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Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

The major complication associated with protein replacement therapy currently used in the treatment of hemophilia B (HB) is the development of antibodies to the infused human Factor IX (hF.IX). We hypothesized that vector-mediated expression of hF.IX, either at a prenatal stage or early in life may lead to tolerance to hF.IX and long-term transgene expression. Fetal, neonatal, and adult F.IX-deficient mice were injected with AAV-1-hF.IX, and the hF.IX levels as well as antibodies to hF.IX in the circulation were assayed. In utero injection followed by postnatal re-administration of adeno-associated virus 1 (AAV-1) vector achieved persistent expression of hF.IX in all animals, with no cellular or humoral immune response to F.IX. Similar results were seen after initial injection in neonatal mice followed by re-administration, whereas all mice injected at the adult stage developed antibodies to hF.IX. In contrast, after administration of AAV-2-hF.IX in the neonatal period, antibodies to hF.IX were formed in all the injected animals. We conclude that in utero or neonatal-stage injection of AAV-1-hF.IX can lead to long-term expression and absence of immune response. The differences in immune response between the AAV-1 and AAV-2 groups suggests that tolerance may be related to differences in bio-distribution, timing of expression, and/or the initial levels of hF.IX expression. This supports the concept of a narrow “window of opportunity” for tolerance induction.

The goal of these studies was to test whether adeno-associated virus (AAV) capsid-specific CD8+ T cells cause loss of hepatic AAV-mediated gene expression in experimental animals. Mice immunized with adenoviral vectors expressing AAV capsid or with AAV vectors developed CD8+ T cells in blood, lymphatic tissues, and liver to epitopes shared between AAV2 and AAV8, and serotype-specific neutralizing antibodies. At the height of the T cells' effector phase, mice were infused with a heterologous AAV vector expressing human factor IX under a hepatocyte-specific promoter. Despite the presence of lytic CD8+ T cells in the liver, hepatic Factor IX expression was sustained and comparable in AAV-preimmune and naïve animals. These results suggest that, in mice, pre-existing CD8+ T cells to AAV capsid do not affect the longevity of AAV-mediated hepatic gene transfer. These results are in contrast to the outcome of a recent gene therapy trial of hemophilia B patients who were treated by hepatic gene transfer of AAV2 vectors expressing Factor IX. The loss of Factor IX expression, accompanied by a rise in liver enzymes and detectable frequencies of circulating AAV capsid-specific T cells, suggested T-cell-mediated destruction of transduced hepatocytes following reactivation of AAV-specific T cells upon AAV transfer.

We set out to analyze the fundamental biological differences between AAV2 and AAV8 that may contribute to their different performances in vivo. High-throughput protein interaction screens were used to identify binding partners for each serotype. Of the >8,000 proteins probed, 115 and 134 proteins were identified that interact with AAV2 and AAV8, respectively. Notably, 76 of these protein interactions were shared between the two serotypes. CDK2/cyclinA kinase was identified as a binding partner for both serotypes in the screen. Subsequent analysis confirmed direct binding of CDK2/cyclinA by AAV2 and AAV8. Inhibition of CDK2/cyclinA resulted in increased levels of vector transduction. Biophysical study of vector particle stability and genome uncoating demonstrated slightly greater thermostability for AAV8 than for AAV2. Heat-induced genome uncoating occurred at the same temperature as particle degradation, suggesting that these two processes may be intrinsically related for adeno-associated virus (AAV). Together, these analyses provide insight into commonalities and divergences in the biology of functionally distinct hepatotropic AAV serotypes.

Adeno-associated virus (AAV) vectors hold the potential to treat a variety of inherited and acquired human diseases. One of the confounding issues regarding the use of AAV in humans is the cellular immune response to the virus vector. While the ability of humoral immunity to interfere with AAV transduction is well-established, the ability of cellular immunity targeting the vector to ablate AAV transduction has only recently been identified. Our laboratory previously described the use of adenovirus expressing AAV capsid as a means to invoke a cellular immune response towards AAV in a mouse model system. These studies identified the immunodominant CD8 T-cell epitopes in Balb/C and C57Bl/6 mice immunized against AAV capsid of serotypes 2 and 8 (Mol Ther. 2005 Dec;12(6):1023-33). Cytolytic potential of these AAV capsid specific T-cells, however, was not investigated. In the current study, we seek to determine whether cellular immune responses reduce the efficiency of AAV transduction.We first evaluated the cytolytic activity of AAV capsid specific T-cells using a sensitive in vivo killing assay. In this assay, naïve murine splenocytes were loaded with either an immunodominant AAV peptide or an irrelevant AAV peptide. To discriminate between the two populations, each was labeled with a different concentration of CFSE dye. The two cell populations were then mixed together in equal numbers and injected into the tail vein of naïve and AAV- immune mice. Nearly equal proportions of both labeled subsets were recovered from naïve mice. In contrast, AAV-immune mice efficiently destroyed splenocytes loaded with the immunodominant AAV peptide. Percent specific lysis was greater than 70% for three out of four immunodominant peptides tested. These results show that AAV immunized mice harbor potent cytotoxic activity directed towards AAV capsid.We subsequently tested the ability of splenocytes from AAV-immune mice to reduce long-term AAV-mediated transgene expression in the liver. Naïve mice were first injected via the portal vein with 5×10 10 vector genomes encapsidated by AAV-2 or AAV-8 capsid. These vectors encoded the human factor IX (hFIX) gene expressed under the control of a liver-specific promoter. Following a delay of 1-2 hours, these mice then received an adoptive transfer of splenocytes harvested from capsid matched AAV-immune mice. Control mice received adoptive transfer of splenocytes from naïve mice. Two months post-infusion, mice that received adoptive transfer from AAV-2 immune mice expressed significantly lower levels of hFIX than mice receiving adoptive transfer of naïve splenocytes (775ng/mL vs. 1771ng/mL, p=.03). Recipients of adoptive transfer from AAV-8 immune mice similarly exhibited reduced levels of hFIX two months post-infusion compared to control mice (606ng/mL vs. 1637ng/mL, p=.04). We conclude from these studies that a cellular immune response specific for AAV capsid can reduce AAV-mediated gene transfer in the liver.

Identifying factors that influence gene transfer efficacy is critical for a successful gene-based clinical study. Here we demonstrate that in vivo AAV-2-mediated gene transfer is efficiently inhibited by unfractionated heparin, but not by a heparin preparation containing mainly low-molecular-weight forms (LMWH). Surprisingly, inhibitors of thrombin or factor Xa (F.Xa) significantly reduced AAV-2 transduction in a dose-dependent manner. These effects were independent of the vector promoter, transgene, or strain of mice. Expression by alternate AAV serotypes 5 and 8 was not affected by anticoagulant drugs, which suggests an AAV-2-specific effect. Moreover, AAV-2-mediated gene expression was diminished in mice with deficiency in thrombin generation (factor IX deficiency) and enhanced in mice with procoagulant phenotype due to factor V Leiden. In addition, inhibitors of F.Xa diminished adenovirus-mediated gene expression. These results demonstrated that coagulation activity itself is critical to ensure optimal viral vector transduction. Since intravascular delivery of vectors often requires the use of anticoagulants, the use of LMWH appears to be safe. These observations are of relevance for approaches using AAV-2 or adenoviral vectors, especially in early phase studies designed to identify the minimum therapeutic doses.

In a Phase I trial for Hemophilia B six subjects underwent infusion of an adeno-associated viral (AAV) vector expressing human Factor IX (F.IX) under the control of a liver specific promoter. One subject in the high dose cohort (5×10e12 vg/kg) attained therapeutic F.IX levels in the range of 5-12% for 5 wks after vector infusion, but these declined to baseline (<1%). The decrease in F.IX levels was accompanied by an asymptomatic transaminitis. The subject never developed a F.IX inhibitor. The decline of F.IX expression in this patient may have been due to immune-mediated destruction of transduced hepatocytes as a result of immune response to vector-encoded F.IX or to residual AAV capsid or to both of these antigens. We hypothesized that pre-existing T cell responses to AAV capsid may be stimulated upon vector administration and that rAAV capsid may be recognized by the immune system as it is cleared from the transduced cells. In order to study the persistence of AAV capsid-derived protein in triggering the immune response, we set up an animal model that would allow us to assess the duration of immunologically detectable AAV capsid in mice injected with an AAV-2 vector via the portal vein. In an adoptive T cell transfer (ATT) experiment, donor mice (C57Bl/6) were immunized with a plasmid expressing AAV-2 capsid, and boosted with an adenoviral vector expressing AAV-2 capsid. Lymphocytes were harvested and transferred to the recipient mice (C57Bl/6). Intracellular cytokine staining documented the presence of AAV-2 capsid-specific CD8+ cells after immunization and the immunodominant CD8 epitope for AAV2 capsid in C57Bl/6 mice was identified. Recipient mice were injected with an AAV2-F.IX vector with a liver-specific promoter (4×10e12 vg/kg) at time points ranging from 1 wk to 3 months prior to T cell transfer. We measured F.IX levels in recipient mice at a series of time points after ATT. In the absence of ATT (control group), mice expressed F.IX at levels in the range of ∼500-1500 ng/ml at steady state (3-4 wks after injection). When T cells were transferred 1 wk after AAV vector injection, F.IX levels at steady state were 20-200 ng/ml, while mice that received T cell transfer at 4 wks after AAV injection showed F.IX levels in the range of 500-1500 ng/ml, i.e. similar to mice that did not receive ATT. Thus transfer of AAV-2-specific T cells at an early time point after AAV injection resulted in reduced F.IX expression, presumably through immune-mediated destruction of hepatocytes containing residual AAV-2 capsid, while ATT at later time points did not reduce F.IX expression compared to levels seen in mice that did not receive T cells. We conclude that AAV-2 capsid sequences may persist in an immunologically detectable form for a period of >1 week but <4 wks after rAAV-2 vector injection. This mouse model will allow further investigation of the effectiveness of transient immune suppression at the time of vector administration and for the period surrounding immunologic persistence of capsid proteins.