RESUMEN
Food allergy (FA) is estimated to impact up to 10% of the population and is a growing health concern. FA results from a failure in the mucosal immune system to establish or maintain immunological tolerance to innocuous dietary antigens, IgE production, and the release of histamine and other mediators upon exposure to a food allergen. Of the different FAs, peanut allergy has the highest incidence of severe allergic responses, including systemic anaphylaxis. Despite the recent FDA approval of peanut oral immunotherapy and other investigational immunotherapies, a loss of protection following cessation of therapy can occur, suggesting that these therapies do not address the underlying immune response driving FA. Our lab has shown that liver-directed gene therapy with an adeno-associated virus (AAV) vector induces transgene product-specific regulatory T cells (Tregs), eradicates pre-existing pathogenic antibodies, and protects against anaphylaxis in several models, including ovalbumin induced FA. In an epicutaneous peanut allergy mouse model, the hepatic AAV co-expression of four peanut antigens Ara h1, Ara h2, Ara h3, and Ara h6 together or the single expression of Ara h3 prevented the development of a peanut allergy. Since FA patients show a reduction in Treg numbers and/or function, we believe our approach may address this unmet need.
Asunto(s)
Dependovirus , Vectores Genéticos , Hipersensibilidad al Cacahuete , Hipersensibilidad al Cacahuete/terapia , Hipersensibilidad al Cacahuete/inmunología , Animales , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Dependovirus/genética , Dependovirus/inmunología , Terapia Genética/métodos , Linfocitos T Reguladores/inmunología , Ratones , Inmunoterapia/métodos , Modelos Animales de Enfermedad , Desensibilización Inmunológica/métodos , Alérgenos/inmunología , Arachis/inmunologíaRESUMEN
Adeno-associated virus (AAV) vectors are used for correcting multiple genetic disorders. Although the goal is to achieve lifelong correction with a single vector administration, the ability to redose would enable the extension of therapy in cases in which initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAbs) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell-targeting protocol using a combination of monoclonal Ab therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression before (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig-mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.
RESUMEN
Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.
Asunto(s)
Activación de Complemento , Proteínas del Sistema Complemento , Dependovirus , Terapia Genética , Vectores Genéticos , Dependovirus/genética , Dependovirus/inmunología , Humanos , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Terapia Genética/métodos , Terapia Genética/efectos adversos , Proteínas del Sistema Complemento/inmunología , Animales , Técnicas de Transferencia de GenRESUMEN
Recombinant adeno-associated viruses (AAVs) have emerged as a widely used gene delivery platform for both basic research and human gene therapy. To ensure and improve the safety profile of AAV vectors, substantial efforts have been dedicated to the vector production process development using suspension HEK293 cells. Here, we studied and compared two downstream purification methods, iodixanol gradient ultracentrifugation versus immuno-affinity chromatography (POROS™ CaptureSelect™ AAVX column). We tested multiple vector batches that were separately produced (including AAV5, AAV8, and AAV9 serotypes). To account for batch-to-batch variability, each batch was halved for subsequent purification by either iodixanol gradient centrifugation or affinity chromatography. In parallel, purified vectors were characterized, and transduction was compared both in vitro and in vivo in mice (using multiple transgenes: Gaussia luciferase, eGFP, and human factor IX). Each purification method was found to have its own advantages and disadvantages regarding purity, viral genome (vg) recovery, and relative empty particle content. Differences in transduction efficiency were found to reflect batch-to-batch variability rather than disparities between the two purification methods, which were similarly capable of yielding potent AAV vectors.
RESUMEN
Loss of oral tolerance (OT) to food antigens results in food allergies. One component of achieving OT is the symbiotic microorganisms living in the gut (microbiota). The composition of the microbiota can drive either pro-tolerogenic or pro-inflammatory responses against dietary antigens though interactions with the local immune cells within the gut. Products from bacterial fermentation, such as butyrate, are one of the main communication molecules involved in this interaction, however, this is released by a subset of bacterial species. Thus, strategies to specifically expand these bacteria with protolerogenic properties have been explored to complement oral immunotherapy in food allergy. These approaches either provide digestible biomolecules to induce beneficial bacteria species (prebiotics) or the direct administration of live bacteria species (probiotics). While this combined therapy has shown positive outcomes in clinical trials for cow's milk allergy, more research is needed to determine if this therapy can be extended to other food allergens.
Asunto(s)
Hipersensibilidad a los Alimentos , Microbioma Gastrointestinal , Microbiota , Hipersensibilidad a la Leche , Probióticos , Bovinos , Animales , Femenino , Hipersensibilidad a los Alimentos/terapia , Hipersensibilidad a la Leche/microbiología , Probióticos/uso terapéutico , BacteriasRESUMEN
Immunotherapies for patients with food allergy have shown some success in limiting allergic responses. However, these approaches require lengthy protocols with repeated allergen dosing and patients can relapse following discontinuation of treatment. The purpose of this study was to test if a single dose of an adeno-associated virus (AAV) vector can safely prevent and treat egg allergy in a mouse model. AAV vectors expressing ovalbumin (OVA) under an ubiquitous or liver-specific promoter were injected prior to or after epicutaneous sensitization with OVA. Mice treated with either AAV8-OVA vector were completely protected from allergy sensitization. These animals had a significant reduction in anaphylaxis mediated by a reduction in OVA-specific IgE titers. In mice with established OVA allergy, allergic responses were mitigated only in mice treated with an AAV8-OVA vector expressing OVA from an ubiquitous promoter. In conclusion, an AAV vector with a liver-specific promoter was more effective for allergy prevention, but higher OVA levels were necessary for reducing symptoms in preexisting allergy. Overall, our AAV gene immunotherapy resulted in an expansion of OVA-specific FoxP3+ CD4+ T cells, an increase in the regulatory cytokine IL-10, and a reduction in the IgE promoting cytokine IL-13.
RESUMEN
Hepatic gene transfer with adeno-associated viral (AAV) vectors shows much promise for the treatment of the X-linked bleeding disorder hemophilia B in multiple clinical trials. In an effort to further innovate this approach and to introduce alternative vector designs with potentially superior features into clinical development, we recently built a vector platform based on AAV serotype 3 because of its superior tropism for human hepatocytes. A vector genome with serotype-matched inverted terminal repeats expressing hyperactive human coagulation factor IX (FIX)-Padua was designed for clinical use that is optimized for translation using hepatocyte-specific codon-usage bias and is depleted of immune stimulatory CpG motifs. Here, this vector genome was packaged into AAV3 (T492V + S663V) capsid for hepatic gene transfer in non-human primates. FIX activity within or near the normal range was obtained at a low vector dose of 5 × 1011 vector genomes/kg. Pre-existing neutralizing antibodies, however, completely or partially blocked hepatic gene transfer at that dose. No CD8+ T cell response against capsid was observed. Antibodies against the human FIX transgene product formed at a 10-fold higher vector dose, albeit hepatic gene transfer was remarkably consistent, and sustained FIX activity in the normal range was nonetheless achieved in two of three animals for the 3-month duration of the study. These results support the use of this vector at low vector doses for gene therapy of hemophilia B in humans.
RESUMEN
Although recombinant adeno-associated virus serotype 8 (AAV8) and serotype 5 (AAV5) vectors have shown efficacy in Phase 1 clinical trials for gene therapy of hemophilia B, it has become increasingly clear that these serotypes are not optimal for transducing primary human hepatocytes. We have previously reported that among the 10 most commonly used AAV serotypes, AAV serotype 3 (AAV3) vectors are the most efficient in transducing primary human hepatocytes in vitro as well as in "humanized" mice in vivo, and suggested that AAV3 vectors expressing human coagulation factor IX (hFIX) may be a more efficient alternative for clinical gene therapy of hemophilia B. In the present study, we extended these findings to develop an AAV3 vector incorporating a compact yet powerful liver-directed promoter as well as optimized hFIX cDNA sequence inserted between two AAV3 inverted terminal repeats. When packaged into an AAV3 capsid, this vector yields therapeutic levels of hFIX in hemophilia B and in "humanized" mice in vivo. Together, these studies have resulted in an AAV3 vector predicted to achieve clinical efficacy at reduced vector doses, without the need for immune-suppression, for clinical gene therapy of hemophilia B.
Asunto(s)
Dependovirus/genética , Factor IX/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Hemofilia B/terapia , Hígado/metabolismo , Animales , Vectores Genéticos/genética , Hemofilia B/genética , Hemofilia B/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Transducción Genética , TransgenesRESUMEN
Hemophilia A is an inherited coagulation disorder resulting in the loss of functional clotting factor VIII (FVIII). Presently, the most effective treatment is prophylactic protein replacement therapy. However, this requires frequent life-long intravenous infusions of plasma derived or recombinant clotting factors and is not a cure. A major complication is the development of inhibitory antibodies that nullify the replacement factor. Immune tolerance induction (ITI) therapy to reverse inhibitors can last from months to years, requires daily or every other day infusions of supraphysiological levels of FVIII and is effective in only up to 70% of hemophilia A patients. Preclinical and recent clinical studies have shown that gene replacement therapy with AAV vectors can effectively cure hemophilia A patients. However, it is unclear how hemophilia patients with high risk inhibitor F8 mutations or with established inhibitors will respond to gene therapy, as these patients have been excluded from ongoing clinical trials. AAV8-coF8 gene transfer in naïve BALB/c-F8e16-/Y mice (BALB/c-HA) results in anti-FVIII IgG1 inhibitors following gene transfer, which can be prevented by transient immune modulation with anti-mCD20 (18B12) and oral rapamycin. We investigated if we could improve ITI in inhibitor positive mice by combining anti-mCD20 and rapamycin with AAV8-coF8 gene therapy. Our hypothesis was that continuous expression of FVIII protein from gene transfer compared to transient FVIII from weekly protein therapy, would enhance regulatory T cell induction and promote deletion of FVIII reactive B cells, following reconstitution. Mice that received anti-CD20 had a sharp decline in inhibitors, which corresponded to FVIII memory B (Bmem) cell deletion. Importantly, only mice receiving both anti-mCD20 and rapamycin failed to increase inhibitors following rechallenge with intravenous FVIII protein therapy. Our data show that B and T cell immune modulation complements AAV8-coF8 gene therapy in naïve and inhibitor positive hemophilia A mice and suggest that such protocols should be considered for AAV gene therapy in high risk or inhibitor positive hemophilia patients.
Asunto(s)
Linfocitos B/inmunología , Factor VIII/genética , Factor VIII/inmunología , Tolerancia Inmunológica , Memoria Inmunológica , Depleción Linfocítica , Sirolimus/farmacología , Traslado Adoptivo , Animales , Linfocitos B/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos/genética , Hemofilia A/genética , Hemofilia A/inmunología , Hemofilia A/terapia , Inmunosupresores/farmacología , Masculino , Ratones , Ratones Transgénicos , Rituximab/farmacología , Especificidad de la EspecieRESUMEN
A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by â¼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.
Asunto(s)
Dependovirus/genética , Antígenos de Histocompatibilidad Clase II/genética , Inmunidad Humoral , Secuencias Repetidas Terminales , Animales , ADN Viral , Regulación hacia Abajo , Regulación de la Expresión Génica , Genes MHC Clase II , Terapia Genética , Vectores Genéticos , Células HEK293 , Antígenos HLA/genética , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Factor Regulador X1/genética , Homología de SecuenciaAsunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Terapia Genética , Inmunidad , Tratamiento Basado en Trasplante de Células y Tejidos/efectos adversos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Terapia Genética/efectos adversos , Terapia Genética/métodos , HumanosRESUMEN
Early preclinical studies in rodents and other species did not reveal that vector or transgene immunity would present a significant hurdle for sustained gene expression. While there was early evidence of mild immune responses to adeno-associated virus (AAV) in preclinical studies, it was generally believed that these responses were too weak and transient to negatively impact sustained transduction. However, translation of the cumulative success in treating hemophilia B in rodents and dogs with an AAV2-F9 vector to human studies was not as successful. Despite significant progress in recent clinical trials for hemophilia, new immunotoxicities to AAV and transgene are emerging in humans that require better animal models to assess and overcome these responses. The animal models designed to address these immune complications have provided critical information to assess how vector dose, vector capsid processing, vector genome, difference in serotypes, and variations in vector delivery route can impact immunity and to develop approaches for overcoming pre-existing immunity. Additionally, a comprehensive dissection of innate, adaptive, and regulatory responses to AAV vectors in preclinical studies has provided a framework that can be utilized for development of immunomodulatory therapies to overcome or bypass immune responses and for developing strategic approaches toward engineering stealth AAV vectors that can circumvent immunity.
RESUMEN
Adeno-associated virus (AAV) vectors are widely used in clinical gene therapy to correct genetic disease by in vivo gene transfer. Although the vectors are useful, in part because of their limited immunogenicity, immune responses directed at vector components have complicated applications in humans. These include, for instance, innate immune sensing of vector components by plasmacytoid dendritic cells (pDCs), which sense the vector DNA genome via Toll-like receptor 9. Adaptive immune responses employ antigen presentation by conventional dendritic cells (cDCs), which leads to cross-priming of capsid-specific CD8+ T cells. In this study, we sought to determine the mechanisms that promote licensing of cDCs, which is requisite for CD8+ T cell activation. Blockage of type 1 interferon (T1 IFN) signaling by monoclonal antibody therapy prevented cross-priming. Furthermore, experiments in cell-type-restricted knockout mice showed a specific requirement for the receptor for T1 IFN (IFNaR) in cDCs. In contrast, natural killer (NK) cells are not needed, indicating a direct rather than indirect effect of T1 IFN on cDCs. In addition, co-stimulation by CD4+ T cells via CD40-CD40L was required for cross-priming, and blockage of co-stimulation but not of T1 IFN additionally reduced antibody formation against capsid. These mechanistic insights inform the development of targeted immune interventions.
Asunto(s)
Cápside/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Interferón Tipo I/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos CD40/metabolismo , Ligando de CD40/metabolismo , Proteínas de la Cápside/inmunología , Dependovirus/inmunología , Eliminación de Gen , Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Modelos Biológicos , Receptor de Interferón alfa y beta/genética , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Adeno-associated virus (AAV) vectors to treat liver-specific genetic diseases are the focus of several ongoing clinical trials. The ability to give a peripheral injection of virus that will successfully target the liver is one of many attractive features of this technology. Although initial studies of AAV liver gene transfer revealed some limitations, extensive animal modeling and further clinical development have helped solve some of these issues, resulting in several successful clinical trials that have reached curative levels of clotting factor expression in hemophilia. Looking beyond gene replacement, recent technologies offer the possibility for AAV liver gene transfer to directly repair deficient genes and potentially treat autoimmune disease.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Ingeniería Genética , Vectores Genéticos/genética , Hígado/metabolismo , Animales , Biotecnología , Cápside/inmunología , Cápside/metabolismo , Dependovirus/inmunología , Modelos Animales de Enfermedad , Edición Génica , Expresión Génica , Ingeniería Genética/métodos , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Genoma Viral , Hepatocitos/metabolismo , Humanos , Modelos Animales , Especificidad de Órganos , Regiones Promotoras GenéticasRESUMEN
In contrast to other diverse therapies for the X-linked bleeding disorder hemophilia that are currently in clinical development, gene therapy holds the promise of a lasting cure with a single drug administration. Near-to-complete correction of hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency) have now been achieved in patients by hepatic in vivo gene transfer. Adeno-associated viral vectors with different viral capsids that have been engineered to express high-level, and in some cases hyperactive, coagulation factors were employed. Patient data support that sustained endogenous production of clotting factor as a result of gene therapy eliminates the need for infusion of coagulation factors (or alternative drugs that promote coagulation), and may therefore ultimately also reduce treatment costs. However, mild liver toxicities have been observed in some patients receiving high vector doses. In some but not all instances, the toxicities correlated with a T-cell response directed against the viral capsid, prompting use of immune suppression. In addition, not all patients can be treated because of preexisting immunity to viral capsids. Nonetheless, studies in animal models of hemophilia suggest that the approach can also be used for immune tolerance induction to prevent or eliminate inhibitory antibodies against coagulation factors. These can form in traditional protein replacement therapy and represent a major complication of treatment. The current review provides a summary and update on advances in clinical gene therapies for hemophilia and its continued development.
Asunto(s)
Terapia Genética/métodos , Hemofilia A/terapia , Hemofilia B/terapia , Animales , Factores de Coagulación Sanguínea/genética , Dependovirus/genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Hemofilia A/genética , Hemofilia B/genética , HumanosRESUMEN
Immune tolerance is a vital component of immunity, as persistent activation of immune cells causes significant tissue damage and loss of tolerance leads to autoimmunity. Likewise, unwanted immune responses can occur in inherited disorders, such as hemophilia and Pompe disease, in which patients lack any expression of protein, during treatment with enzyme replacement therapy, or gene therapy. While the liver has long been known as being tolerogenic, it was only recently appreciated in the last decade that liver directed adeno-associated virus (AAV) gene therapy can induce systemic tolerance to a transgene. In this review, we look at the mechanisms behind liver induced tolerance, discuss different factors influencing successful tolerance induction with AAV, and applications where AAV mediated tolerance may be helpful.