B cell focused transient immune suppression protocol for efficient AAV readministration to the liver.

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.

Complement System Response to Adeno-Associated Virus Vector Gene Therapy.

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.

Manipulating the microbiome to enhance oral tolerance in food allergy.

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.

Single-dose AAV vector gene immunotherapy to treat food allergy.

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.

Coagulation factor IX gene transfer to non-human primates using engineered AAV3 capsid and hepatic optimized expression cassette.

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.

Development of a Clinical Candidate AAV3 Vector for Gene Therapy of Hemophilia B.

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.

B Cell Depletion Eliminates FVIII Memory B Cells and Enhances AAV8-coF8 Immune Tolerance Induction When Combined With Rapamycin.

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.

Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response.

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 ∼93% 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.

Immune Response Mechanisms against AAV Vectors in Animal Models.

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.

Type I IFN Sensing by cDCs and CD4+ T Cell Help Are Both Requisite for Cross-Priming of AAV Capsid-Specific CD8+ T Cells.

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.

AAV-Mediated Gene Delivery to the Liver: Overview of Current Technologies and Methods.

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.

Liver induced transgene tolerance with AAV vectors.

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.

Update on clinical gene therapy for hemophilia.

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.

Gene Therapy-Induced Antigen-Specific Tregs Inhibit Neuro-inflammation and Reverse Disease in a Mouse Model of Multiple Sclerosis.

The devastating neurodegenerative disease multiple sclerosis (MS) could substantially benefit from an adeno-associated virus (AAV) immunotherapy designed to restore a robust and durable antigen-specific tolerance. However, developing a sufficiently potent and lasting immune-regulatory therapy that can intervene in ongoing disease is a major challenge and has thus been elusive. We addressed this problem by developing a highly effective and robust tolerance-inducing in vivo gene therapy. Using a pre-clinical animal model, we designed a liver-targeting gene transfer vector that expresses full-length myelin oligodendrocyte glycoprotein (MOG) in hepatocytes. We show that by harnessing the tolerogenic nature of the liver, this powerful gene immunotherapy restores immune tolerance by inducing functional MOG-specific regulatory T cells (Tregs) in vivo, independent of major histocompatibility complex (MHC) restrictions. We demonstrate that mice treated prophylactically are protected from developing disease and neurological deficits. More importantly, we demonstrate that when given to mice with preexisting disease, ranging from mild neurological deficits to severe paralysis, the gene immunotherapy abrogated CNS inflammation and significantly reversed clinical symptoms of disease. This specialized approach for inducing antigen-specific immune tolerance has significant therapeutic potential for treating MS and other autoimmune disorders.

Evaluation of engineered AAV capsids for hepatic factor IX gene transfer in murine and canine models.

BACKGROUND: Adeno-associated virus (AAV) gene therapy vectors have shown the best outcomes in human clinical studies for the treatment of genetic diseases such as hemophilia. However, these pivotal investigations have also identified several challenges. For example, high vector doses are often used for hepatic gene transfer, and cytotoxic T lymphocyte responses against viral capsid may occur. Therefore, achieving therapy at reduced vector doses and other strategies to reduce capsid antigen presentation are desirable. METHODS: We tested several engineered AAV capsids for factor IX (FIX) expression for the treatment of hemophilia B by hepatic gene transfer. These capsids lack potential phosphorylation or ubiquitination sites, or had been generated through molecular evolution. RESULTS: AAV2 capsids lacking either a single lysine residue or 3 tyrosine residues directed substantially higher coagulation FIX expression in mice compared to wild-type sequence or other mutations. In hemophilia B dogs, however, expression from the tyrosine-mutant vector was merely comparable to historical data on AAV2. Evolved AAV2-LiC capsid was highly efficient in hemophilia B mice but lacked efficacy in a hemophilia B dog. CONCLUSIONS: Several alternative strategies for capsid modification improve the in vivo performance of AAV vectors in hepatic gene transfer for correction of hemophilia. However, capsid optimization solely in mouse liver may not predict efficacy in other species and thus is of limited translational utility.

An Immune-Competent Murine Model to Study Elimination of AAV-Transduced Hepatocytes by Capsid-Specific CD8+ T Cells.

Multiple independent adeno-associated virus (AAV) gene therapy clinical trials for hemophilia B, utilizing different AAV serotypes, have reported a vector dose-dependent loss of circulating factor IX (FIX) protein associated with capsid-specific CD8+ T cell (Cap-CD8) elimination of transduced hepatocytes. Hemophilia B patients who develop transient transaminitis and loss of FIX protein may be stabilized with the immune-suppressive (IS) drug prednisolone, but do not all recover lost FIX expression, whereas some patients fail to respond to IS. We developed the first animal model demonstrating Cap-CD8 infiltration and elimination of AAV-transduced hepatocytes of immune-deficient mice. Here, we extend this model to an immune-competent host where Cap-CD8 transfer to AAV2-F9-treated mice significantly reduced circulating and hepatocyte FIX expression. Further, we studied two high-expressing liver tropic AAV2 variants, AAV2-LiA and AAV2-LiC, obtained from a rationally designed capsid library. Unlike AAV2, Cap-CD8 did not initially reduce circulating FIX levels for either variant. However, FIX levels were significantly reduced in AAV2-LiC-F9-treated, but not AAV2-LiA-F9-treated, mice at the study endpoint. Going forward, the immune-competent model may provide an opportunity to induce immunological memory directed against a surrogate AAV capsid antigen and study recall responses following AAV gene transfer.

Combination therapy for inhibitor reversal in haemophilia A using monoclonal anti-CD20 and rapamycin.

Development of antibodies (inhibitors) against coagulation factor VIII (FVIII) is a major complication of intravenous replacement therapy in haemophilia A (HA). Current immune tolerance induction (ITI) regimens are not universally effective. Rituximab, a B cell-depleting antibody against CD20, has shown mixed results for inhibitor reversal in patients. This study aims to develop a combinatorial therapy for inhibitor reversal in HA, using anti-murine CD20 (anti-mCD20) antibody and rapamycin, which targets both B and T cell responses. Additionally, it extensively characterises the role of the IgG backbone in B cell depletion by anti-CD20 antibodies. For this, inhibitors were generated in BALB/c-HA mice by weekly IV injection of FVIII. Subsequently, anti-mCD20 (18B12) with IgG2a or IgG1 backbone was injected IV in two doses three weeks apart and B cell depletion and recovery was characterised. Rapamycin was administered orally 3x/week (for 1 month) while continuing FVIII injections. Altering the IgG backbone of anti-mCD20 from IgG2a to IgG1 reduced overall depletion of B cells (including memory B cells), and marginal zone, B-10, and B-1b cells were specifically unaffected. While neither antibody was effective alone, in combination with rapamycin, anti-mCD20 IgG2a but not IgG1 was able to reverse inhibitors in HA mice. This regimen was particularly effective for starting titres of ~10 BU. Although IgG1 anti-mCD20 spared potentially tolerogenic B cell subsets, IgG2a directed sustained hyporesponsiveness when administered in conjunction with rapamycin. This regimen represents a promising treatment for inhibitor reversal in HA, as both of these compounds have been extensively used in human patients.

Potential for cellular stress response to hepatic factor VIII expression from AAV vector.

Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity.