Andrea Labelle

Ex Vivo Gene Therapy for Hemophilia A That Enhances Safe Delivery and Sustained In Vivo Factor VIII Expression from Lentivirally Engineered Endothelial Progenitors

Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate long‐term safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of preclinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression. Circulating blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood and transduced with a lentiviral vector encoding the canine FVIII transgene. To enhance safety, these cells were implanted subcutaneously in a Matrigel scaffold, and the efficacy of this strategy was compared with i.v. delivery of engineered BOECs in nonhemophilic nonobese diabetic/severe combined immunodeficiency mice. Therapeutic levels of FVIII persisted for 15 weeks, and these levels of stable expression were extended to 20 weeks when the cytomegalovirus promoter was replaced with the thrombomodulin regulatory element. Subsequent studies in immunocompetent hemophilic mice, pretreated with tolerizing doses of FVIII or with transient immunosuppression, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels. This loss of transgene expression appears to be due to the disappearance of the implanted cells. The animals treated with either of the two tolerizing regimens did not develop anti‐FVIII antibodies. Biodistribution analysis demonstrated that BOECs were retained inside the subcutaneous implants. These results indicate, for the first time, that genetically modified endothelial progenitor cells implanted in a subcutaneous scaffold can provide sustained therapeutic levels of FVIII and are a promising and safe treatment modality for hemophilia A.

Anti-CD3 prevents factor VIII inhibitor development in hemophilia A mice by a regulatory CD4+CD25+-dependent mechanism and by shifting cytokine production to favor a Th1 response

Non-Fc-receptor binding anti-CD3 Ab therapy, in the setting of several different autoimmune disorders, can induce antigen-specific and long-lasting immunologic tolerance. Because factor VIII (FVIII) inhibitor formation is the most serious treatment-related complication for hemophilia A patients, we tested the efficacy of anti-CD3 to prevent FVIII inhibitor formation in hemophilia A BALB/c and C57BL/6 mice. A short course of low-dose anti-CD3 significantly increased expression of CD25 and the proportion of CD4+CD25+ regulatory T cells in the spleen and potently prevented the production of inhibitory and non-neutralizing anti-FVIII antibodies in both strains of mouse. Depleting the CD4+CD25+ cells during anti-CD3 therapy completely ablated tolerance to FVIII. Further phenotypic characterization of regulatory cells in tolerant mice showed a consistently higher number of CD4+GITR+ and CD4+FoxP3+ cells in both strains of mice. In addition, in tolerant C57BL/6 mice we observed an increase in CD4+CD25+ CTLA-4+ and CD4+CD25+mTGF-beta1+ cells. Finally, in vitro cytokine profiling demonstrated that splenocytes from tolerant BALB/c and C57BL/6 were polarized toward a Th1-immune response. Taken together, these findings indicate that anti-CD3 induces tolerance to FVIII and that the mechanism(s) regulating this response almost certainly occurs through the generation of several distinct regulatory T-cell lineages and by influencing cytokine production and profile.

A MicroRNA-regulated and GP64-pseudotyped Lentiviral Vector Mediates Stable Expression of FVIII in a Murine Model of Hemophilia A

The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system.

A murine model for induction of long-term immunologic tolerance to factor VIII does not require persistent detectable levels of plasma factor VIII and involves contributions from Foxp3+ T regulatory cells

Under certain instances, factor VIII (FVIII) stimulates an immune response, and the resulting neutralizing antibodies present a significant clinical challenge. Immunotherapies to re-establish or induce long-term tolerance would be beneficial, and an in-depth knowledge of mechanisms involved in tolerance induction is essential to develop immune-modulating strategies. We have developed a murine model system for studying mechanisms involved in induction of immunologic tolerance to FVIII in hemophilia A mice. We used lentiviral vectors to deliver the canine FVIII transgene to neonatal hemophilic mice and demonstrated that induction of long-term FVIII tolerance could be achieved. Hemophilia A mice are capable of mounting a robust immune response to FVIII after neonatal gene transfer, and tolerance induction is dependent on the route of delivery and type of promoter used. High-level expression of FVIII was not required for tolerance induction and, indeed, tolerance developed in some animals without evidence of detectable plasma FVIII. Tolerance to FVIII could be adoptively transferred to naive hemophilia recipient mice, and FVIII-stimulated splenocytes isolated from tolerized mice expressed increased levels of interleukin-10 and decreased levels of interleukin-6 and interferon-gamma. Finally, induction of FVIII tolerance mediated by this protocol is associated with a FVIII-expandable population of CD4(+)CD25(+)Foxp3(+) regulatory T cells.

Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain

Summary.  Background: The development of anti‐factor VIII (FVIII) neutralizing antibodies (inhibitors) is a significant obstacle to FVIII replacement therapy. Objective: As mucosal administration of an antigen may induce immune tolerance we have evaluated the efficacy of mucosal antigen exposure to achieve tolerance to FVIII. Methods: We investigated the effects of oral and nasal administration of the purified FVIII C2 domain (FVIII‐C2) to FVIII‐deficient BALB/c mice prior to FVIII protein challenge. Mice received oral or nasal doses of FVIII‐C2, followed by a subcutaneous challenge of either FVIII‐C2 or FVIII. The development of anti‐FVIII inhibitors, cytokine production by splenocytes in vitro, and adoptive transfer assays were analyzed. Results and Conclusions: Mucosal administration of FVIII‐C2 decreases the titer of anti‐FVIII‐C2 inhibitors after FVIII‐C2 challenge, and decreases the percentage of FVIII‐C2 specific antibodies after challenge with full‐length FVIII. Tolerance induction to FVIII‐C2 is associated with increased IL‐10 production by splenocytes in vitro, and can be adoptively transferred to naïve mice. This study is the first to demonstrate that tolerance to the FVIII‐C2 domain can be induced via the mucosal route. Based on these results, the potential use of FVIII‐specific mucosal tolerance induction as an immunotherapy treatment for anti‐FVIII inhibitor development warrants further investigation.

Reduction of the immune response to factor VIII mediated through tolerogenic factor VIII presentation by immature dendritic cells

Summary.  Background: The development of neutralizing antibodies to factor FVIII (FVIII) represents the most serious complication in the treatment of hemophilia A. Objective: We have explored the potential of using immature dendritic cells (iDCs) to present FVIII in a tolerogenic manner to T cells. Methods: The iDCs were isolated from hemophilic murine bone marrow and pulsed with canine cFVIII (cFVIII‐iDCs) in the presence or absence of the NFκB pathway blocking compound Andrographolide (Andro‐cFVIII‐iDCs). Three weekly intravenous infusions of one million cFVIII pulsed‐iDCs were administered to a group of five hemophilic Balb/c mice. Anti‐FVIII antibody levels were monitored by functional Bethesda assay after four weekly intravenous challenges with 2 IU of cFVIII. Results: We have shown that cFVIII in the presence or absence of Andro is efficiently taken up by iDCs and that this process does not result in the maturation of DCs or the activation of co‐cultured T cells. Following repeated infusion of the cFVIII‐iDCs and Andro‐cFVIII‐iDCs into hemophilic mice, which were subsequently challenged with cFVIII, long‐term reductions of FVIII inhibitors of 25% and 40%, respectively, were documented. Studies of cytokine release and T‐cell phenotypes indicate that the mechanisms responsible for reducing immunologic responsiveness to cFVIII appear to involve an expansion of Foxp3 T regulatory cells in the case of cFVIII‐iDC infusion and the elaboration of the immunosuppressive cytokines IL‐10 and TGF‐β following andrographolide‐treated cFVIII‐iDCs. Conclusions: This study shows that tolerogenic presentation of cFVIII to the immune system can significantly reduce immunogenicity of the protein.

610. Evaluation of Three Reticuloendothelial System Blocking Modalities on the Outcome of Adenoviral Mediated Gene Therapy in Hemophilic Mice

Gene therapy is an attractive potential treatment for hemophilia A and replication deficient adenovirus has been a commonly used vector for hepatic transgene delivery. However, a major obstacle has been the activation the innate and adaptive immunity when applied in vivo. The virus localizes in the liver within one hour after IV administration and is taken up by liver macrophages. This induces inflammatory cytokines that limit the efficiency of transduction and causes substantial loss of the vector genome. We aimed to evaluate the influence of three modalities of blocking the reticuloendothelial system on the outcome of Ad mediated gene delivery in FVIII KO mi Transfusion of chilled platelets (1 |[times]| 108 platelets) either 1h before (group I) or simultaneously with (group II) Ad. Immunoglobulin preparation (IVIG-50 mg) and the macrophage depleting drug Clodronate (200 uL clodronate liposome). In all mice, we used an E1/E3 deleted with canine FVIII. HNF (1 |[times]| 1011 viral particles). Assessment of the outcome of gene therapy was done by FVIII assay, liver transaminase (ALT) levels, TNF alpha, FVIII inhibitors and platelet count. Our study showed that FVIII expression levels that peaked at 1 week post Ad injection was significantly higher with the clodronate group (n=5, p=0.005) and significantly lower in mice receiving IVIG (n=5, p=0.0001) when compared to the group that received Ad only. In both groups, the FVIII antibodies were significantly lower when compared to the group that received Ad alone. The two chilled platelet groups did not show significant effects on FVIII levels or FVIII inhibitor incidence. The ALT significantly elevated at day 8 in the two groups receiving chilled platelets (group I; n= 5 p=0.01) (group II; n= 3, p=0.002).There was a significant reduction of ALT in the IVIG group (n=5, p= 0.002) when compared to the Ad only group. TNF alpha showed a significant elevation 3 hours post injection in the group receiving IVIG (n=5, p=0.04) and a significant reduction in the Chilled/Ad simultaneously group (n=3, p=0.01). At 1W, a significant reduction was obtained in the clodronate group only (n=5, p= 0.02) and no effect among the other groups. Thrombocytopenia was least evident with the clodronate and IVIG groups and most evident with the two chilled platelet groups (group I: n=5, p= 0.00001)(group II: n=3, 0.002) with longer duration of thrombocytopenia in group II. These data show that there is a potential for using the clodronate in improving the outcome of gene therapy since it not only enhances the expression of FVIII but also significantly reduces the adaptive immune response to FVIII. The IVIG preparation may have a positive influence in modulating the immune response to Ad, however a major obstacle is the limitation of the gene expression presumably due to neutralizing anti- adenovirus antibodies. Although the chilled platelets may play a minor role in blocking liver macrophages, this strategy does not improve the FVIII expression levels and has shown adverse effects in terms of liver injury as well as thrombocytopenia.

410. Adenovirus Activates Mouse Platelets and Induces Platelet Leucocyte Association

Adenovirus vectors have been used extensively in pre-clinical and clinical studies as agents for gene delivery. Acute, transient thrombocytopenia is a known adverse effect of adenovirus vector administration with a mechanism that currently remains unresolved. The fall in platelets appears 24 hours following IV administration of adenovector and is vector dose dependent. We have initiated experiments to investigate the mechanism of Ad induced thrombocytopenia. We examined the expression of the platelet activation marker P-selectin (CD-62p) on mouse platelets after incubation with adenovirus in vitro by means of flow cytometry. An indirect immunoflourescence approach was used with anti-mouse CD 62p monoclonal antibody and an FITC labeled rat antimouse-IgG. An appropriate negative isotype control and positive platelet activation control using thrombin were incorporated into these studies. The index of platelet activation (IPA), was used to quantify the activation effect of Ad on the platelets. This is the product of the mean channel fluorescence and the percentage of positive cells. We also examined Platelet leucocyte association (PLA) using the direct immunoflourescence and double colour staining of platelets and leucocytes using FITC labeled anti-mouse CD41 (platelets) and PE labeled anti-mouse CD 45 (common leucocyte antigen) respectively. Our study has shown that the mean IPA for platelets stimulated by adenovirus was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. PLA visualisation was also confirmed by means of direct fluorescence microscopy. These preliminary in vitro results indicate that the Adenovirus activates mouse platelets as it induces degranulation and expression of P-selectin and supports the recruitment of leucocytes presumably through an interaction with the leucocyte P-selectin glycoprotein ligand-1. This may have a role in mediating the accelerated platelet clearance by the reticuloendothelial system following adenovirus administration. In addition, the interaction with leucocytes may also stimulate pro-coagulant microparticle generation and thus lead to systemic hemostatic activation. To our knowledge, this is the first report that identifies Adenovirus as an inducer for mouse platelet activation and as a promoter for platelet leucocyte association. However, further in vitro and in vivo studies are still needed to better understand the mechanism of Ad induced thrombocytopenia; a complication that significantly limits the application of Ad mediated gene therapy.