopes will make it possible to monitor cellular responses to the AAV capsid in vivo, facilitating mechanistic studies critical to defining how cellular immunity to the AAV capsid arises and, ultimately, how the generation of capsid-specific T cells can be avoided to ensure safety in a gene therapy setting.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Capsid/immunology , Dependovirus/immunology , Epitopes, T-Lymphocyte/immunology , Genetic Vectors/immunology , H-2 Antigens/immunology , Animals , Enzyme-Linked Immunosorbent Assay , Epitopes, T-Lymphocyte/genetics , Genetic Vectors/genetics , H-2 Antigens/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Transgenes/physiology
10.
J Immunol ; 182(10): 6051-60, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19414756

ABSTRACT

The immunological sequelae of adeno-associated virus (AAV)-mediated gene transfer in vivo is quite complex. In murine models, most AAV capsids are associated with minimal or dysfunctional T cell responses to antigenic transgene products. In this study we compared T cell activation against AAV2/8 and AAV2/rh32.33 vectors expressing nuclear-targeted LacZ (nLacZ), GFP, or firefly luciferase in murine skeletal muscle. We show that, unlike AAV8, AAVrh32.33 yields qualitatively and quantitatively robust T cell responses to both the capsid and transgene product. AAV2/rh32.33.CB.nLacZ, but not AAV2/8, drives a high degree of cellular infiltration and a loss of detectable transgene expression in C57BL/6 mice. However, cellular immunity to AAVrh32.33 is ablated in the absence of CD4, CD40L, or CD28, permitting stable beta-galactosidase expression. Treatment of CD40L(-/-) mice with the CD40 agonist, FGK45, failed to restore the CD8 response to AAV2/rh32.33.nLacZ, suggesting that additional factors are involved. Our results suggest that specific domains within the AAVrh32.33 capsid augment the adaptive response to both capsid and transgene Ags in a CD4-dependent pathway involving CD40L signaling and CD28 costimulation. Structural comparison of the AAV8 and rh32.33 capsids has identified key differences that may drive differential immunity by affecting tropism, Ag presentation or the activation of innate immunity. This murine model of AAV-mediated cytotoxicity allows us to delineate the mechanism of viral immune activation, which is relevant to the translation of AAV technology in higher order species.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Capsid/immunology , Dependovirus/immunology , Genetic Vectors/immunology , Lymphocyte Activation/immunology , Animals , CD28 Antigens/genetics , CD28 Antigens/immunology , CD4 Antigens/immunology , CD40 Antigens/immunology , CD40 Ligand/deficiency , CD40 Ligand/genetics , CD40 Ligand/immunology , Green Fluorescent Proteins/genetics , Lac Operon , Luciferases, Firefly/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Muscle, Skeletal/immunology , Muscle, Skeletal/metabolism , Transgenes/immunology
11.
Cell Res ; 17(11): 904-18, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17923863

ABSTRACT

Central tolerance in the thymus is the primary mechanism for deleting autoreactive T cells. Despite this, escape of self-reactive T lymphocytes into the periphery reveals the threat of autoimmunity. To compensate for its imperfection, the thymus also produces a naturally occurring subset of Foxp3+ CD4+ CD25+ regulatory T cells with suppressive function, capable of controlling autoreactive cells. Foxp3 (forkhead box P3), the lineage-specific marker for this subset of cells, is crucial to their thymic development and peripheral function, and yet the transcriptional program driven by Foxp3 was until now largely undefined. Emerging evidence has provided insight into its role: from the ability of Foxp3 to cooperate with other transcription factors such as NFAT, to the genome-wide characterization of target genes directly bound and regulated by Foxp3. Here we discuss the discovery of naturally occurring regulatory T cells - their phenotype, development, maintenance, and function - largely as they are defined by the lineage-specific marker, Foxp3.


Subject(s)
Antigens, Differentiation/immunology , Forkhead Transcription Factors/immunology , Gene Expression Regulation/physiology , Immune Tolerance/physiology , NFATC Transcription Factors/immunology , Thymus Gland/immunology , Animals , Antigens, Differentiation/metabolism , Forkhead Transcription Factors/metabolism , Humans , NFATC Transcription Factors/metabolism , T-Lymphocytes, Regulatory , Thymus Gland/growth & development , Thymus Gland/metabolism
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