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Find video protocols related to scientific articles indexed in Pubmed.
Ex Vivo Expanded Autologous Polyclonal Regulatory T Cells Suppress Inhibitor Formation in Hemophilia.
Mol Ther Methods Clin Dev
PUBLISHED: 11-04-2014
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Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft vs host disease in bone marrow transplantation. Here we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e. hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express GFP-marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 80-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within two weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.
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A David promoter with Goliath strength.
Blood
PUBLISHED: 05-17-2014
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In this issue of Blood, Nair et al report on a computationally derived hepatocyte-specific cis-regulatory module (HS-CRM8) to accomplish high tissue-specific expression in gene therapy for hemophilia. When HS-CRM8 is introduced upstream of a minimal liver-specific promoter in an adenoassociated virus (AAV) that expresses a codon-optimized hyperactive human factor IX (FIX) mutant (FIX Padua), it provides a >1 log increase in systemic FIX protein levels and supraphysiological activity over a range of vector doses.
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Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells.
Blood
PUBLISHED: 05-13-2014
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Hemophilia A is the X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). To address serious complications of inhibitory antibody formation in current replacement therapy, we created tobacco transplastomic lines expressing FVIII antigens, heavy chain (HC) and C2, fused with the transmucosal carrier, cholera toxin B subunit. Cholera toxin B-HC and cholera toxin B-C2 fusion proteins expressed up to 80 or 370 µg/g in fresh leaves, assembled into pentameric forms, and bound to GM1 receptors. Protection of FVIII antigen through bioencapsulation in plant cells and oral delivery to the gut immune system was confirmed by immunostaining. Feeding of HC/C2 mixture substantially suppressed T helper cell responses and inhibitor formation against FVIII in mice of 2 different strain backgrounds with hemophilia A. Prolonged oral delivery was required to control inhibitor formation long-term. Substantial reduction of inhibitor titers in preimmune mice demonstrated that the protocol could also reverse inhibitor formation. Gene expression and flow cytometry analyses showed upregulation of immune suppressive cytokines (transforming growth factor ? and interleukin 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4(+)CD25(+) and CD4(+)CD25(-) T cells that potently suppressed anti-FVIII formation. In sum, these data support plant cell-based oral tolerance for suppression of inhibitor formation against FVIII.
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Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants.
Mol. Ther.
PUBLISHED: 03-19-2014
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Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of "virtual family shuffling" to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8?×?10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8.
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Immune tolerance induction to factor IX through B cell gene transfer: TLR9 signaling delineates between tolerogenic and immunogenic B cells.
Mol. Ther.
PUBLISHED: 03-03-2014
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A subset of patients with severe hemophilia B, the X-linked bleeding disorder resulting from absence of coagulation factor IX (FIX), develop pathogenic antibody responses during replacement therapy. These inhibitors block standard therapy and are often associated with anaphylactic reactions to FIX. Established clinical immune tolerance induction protocols often fail for FIX inhibitors. In a murine model of this immune complication, retrovirally transduced primary B cells expressing FIX antigen fused with immunoglobulin-G heavy chain prevented antibody formation to FIX and was also highly effective in desensitizing animals with preexisting response. In contrast, transplant of B cells that received the identical expression cassette via nucleofection of plasmid vector substantially heightened antibody formation against FIX, a response that could be blocked by toll-like receptor 9 (TLR9) inhibition. While innate responses to TLR4 activation or to retrovirus were minimal in B cells, plasmid DNA activated TLR9, resulting in CpG-dependent NF-?B activation/IL-6 expression and adaptor protein 3 dependent, CpG-independent induction of IFN-I. Neither response was seen in TLR9-deficient B cells. Therefore, TLR9 signaling in B cells, in particular in response to plasmid vector, is highly immunogenic and has to be avoided in design of tolerance protocols.
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Portal vein delivery of viral vectors for gene therapy for hemophilia.
Methods Mol. Biol.
PUBLISHED: 02-22-2014
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The liver is a very complex organ with a large variety of functions, making it an attractive organ for gene replacement therapy. Many genetic disorders can be corrected by delivering gene products directly into the liver using viral vectors. In this chapter, we will describe gene delivery via portal vein administration in mice and dogs to correct the blood coagulation disorder hemophilia B. Although there are multiple delivery routes for both viral and non-viral vectors in animals, portal vein administration delivers vectors directly and efficiently into the liver. Complete correction of murine hemophilia B and multi-year near-correction of canine hemophilia B have been achieved following portal vein delivery of adeno-associated viral (AAV) vectors expressing factor IX from hepatocyte-specific promoters. Peripheral vein injection can lead to increased vector dissemination to off-target organ such as the lung and spleen. Below, we will describe portal vein injection delivery route via laparotomy.
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Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B.
J Transl Med
PUBLISHED: 01-23-2014
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Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX).
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Immune responses and hypercoagulation in ERT for Pompe disease are mutation and rhGAA dose dependent.
PLoS ONE
PUBLISHED: 01-01-2014
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Enzyme replacement therapy (ERT) with recombinant human acid-?-glucosidase (rhGAA) is the only FDA approved therapy for Pompe disease. Without ERT, severely affected individuals (early onset) succumb to the disease within 2 years of life. A spectrum of disease severity and progression exists depending upon the type of mutation in the GAA gene (GAA), which in turn determines the amount of defective protein produced and its enzymatic activity. A large percent of the early onset patients are also cross reactive immunological material negative (CRIM-) and develop high titer immune responses to ERT with rhGAA. New insights from our studies in pre-clinical murine models reveal that the type of Gaa mutation has a profound effect on the immune responses mounted against ERT and the associated toxicities, including activation of clotting factors and disseminated intravascular coagulation (DIC). Additionally, the mouse strain affects outcomes, suggesting the influence of additional genetic components or modifiers. High doses of rhGAA (20 mg/kg) are currently required to achieve therapeutic benefit. Our studies indicate that lower enzyme doses reduce the antibody responses to rhGAA, reduce the incidence of immune toxicity and avoid ERT-associated anaphylaxis. Therefore, development of rhGAA with increased efficacy is warranted to limit immunotoxicities.
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Glucocorticoid-Induced TNF Receptor Family-Related Protein Ligand is Requisite for Optimal Functioning of Regulatory CD4(+) T Cells.
Front Immunol
PUBLISHED: 01-01-2014
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Glucocorticoid-induced tumor necrosis factor receptor family-related protein (TNFRSF18, CD357) is constitutively expressed on regulatory T cells (Tregs) and is inducible on effector T cells. In this report, we examine the role of glucocorticoid-induced TNF receptor family-related protein ligand (GITR-L), which is expressed by antigen presenting cells, on the development and expansion of Tregs. We found that GITR-L is dispensable for the development of naturally occurring FoxP3(+) Treg cells in the thymus. However, the expansion of Treg in GITR-L (-/-) mice is impaired after injection of the dendritic cells (DCs) inducing factor Flt3 ligand. Furthermore, DCs from the liver of GITR-L (-/-) mice were less efficient in inducing proliferation of antigen-specific Treg cells in vitro than the same cells from WT littermates. Upon gene transfer of ovalbumin into hepatocytes of GITR-L (-/-)FoxP3(GFP) reporter mice using adeno-associated virus (AAV8-OVA) the number of antigen-specific Treg in liver and spleen is reduced. The reduced number of Tregs resulted in an increase in the number of ovalbumin specific CD8(+) T effector cells. This is highly significant because proliferation of antigen-specific CD8(+) cells itself is dependent on the presence of GITR-L, as shown by in vitro experiments and by adoptive transfers into GITR-L (-/-) Rag (-/-) and Rag (-/-) mice that had received AAV8-OVA. Surprisingly, administering ?CD3 significantly reduced the numbers of FoxP3(+) Treg cells in the liver and spleen of GITR-L (-/-) but not WT mice. Because soluble Fc-GITR-L partially rescues ?CD3 induced in vitro depletion of the CD103(+) subset of FoxP3(+)CD4(+) Treg cells, we conclude that expression of GITR-L by antigen presenting cells is requisite for optimal Treg-mediated regulation of immune responses including those in response during gene transfer.
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Glucocorticoid-induced TNF receptor family-related protein ligand regulates the migration of monocytes to the inflamed intestine.
FASEB J.
PUBLISHED: 10-09-2013
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Glucocorticoid-induced TNF receptor family-related protein (GITR) regulates the function of both T cells and antigen-presenting cells (APCs), while the function of GITR ligand (GITR-L) is largely unknown. Here we evaluate the role of GITR-L, whose expression is restricted to APCs, in the development of enterocolitis. On injecting naive CD4(+) T cells, GITR-L(-/-)Rag(-/-) mice develop a markedly milder colitis than Rag(-/-) mice, which correlates with a 50% reduction of Ly6C(+)CD11b(+)MHCII(+) macrophages in the lamina propria and mesenteric lymph nodes. The same result was observed in ?CD40-induced acute colitis and during peritonitis, suggesting an altered monocyte migration. In line with these observations, the number of nondifferentiated monocytes was approximately 3-fold higher in the spleen of GITR-L(-/-)Rag(-/-) mice than in Rag(-/-) mice after ?CD40 induction. Consistent with the dynamic change in the formation of an active angiotensin II type 1 receptor (AT1) dimer in GITR-L(-/-) splenic monocytes during intestinal inflammation, the migratory capability of splenic monocytes from GITR-L-deficient mice was impaired in an in vitro transwell migration assay. Conversely, ?GITR-L reduces the number of splenic Ly6C(hi) monocytes, concomitantly with an increase in AT1 dimers. We conclude that GITR-L regulates the number of proinflammatory macrophages in sites of inflammation by controlling the egress of monocytes from the splenic reservoir.-Liao, G., van Driel, B., Magelky, E., OKeeffe, M. S., de Waal Malefyt, R., Engel, P., Herzog, R. W., Mizoguchi, E., Bhan, A. K., Terhorst, C. Glucocorticoid-induced TNF receptor family-related protein ligand regulates the migration of monocytes to the inflamed intestine.
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Effective gene therapy for haemophilic mice with pathogenic factor IX antibodies.
EMBO Mol Med
PUBLISHED: 04-08-2013
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Formation of pathogenic antibodies is a major problem in replacement therapies for inherited protein deficiencies. For example, antibodies to coagulation factors (inhibitors) seriously complicate treatment of haemophilia. While immune tolerance induction (ITI) protocols have been developed, inhibitors against factor IX (FIX) are difficult to eradicate due to anaphylactic reactions and nephrotic syndrome and thus substantially elevate risks for morbidity and mortality. However, hepatic gene transfer with an adeno-associated virus (AAV) serotype 8 vector expressing FIX (at levels of ?4% of normal) rapidly reversed pre-existing high-titre inhibitors in haemophilia B mice, eliminated antibody production by B cells, desensitized from anaphylaxis (even if protein therapy was resumed) and provided long-term correction. High levels of FIX protein suppressed memory B cells and increased Treg induction, indicating direct and indirect mechanisms of suppression of inhibitor formation. Persistent presence of Treg was required to prevent relapse of antibodies. Together, these data suggest that hepatic gene transfer-based ITI provides a safe and effective alternative to eradicate inhibitors. This strategy may be broadly applicable to reversal of antibodies in different genetic diseases.
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Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.
Blood
PUBLISHED: 01-16-2013
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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.
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Differential type I interferon-dependent transgene silencing of helper-dependent adenoviral vs. adeno-associated viral vectors in vivo.
Mol. Ther.
PUBLISHED: 01-15-2013
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We previously dissected the components of the innate immune response to Helper-dependent adenoviral vectors (HDAds) using genetic models, and demonstrated that multiple pattern recognition receptor signaling pathways contribute to this host response to HDAds in vivo. Based on analysis of cytokine expression profiles, type I interferon (IFN) mRNA is induced in host mouse livers at 1 hour post-injection. This type I IFN signaling amplifies cytokine expression in liver independent of the nature of vector DNA sequences after 3 hours post-injection. This type I IFN signaling in response to HDAds administration contributes to transcriptional silencing of both HDAd prokaryotic and eukaryotic DNA in liver. This silencing occurs early and is mediated by epigenetic modification as shown by in vivo chromatin immunoprecipitation (ChIP) with anti-histone deacetylase (HDAC) and promyelocytic leukemia protein (PML). In contrast, self-complementary adeno-associated viral vectors (scAAVs) showed significantly lower induction of type I IFN mRNA in liver compared to HDAds at both early and late time points. These results show that the type I IFN signaling dependent transgene silencing differs between AAV and HDAd vectors after liver-directed gene transfer.
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B-Cell depletion and immunomodulation before initiation of enzyme replacement therapy blocks the immune response to acid alpha-glucosidase in infantile-onset Pompe disease.
J. Pediatr.
PUBLISHED: 01-08-2013
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To evaluate whether B-cell depletion before enzyme replacement therapy (ERT) initiation can block acid alpha-glucosidase (GAA) antibody responses and improve clinical outcomes.
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A robust strategy for negative selection of Cre-loxP recombination-based excision of transgenes in induced pluripotent stem cells.
PLoS ONE
PUBLISHED: 01-01-2013
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Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In this study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1? promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. A reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies.
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Measuring immune responses to recombinant AAV gene transfer.
Methods Mol. Biol.
PUBLISHED: 10-29-2011
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Following AAV-based gene transfer, the occurrence of adaptive immune responses specific to the vector or the transgene product is a major roadblock to successful clinical translation. These responses include antibodies against the AAV capsid, which can be neutralizing and therefore prevent the ability to repeatedly administer the vector, and CD8(+) cytotoxic T lymphocytes, which can eliminate transduced cells. In addition, humans may have both humoral and cellular preexisting immunity, as a result from natural infection with parent virus or related serotypes. The need for assays to detect and measure these anti-capsid immune responses in humans and in experimental animals is profound. Here, ELISPOT, immunocapture (ELISA), and neutralization assays are explained and provided in detail. Furthermore, such techniques can readily be adapted to monitor and quantify immune responses against therapeutic transgene products encoded by the vector genome.
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Glucocorticoid-induced tumor necrosis factor receptor family-related protein regulates CD4(+)T cell-mediated colitis in mice.
Gastroenterology
PUBLISHED: 06-24-2011
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The glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR; also called TNFRSF18 or CD357) regulates the T cell-mediated immune response and is present on surfaces of regulatory T (Treg) cells and activated CD4(+) T cells. We investigated the roles of GITR in the development of colitis in mice.
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Prevention and Reversal of Antibody Responses Against Factor IX in Gene Therapy for Hemophilia B.
Front Microbiol
PUBLISHED: 06-20-2011
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Intramuscular (IM) administration of an adeno-associated viral (AAV) vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency). However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4(+) T cell epitope) caused an induction of regulatory T cells (Treg) with a concomitant apoptosis of antigen-specific effector T cells (Nayak et al., 2009). This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX) in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion). Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1-hF.IX vector resulted in inhibitors of on average 8-10?BU within 1?month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specific antibodies to <2?BU, which lasted for >4.5?months. Systemic hF.IX expression increased from undetectable to >200?ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.
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Innate Immune Responses to AAV Vectors.
Front Microbiol
PUBLISHED: 06-08-2011
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Gene replacement therapy by in vivo delivery of adeno-associated virus (AAV) is attractive as a potential treatment for a variety of genetic disorders. However, while AAV has been used successfully in many models, other experiments in clinical trials and in animal models have been hampered by undesired responses from the immune system. Recent studies of AAV immunology have focused on the elimination of transgene-expressing cells by the adaptive immune system, yet the innate immune system also has a critical role, both in the initial response to the vector and in prompting a deleterious adaptive immune response. Responses to AAV vectors are primarily mediated by the TLR9-MyD88 pathway, which induces the production of pro-inflammatory cytokines by activating the NF-?B pathways and inducing type I IFN production; self-complementary AAV vectors enhance these inflammatory processes. Additionally, the alternative NF-?B pathway influences transgene expression in cells transduced by AAV. This review highlights these recent discoveries regarding innate immune responses to AAV and discusses strategies to ablate these potentially detrimental signaling pathways.
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Pompe disease gene therapy.
Hum. Mol. Genet.
PUBLISHED: 04-25-2011
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Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.
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The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver.
Blood
PUBLISHED: 04-07-2011
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Although adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-?, monocyte chemotactic protein-1, IFN-? inducible protein-10, and IFN-?/? expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.
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High Efficiency Ex Vivo Gene Transfer to Primary Murine B Cells Using Plasmid or Viral Vectors.
J Genet Syndr Gene Ther
PUBLISHED: 03-15-2011
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Primary autologous B-lymphocytes, following ex vivo gene transfer and re-implantation, have been successfully utilized to prevent autoimmune disease and adaptive responses to therapeutic proteins in several animal models. However, efficient gene transfer to primary B cells requires use of retroviral vectors, which increase the risk of insertional mutagenesis. Here, we evaluated several alternative gene transfer approaches. Resting splenic B cells were purified and activated with LPS, and ex vivo GFP gene transfer was performed by means of nucleofection, lipofectamine, adenoviral infection, or murine retroviral infection. The Adenoviral (Ad) vectors were added to B cell cultures with or without calcium phosphate precipitation. For transfection and nucleofection, naked plasmid DNA was utilized. Nucleofection technology represents a modified electroporation technique for effective transfer of nucleic acids to the nucleus and thus enhances the efficiency of transfer particularly for primary cells. Efficiency of ex vivo gene transfer was determined by flow cytometry using GFP, CD19, and a vital dye as markers. Nucleofection yielded the highest level of gene transfer with 60-65% of B cells being GFP+. Efficiencies were 30-35% for retrovirus, 20% for Ad5/11, 15% for Ad5/35, and 5% for lipofectamine-mediated transfection. Calcium phosphate precipitation increased efficiencies for Ad vectors to 30% (Ad5/11) and 25% (Ad5/35). Lipofectamin caused the greatest cell death at 80%, followed by nucleofection (35%), and viral vector (10-15% in each case). For all methods, gene transfer efficiencies were nearly identical for B cells from C57BL/6 or C3H/HeOuJ mice. In conclusion, recent advances in gene transfer technologies provide alternatives to retroviral vectors for primary B cells. If stable gene transfer is desired, non-integrating vector systems may be combined with transposon- or phage integrase-based systems or future site-specific systems to achieve integration into the host B cell genome.
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Nonredundant roles of IL-10 and TGF-? in suppression of immune responses to hepatic AAV-factor IX gene transfer.
Mol. Ther.
PUBLISHED: 03-08-2011
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Hepatic gene transfer using adeno-associated viral (AAV) vectors has been shown to efficiently induce immunological tolerance to a variety of proteins. Regulatory T-cells (Treg) induced by this route suppress humoral and cellular immune responses against the transgene product. In this study, we examined the roles of immune suppressive cytokines interleukin-10 (IL-10) and transforming growth factor-? (TGF-?) in the development of tolerance to human coagulation factor IX (hF.IX). Interestingly, IL-10 deficient C57BL/6 mice receiving gene transfer remained tolerant to hF.IX and generated Treg that suppressed anti-hF.IX formation. Effects of TGF-? blockade were also minor in this strain. In contrast, in C3H/HeJ mice, a strain known to have stronger T-cell responses against hF.IX, IL-10 was specifically required for the suppression of CD8(+) T-cell infiltration of the liver. Furthermore, TGF-? was critical for tipping the balance toward an regulatory immune response. TGF-? was required for CD4(+)CD25(+)FoxP3(+) Treg induction, which was necessary for suppression of effector CD4(+) and CD8(+) T-cell responses as well as antibody formation. These results demonstrate the crucial, nonredundant roles of IL-10 and TGF-? in prevention of immune responses against AAV-F.IX-transduced hepatocytes.
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Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-14-2011
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Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-?B, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-?B during its life cycle. We used small molecule modulators of NF-?B in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-?B activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-?B inhibitors, Bay11, which blocks both the canonical and the alternative NF-?B pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-?B pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-?B pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-?B pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-?B pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-?B inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.
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High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.
Mol. Ther.
PUBLISHED: 08-24-2010
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Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.
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Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-29-2010
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To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin beta-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20-25% of control animals survived after six to eight F.IX doses, 90-93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyers patches in the ileum. Within 2-5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5-80 microg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (approximately 40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.
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Two decades of clinical gene therapy--success is finally mounting.
Discov Med
PUBLISHED: 03-03-2010
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Human gene therapy has made substantial progress since the initiation of the first clinical trials 20 years ago. Here, we summarized important applications of gene transfer protocols in the treatment of various human diseases using different viral vectors. Recent successful trials on the treatment of ocular diseases and inherited immune deficiencies are particularly encouraging and have raised hopes that human gene therapy as a standard treatment option will finally become a reality. While immune responses and insertional mutagenesis pose obstacles for this novel form of molecular medicine, continuous progress suggests that a wider range of diseases can be treated with gene therapy in the future.
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GITR engagement preferentially enhances proliferation of functionally competent CD4+CD25+FoxP3+ regulatory T cells.
Int. Immunol.
PUBLISHED: 02-05-2010
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Naturally occurring regulatory T cells (Treg) express high levels of glucocorticoid-induced tumour necrosis factor receptor (GITR). However, studies of the role of GITR in Treg biology has been complicated by the observation that upon activation effector CD4(+) T (Teff) cells also express the receptor. Here, we dissect the contribution of GITR-induced signaling networks in the expansion and function of FoxP3(+) Treg. We demonstrate that a high-affinity soluble Fc-GITR-L dimer, in conjugation with alphaCD3, specifically enhances in vitro proliferation of Treg, which retain their phenotypic markers (CD25 and FoxP3) and their suppressor function, while minimally affecting Teff cells. Furthermore, Fc-GITR-L does not impair Teff susceptibility to suppression, as judged by cocultures employing GITR-deficient and GITR-sufficient CD4(+) T-cell subsets. Notably, this expansion of Treg could also be seen in vivo, by injecting FoxP3-IRES-GFP mice with Fc-GITR-L even in the absence of antigenic stimulation. In order to test the efficacy of these findings therapeutically, we made use of a C3H/HeJ hemophilia B-prone mouse model. The use of liver-targeted human coagulation factor IX (hF.IX) gene therapy in this model has been shown to induce liver toxicity and the subsequent failure of hF.IX expression. Interestingly, injection of Fc-GITR-L into the hemophilia-prone mice that were undergoing liver-targeted hF.IX gene therapy increased the expression of F.IX and reduced the anticoagulation factors. We conclude that GITR engagement enhances Treg proliferation both in vitro and in vivo and that Fc-GITR-L may be a useful tool for in vivo tolerance induction.
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Gene therapy approaches to induce tolerance in autoimmunity by reshaping the immune system.
Curr Opin Investig Drugs
PUBLISHED: 10-31-2009
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Gene therapy as a concept usually seeks to replace a defective or missing protein in order to restore a desired function. The concept typically implies that diseases amenable to this approach are of monogenic origin; indeed, monogenic disorders have received the most research attention. Polygenic diseases are conceptually different in terms of formulating a gene therapy strategy, and include autoimmune diseases in the category. For the treatment of autoimmune diseases by gene therapy approaches, the rationale is not to replace missing or malfunctioning proteins, but to deliver proteins, by either ex vivo or in vivo gene transfer, in an attempt to modify the response of the immune system. In addition, the strategy for the induction of immune tolerance can be either a broad approach, in which large parts of the immune response are reset, or can be tailored to be more specific, focusing on autoantigen-directed effector cells. Gene therapy protocols for autoimmune disease are still largely experimental. For each type of autoimmune disease, the risk-to-benefit ratio varies, depending in part on the severity of disease. It is unclear when gene therapy approaches will be evaluated in clinical trials; however, as progress in safety and advances in understanding true antigen-specific tolerance occur, gene therapy is likely to become available for clinical use.
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Evading the immune response upon in vivo gene therapy with viral vectors.
Curr. Opin. Mol. Ther.
PUBLISHED: 10-07-2009
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Gene therapy has the potential to provide minimally invasive and long-term treatment for many inherited disorders that otherwise have poor prognoses and limited treatment options. The sustained therapeutic correction of genetic disease by viral gene transfer has been accomplished in patients with severe immune deficiencies, or by the transduction of an immune privileged site for the treatment of ocular disease. For other diseases and target tissues, immune responses to vectors or transgene products often present major obstacles for therapy. Innate and adaptive immunity, sometimes including pre-existing or memory responses, may contribute by varying degrees to immune-mediated rejection and immunotoxicity. This review provides an overview of the immune responses to in vivo gene transfer with the most commonly used viral gene therapy vectors, and discusses strategies and protocols employed in evading the immune system in order to provide optimal gene therapy.
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Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B.
Mol. Ther.
PUBLISHED: 07-14-2009
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Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.
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Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.
PLoS ONE
PUBLISHED: 05-20-2009
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Hepatic gene transfer, in particular using adeno-associated viral (AAV) vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.
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Hepatic gene transfer as a means of tolerance induction to transgene products.
Curr Gene Ther
PUBLISHED: 04-10-2009
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The liver is a preferred target organ for gene therapy not only for liver-specific diseases but also for disorders that require systemic delivery of a protein. Diseases that could benefit from hepatic gene transfer include hemophilia, metabolic disorders, lysosomal storage disorders, and others. For a successful delivery of the transgene and sustained expression, the protocol must avoid immune responses in order to be efficacious. A growing number of studies have demonstrated that liver-directed transfer can induce transgene product-specific immune tolerance. Tolerance obtained via this route requires optimal engineering of the vector to eliminate transgene expression in antigen presenting cells while restricting high levels of therapeutic expression to hepatocytes. Innate immune responses may prevent tolerance induction, cause toxicity, and have to be minimized. Discussed in our review is the crucial role of CD4(+)CD25(+) regulatory T cells in tolerance to the hepatocyte-derived gene product, the immunobiology of the liver and our current understanding of its tolerogenic properties, current and proposed research as to the mechanisms behind the livers unique cellular environment, as well as development of the tools for tolerance induction such as advanced vector systems.
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Improved induction of immune tolerance to factor IX by hepatic AAV-8 gene transfer.
Hum. Gene Ther.
PUBLISHED: 03-25-2009
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Gene therapy for hemophilia B has been shown to result in long-term expression and immune tolerance to factor IX (F.IX) after in vivo transduction of hepatocytes with adeno-associated viral (AAV-2) vectors in experimental animals. An optimized protocol was effective in several strains of mice with a factor 9 gene deletion (F9(-/-)). However, immune responses against F.IX were repeatedly observed in C3H/HeJ F9(-/-) mice. We sought to establish a gene transfer protocol that results in sustained expression without a requirement for additional manipulation of the immune system. Compared with AAV-2, AAV-8 was more efficient in transgene expression and induction of tolerance to F.IX in three different strains of wild-type mice. At equal vector doses, AAV-8 induced transgene product-specific regulatory CD4(+)CD25(+)FoxP3(+) T cells at significantly higher frequency. Moreover, sustained correction of hemophilia B in C3H/HeJ F9(-/-) mice without antibody formation was documented in all animals treated with > or =4 x 10(11) vector genomes (VG)/kg and in 80% of mice treated with 8 x 10(10) VG/kg. Therefore, it is possible to develop a gene transfer protocol that reliably induces tolerance to F.IX largely independent of genetic factors. A comparison with other studies suggests that additional parameters besides plateau levels of F.IX expression contributed to the improved success rate of tolerance induction.
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Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy.
Blood
PUBLISHED: 02-10-2009
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Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.
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Development of Novel Recombinant AAV Vectors and Strategies for the Potential Gene Therapy of Hemophilia.
J Genet Syndr Gene Ther
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Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV), have gained attention as a potentially safe and useful alternative to the more commonly used retroviral and adenoviral vectors. AAV vectors are currently in use in Phase I/II clinical trials for gene therapy of a number of diseases such as cystic fibrosis, ?-1 antitrypsin deficiency, muscular dystrophy, Battens disease, and Parkinsons disease, and have shown efficacy in patients with Lebers congenital amaurosis, and hemophilia B. For patients with hemophilia B, however, relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as significant fraction of the vectors fails to traffic efficiently to the nucleus, and is targeted for degradation by the host cell proteasome machinery. With a better understanding of the various steps in the life cycle of AAV vectors, strategies leading to the development of novel AAV vectors that are capable of high-efficiency transduction at lower doses are needed. In this review, we summarize our strategies to develop novel AAV vectors for the potential gene therapy of both hemophilia B and hemophilia A, based on our recent studies on the basic molecular biology of AAV. These strategies, including the development of novel AAV vectors by site-directed mutagenesis of critical surface-exposed tyrosine residues on AAV2 capsids to circumvent the ubiquitination step and the use of different AAV serotypes and self-complementary (sc) AAV2 vectors, and their use as helper vectors to circumvent the obstacles of second-strand DNA synthesis of single-stranded (ss) AAV, should dramatically accelerate the progress towards the potential gene therapy of both hemophilia A and hemophilia B.
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Mechanism of oral tolerance induction to therapeutic proteins.
Adv. Drug Deliv. Rev.
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Oral tolerance is defined as the specific suppression of humoral and/or cellular immune responses to an antigen by administration of the same antigen through the oral route. Due to its absence of toxicity, easy administration, and antigen specificity, oral tolerance is a very attractive approach to prevent unwanted immune responses that cause a variety of diseases or that complicate treatment of a disease. Many researchers have induced oral tolerance to efficiently treat autoimmune and inflammatory diseases in different animal models. However, clinical trials yielded limited success. Thus, understanding the mechanisms of oral tolerance induction to therapeutic proteins is critical for paving the way for clinical development of oral tolerance protocols. This review will summarize progress on understanding the major underlying tolerance mechanisms and contributors, including antigen presenting cells, regulatory T cells, cytokines, and signaling pathways. Potential applications, examples for therapeutic proteins and disease targets, and recent developments in delivery methods are discussed.
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Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells.
Adv. Drug Deliv. Rev.
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Among 12billion injections administered annually, unsafe delivery leads to >20million infections and >100million reactions. In an emerging new concept, freeze-dried plant cells (lettuce) expressing vaccine antigens/biopharmaceuticals are protected in the stomach from acids/enzymes but are released to the immune or blood circulatory system when plant cell walls are digested by microbes that colonize the gut. Vaccine antigens bioencapsulated in plant cells upon oral delivery after priming, conferred both mucosal and systemic immunity and protection against bacterial, viral or protozoan pathogens or toxin challenge. Oral delivery of autoantigens was effective against complications of type 1 diabetes and hemophilia, by developing tolerance. Oral delivery of proinsulin or exendin-4 expressed in plant cells regulated blood glucose levels similar to injections. Therefore, this new platform offers a low cost alternative to deliver different therapeutic proteins to combat infectious or inherited diseases by eliminating inactivated pathogens, expensive purification, cold storage/transportation and sterile injections.
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Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy.
PLoS ONE
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The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors) against factor VIII (FVIII). The current method for eradicating inhibitors, termed immune tolerance induction (ITI), is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8) gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.
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Mapping the T helper cell response to acid ?-glucosidase in Pompe mice.
Mol. Genet. Metab.
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Pompe disease is a neuromuscular disease caused by an inherited deficiency of the lysosomal enzyme acid ?-glucosidase (GAA). The resulting accumulation of glycogen causes muscle weakness with the severe form of the disease resulting in death by cardiorespiratory failure in the first year of life. The only available treatment, enzyme replacement therapy (ERT) with recombinant GAA (rhGAA), is severely hampered by antibody responses that reduce efficacy and cause immunotoxicities. Currently, Pompe mice represent the only pre-clinical model for development of new treatments and for immunological studies. While antibody formation following ERT in this model has been described, the underlying T cell response has not been studied. In order to define the T helper response to rhGAA in Pompe mice, immunodominant CD4(+) T cell epitopes were mapped in GAA(-/-) 129SVE mice using ELISpot. Additionally, cytokine responses and antibody formation against rhGAA during ERT were measured. Among the three CD4(+) T cell epitopes identified, only epitope IFLGPEPKSVVQ, predicted to be the strongest MHC II binder, consistently contributed to IL-4 production. Frequencies of IL-4 producing T cells were considerably higher than those of IL-17 or IFN-? producing cells, suggesting a predominantly Th2 cell mediated response. This is further supported by IgG1 being the prevalent antibody subclass against rhGAA during ERT and consistent with prior reports on IgE formation and anaphylaxis in this model. These results will facilitate mechanistic studies of the immune response to rhGAA in Pompe mice during development of new therapies and tolerance protocols.
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Liver-Directed Adeno-Associated Viral Gene Therapy for Hemophilia.
J Genet Syndr Gene Ther
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Hemophilia A and B are monogenic bleeding disorders resulting from loss of functional coagulation factors VIII or IX, respectively. Prophylactic treatment requires frequent intravenous injections of exogenous factor VIII (F.VIII) or factor IX (F.IX), due to the short half-life of both factors. Hemophilia patients are at risk of developing neutralizing antibodies to F.VIII (~25-30%) or F.IX (~2-4%), which require the use of expensive bypass agents and immune tolerance induction protocols. Viral vector mediated liver gene transfer of F.VIII or F.IX offers an alternative treatment for hemophilia with easily defined clinical endpoints and no need for strict regulation of coagulation factor expression, as both proteins circulate as inactive zymogens. Adeno-associated viral (AAV) vectors are derived from a non-pathogenic human virus that efficiently transduce non-dividing cells, such as hepatocytes, and provide stable transgene expression. In vivo liver gene transfer of AAV-F.VIII and -F.IX vectors has restored hemostasis in murine and canine hemophilia models long-term, and has also been shown to induce immune tolerance. Consequently, two Phase I/II clinical trials have been conducted, based on hepatic AAV-FIX gene transfer to patients with severe hemophilia B. The first trial, utilizing serotype 2, demonstrated transient correction, which was limited by a cellular immune response against the viral capsid. However, sustained therapeutic expression has been achieved in a second trial, using AAV8 for expression of a codon-optimized F.IX transgene. Translation of F.VIII gene transfer studies into the clinic may require additional optimization of gene transfer and vector to effectively express the larger cDNA of F.VIII.
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