Article Published in the Author Account of

Alexandra Sharland

Liver-directed Gene Expression Using Recombinant AAV 2/8 Vectors — a Tolerogenic Strategy for Gene Delivery?

Abstract: Vectors based on recombinant adeno-associated virus (AAV) 2/8 hold considerable promise for use in human gene therapy. These vectors are safe, and have minimal immunostimulatory properties. Their combination with efficient, liver-specific promoters allows high-level transgene expression in the hepatocytes of small and large animals. In small animal models, this high level of liver expression results in tolerance to the transgene products. Tolerance to transgene products may also be achievable using these vectors for human gene therapy, but the HLA diversity (and thus variability in T cell recognition of transgene products) and high frequency of prior natural exposure to AAV in human populations impose additional challenges that must be overcome in order for this strategy to succeed.



Rationale for Gene Delivery to the Liver

The liver is required to perform a remarkable number of tasks to maintain metabolism, energy storage and release, plasma protein secretion, and detoxification of harmful metabolites and xenobiotics. Given the diverse range of hepatic functions, the liver is therefore an appealing gene therapy target for correction of inborn errors of metabolism (Alexander et al., 2008) and coagulopathies (Nathwani et al., 2003). In addition to the correction of various genetic deficits, there is increasing interest in the potential use of gene delivery to hepatocytes to express therapeutic proteins in other situations. For instance, in the context of organ and tissue transplantation, the liver may be used to express immunomodulatory molecules which might attenuate ischemia-reperfusion injury and rejection responses, and/or promote graft tolerance (Laurence et al., 2009a). The increasing efficiency of liver gene delivery is also presenting opportunities for the treatment of infectious diseases (Khaliq et al., 2010). A gene transfer vector well suited to the task of liver gene therapy is adeno-associated virus (AAV), a vector with high gene transfer efficiencies and limited immunostimulatory properties.

Brief Overview of AAV Biology

AAV is a non-pathogenic human parvovirus isolated over 40 years ago (Atchison et al., 1965; Hoggan et al., 1966). The replicative cycle of the virus is dependent on co-infection with a helper virus, usually adenovirus (Atchison et al., 1965), although members of the herpes virus family are also suitable (Atchison, 1970). In the absence of the helper virus, AAV proviruses persist episomally in post-mitotic cells (Muzyczka, 1992) or integrate into the genome of mitotic cells with preference for a specific site on chromosome 19 (Kotin et al., 1990; Samulski et al., 1991). The viral genome is single-stranded DNA of approximately 4,700 nucleotides (Rose et al., 1969; Srivastava et al., 1983) and encodes two large open reading frames, Rep and Cap. Flanking the genome are 145-nucleotide inverted terminal repeat (ITR) sequences required for site-specific integration, genome replication, and encapsidation (Lusby et al., 1980; Samulski et al., 1983).

AAV possesses many qualities ideally suited for gene transfer. It is non-pathogenic and unable to complete its virus cycle without co-infection with a helper virus. Vector safety is further enhanced by removal of all virus coding sequences as only the 145-nucleotide ITRs are required for genome packaging. The capacity of AAV to establish a latent infection combined with AAV’s minimal activation of innate immune pathways enable long-term transgene persistence in immuno-competent hosts (Kessler et al., 1996; Xiao et al., 1996).

Retention of the ITR and replacement of the virus coding sequences with promoter elements, transgene, and poly-adenylation signal conveniently enables packaging of the expression cassette into virus capsids and, since the pioneering studies to develop AAV for gene delivery (Hermonat and Muzyczka, 1984; Tratschin et al., 1984), many problems have been effectively overcome. The generation of wild-type AAV and adenovirus during vector packaging has been eliminated by the separation of AAV replicative and structural genes onto different plasmids and substitution of adenovirus co-infection with plasmid co-transfection (Xiao et al., 1998). One of the most significant advances has been the demonstration that vector constructs can be “cross-packaged” into capsids of alternative serotypes (Gao et al., 2005) effectively altering vector tropism. The best characterized capsid serotypes have been 1-11, which have revealed a diverse range of tissue tropisms for terminally differentiated or post-mitotic tissues, including liver, muscle, brain, lung, and eye (Gao et al., 2005). The efficiency of gene transfer in many systems is impressive, with some animal studies showing genetic modification of every target cell in a living organism (Gao et al., 2002; Gregorevic et al., 2004; Wang et al., 2005).

Spectrum of Immune Responses to AAV-mediated Gene Delivery

AAV-mediated introduction of a foreign gene exposes the host to two sources of potentially antigenic protein; the viral capsid and the protein(s) encoded by the transgene. The immune response to these proteins may be ignorance, tolerance, or a productive response, leading to their neutralization or elimination. The outcome is influenced by the biology of the vector, the extent of vector biodistribution and of transgene expression, the nature of the transgene, the host’s genetic background, and previous history of exposure to viral proteins and/or transgene products.

Humoral and Cell-mediated Responses Against AAV Capsid

Studies of AAV-mediated gene transfer have consistently described a neutralizing antibody response against viral capsid antigens (Halbert et al., 1997; Halbert et al., 1998). These antibodies reduce the efficiency of gene expression upon subsequent administration of the vector, but have not been reported to damage transduced cells which may be displaying capsid-derived peptides at the cell surface. In order for a humoral response to be generated against capsid proteins, these proteins must bind to the B cell antigen receptor and also be taken up and processed by dendritic cells (DC), with the resulting peptides presented to CD4+ T helper cells in conjunction with MHC class II. Interaction of activated T and B cells results in B cell survival, and antibody production.

For an anti-capsid cytotoxic T cell-mediated response to be primed, capsid proteins must enter the class I antigen-presentation pathway in DC. This can occur whenever there is uptake of the vector into DC, even if the transgene is not expressed in this cell type. Binding of the capsid to the extracellular matrix molecule heparan sulphate proteoglycan (HSPG) facilitates uptake of the virion by DC, and directs internalized virus into the class I pathway (cross-presentation). Recent work has highlighted differences between the capsids of various AAV serotypes in their ability to bind HSPG, and thus to stimulate anti-capsid responses. AAV2, the prototypic AAV serotype, has HSPG-binding activity, whereas AAV8 lacks this property (Vandenberghe et al., 2006). Other aspects of capsid structure influence T cell immunity against both capsid and transgene products (Mays et al., 2009). The capsid of AAV rh32.33, an engineered vector phylogenetically similar to AAV4, drives vigorous IFN-γ-producing T cell responses against both capsid and transgene-derived antigens, leading to the elimination of transgene expression within 60 days (Mays et al., 2009). Blockade of CD4+ T cell help by CD4+ depletion or by interruption of CD40-CD40L interactions completely ablated capsid and transgene-specific CD8+ T cell responses in this model. With one exception, discussed below, AAV8 failed to trigger a response against a panel of different transgenes in multiple mouse strains tested (Mays et al., 2009).

Most AAV vectors do not have strong adjuvant properties which would promote a vigorous primary cell-mediated response, but prior natural infection with an AAV and concomitant Adenovirus may result in memory cells specific for capsid proteins, which can be activated upon re-exposure to AAV alone (Manno et al., 2006). Whether or not these cells can destroy transduced target cells depends upon the extent and persistence of viral peptides bound to the target cell surface, and the presence of any immunomodulatory cell populations able to suppress cell-mediated immunity.

Requirements for Productive Immune Responses Against Transgene Products

Theoretically, generation of a CD8+ T cell response against the transgene product(s) could result either from their expression in DC, or in a compartment from which they could be captured, internalized, and directed into the DC class I antigen-presentation pathway. In practice, vector-mediated transduction of DC appears necessary for cell-mediated responses against most transgene products to occur (Jooss et al., 1998; Mercier et al., 2002). Post-entry blocks usually prevent transgene expression from AAV vectors that have entered DC, even when the promoter is compatible with DC expression (Jooss et al., 1998), with the result that T cell responses against transgene products delivered by low-immunogenicity AAV vectors are rarely observed. One exception involved AAV8-mediated green fluorescent protein (GFP) expression in Balb/c mice, where display of a strong immunodominant epitope of GFP on H-2d overcame the threshold for CD8+ T cell activation, resulting in a substantial GFP-specific response (Mays et al., 2009).

Expression of a soluble transgene product, or one which can be shed from the cell surface, may also lead to generation of an antibody response. Antibody responses to coagulation factors, α-1 antitrypsin, lysosomal enzymes, and other antigens have been demonstrated following gene transfer (Dobrzynski et al., 2004). Antibody formation is influenced by the genetic background of the host, and is most likely to occur if the host is completely lacking the transgene product (Fields et al., 2001). Consequences of a neutralizing antibody response include blockade of the therapeutic effect of the secreted protein and interference with subsequent conventional therapy using the same protein antigen.

Sustained Transgene Expression After AAV-mediated Gene Delivery — Ignorance or Tolerance?

Low-immunogenicity AAV vectors have been used to achieve sustained transgene expression, often accompanied by correction of a disease phenotype, in a range of target tissues, including skeletal muscle, CNS, and liver. However, sustained gene expression is not necessarily synonymous with immunological tolerance. Tolerance implies an active process, whereby the recipient’s lymphoid cells recognize an antigen, but are prevented from mounting a productive response against it. This may be because the responding cells are deleted, rendered dysfunctional (anergic), or are able to function when in isolation, but have their function suppressed by another population of (regulatory) cells.

Muscle expression of a truncated version of the HIV-1 gag gene by rAAV2 vectors pseudotyped with capsids from AAV serotypes 1, 2, 5, 7, 8, and 9 leads to the production of gag-specific CD8+ T cells (Lin et al., 2007). Whilst these cells do not destroy rAAV-transduced muscle cells, they are able to lyse cells pulsed with the gag peptide when these are adoptively-transferred into immunized recipients (Lin et al., 2007). Gag-specific CD8+ T cells in this model fail to proliferate upon boosting with the same antigen delivered by an immunogenic adenoviral or Vaccinia-derived vector, and show some features of exhaustion including upregulation of the negative costimulatory receptor PD-1. However, proliferative capacity was not restored by PD-1 blockade, nor was the presence of regulatory T cells able to be demonstrated. The authors hypothesized that a state of “partial exhaustion” of these cells was the result of prolonged exposure to the antigen (Lin et al., 2007).

AAV2-mediated expression of the LacZ gene product β-galactosidase (β-gal) in mouse skeletal muscle persists as long as the transgene product remains hidden from the recipient’s immune system. Subsequent administration of an adenoviral vector encoding LacZ to the contralateral muscle instigates a cytotoxic T cell-mediated response against β-gal, which results in the destruction of all cells expressing this transgene, whether transduced by the AAV or the adenoviral vector (Jooss et al., 1998). Persistence of β-gal expression after AAV-administration in this model was thus an example of ignorance rather than true tolerance. In contrast to this, AAV-mediated liver-specific gene delivery has been demonstrated to induce tolerance to a number of transgene products, both expressed in the intracellular compartment, and secreted. This will be discussed further, below.

The “Liver Tolerance Effect”

Expression of a foreign antigen in the liver often leads to immunological non-reactivity to the antigen while presentation of the same antigen by other routes results in an immune response. This is termed the “liver tolerance effect” and is supported by the following findings: 1) oral tolerance, where ingestion induces tolerance to an antigen that can stimulate a response after sub-cutaneous or intra-venous injection, depends on the liver; 2) portal venous tolerance, where injection of an antigen into the liver via the portal vein leads to tolerance compared to other routes; 3) liver transplant tolerance, where transplanted livers are much less likely to be rejected than heart or kidney transplants and can even prevent rejection of these organs in animal models; and 4) persistence of viral infections that target the liver such as hepatitis B and C. This hypo-reactivity of the immune response to antigens expressed in the liver suggests that expression vectors that target the gene of interest to the liver are less likely to induce an immune response than those that target other tissues.

Oral ingestion of antigen often results in non-responsiveness of both antibody and delayed-type hypersensitivity (cell-mediated) responses to that antigen on subsequent challenges whereas intravenous or subcutaneous injection often induces an immune response (Chase, 1946). Subsequently this was shown to depend on the liver as removal of the liver from the portal circulation (Cantor and Dumont, 1967; Callery et al., 1989; Yang et al., 1994) or a dysfunctional liver (Thomas et al., 1976) prevents induction of oral tolerance. Furthermore, injection of antigen directly into the portal vein results in non-responsiveness while injection into the vena cava often does not and may even result in priming (Triger et al., 1973). Numerous investigators have confirmed that intraportal inoculation results in tolerance (Qian et al., 1985; Nakano et al., 1992) showing the importance of liver, not the gut, in this process.

A clear demonstration that antigen presentation by the liver can induce immunological tolerance comes from liver transplantation. In animal models, livers transplanted across complete major histocompatibility complex (MHC) barriers are accepted without requiring treatment (Zimmermann et al., 1979) and rapidly induce donor-specific tolerance (Kamada et al., 1981). This is seen consistently in a range of species including mice, rats, pigs, and primates (Bishop et al., 2002). In clinical transplantation, liver transplant patients have less acute and chronic rejection than recipients of organs such as kidneys or hearts (Bishop and McCaughan, 2001; Bishop et al., 2002). Moreover, in contrast to recipients of kidney or heart transplants, a significant proportion of liver transplant patients with stable function can be completely removed from all immunosuppression, a state termed “operational tolerance” (Lerut and Sanchez-Fueyo, 2006).

Many mechanisms of the liver tolerance effect have been proposed (Benseler et al., 2007). Two are of special relevance here: the large size of the liver, which exhausts the immune response (Sun et al., 1996; Wang et al., 1999a) and the unique fenestrated endothelium of the liver that allows direct contact between naïve lymphocytes and hepatocytes (Bowen et al., 2004). The vascular endothelium lining liver sinusoids, which is exposed to the blood entering the liver from the gut via the portal vein, is different from other endothelia. Liver sinusoidal endothelium is fenestrated, with many small holes that allow direct contact between recirculating lymphocytes in the blood and hepatocytes. As only antigen-experienced (memory) T lymphocytes, not naïve T cells, are able to traverse normal vascular endothelium to contact parenchymal cells, the liver provides a unique environment where naïve T cells can directly contact hepatocytes (Bertolino et al., 2002). As hepatocytes do not express all the molecules necessary for complete activation, responding T cells are not maximally activated, and die or become unresponsive.

These two properties of the liver suggest that approaches that result in the expression of antigen in the greatest number of hepatocytes might be most likely to lead to unresponsiveness, both to the antigenic components of the vector and to the construct that it expresses.

Means of Achieving Liver-specific Expression

Targeting gene delivery and restricting transgene expression to the intended tissues is paramount in many gene therapy applications. The precision of gene delivery and expression can be guided and/or regulated at multiple levels, with route of vector delivery being the first consideration. Portal vein injection is the most direct approach in specifically targeting the liver although intrasplenic injection is used in some animal models as a convenient alternative to access the portal circulatory system. We have also found that a simple injection into the murine intraperitoneal cavity is an effective route for AAV gene delivery to the liver (Cunningham et al., 2008).

Choice of capsid for vector packaging is a second consideration that can guide vector to the intended tissue. AAV serotype 8 is highly liver tropic with impressive gene transduction in the livers of mice (Cunningham et al., 2008; Cunningham et al., 2009), rats (Laurence et al., 2009b), and Rhesus macaques (Wang et al., 2010). Furthermore, the serotype 8 capsid induces faster kinetics of transgene expression in the liver compared to other capsid serotypes (Thomas et al., 2004). Such a feature is appealing when rapid induction of transgene expression is required for a therapeutic end-point.

The tissue tropism of serotype 8 capsid is not confined to the liver (Wang et al., 2005). Consequently there is opportunity for this vector to have “off-target” effects and to deliver transgenes to non-hepatic tissue. Use of liver-specific promoters to drive transgene expression reduces the chances of protein synthesis outside the liver. For example, liver-specific promoters engineered into AAV vectors have been based on thyroid hormone-binding globulin promoter (Wang et al., 1999b) and α-1-anti-trypsin promoter (Cunningham et al., 2009). Not only does a liver-specific promoter strategy enhance the safety of the vector but it also improves the likelihood of tolerance induction to the transgene product.

AAV-mediated Liver Expression of Various Transgenes Results In Tolerance

Liver-directed transgene delivery in mouse models has been shown to result in tolerance to a number of expressed proteins, both secreted (α-1 anti-trypsin, chicken ovalbumin, Factor IX) (Breous et al., 2009; Dobrzynski et al., 2006; Dobrzynski et al., 2004) and cytosolic (β-galactosidase) (Martino et al., 2009). Studies using transgenic mice for the D011.10 T cell receptor which specifically recognizes a peptide of chicken ovalbumin (OVA) demonstrated antigen-specific CD4+ T cell anergy and deletion following AAV-mediated liver-specific expression of OVA. The proportion of thymocytes expressing the D011.10 receptor declined over time, suggesting that central, as well as peripheral, deletion was occurring. Moreover, these mice failed to produce anti-OVA antibodies after a subsequent challenge with OVA, implying that the absence of T cell help resulted in operational B cell tolerance. Similarly, wild-type mice did not produce antibodies upon challenge with Factor IX (F.IX) following AAV-mediated expression of this antigen in the liver (Dobrzynski et al., 2004).

Cytotoxic T cell responses to transgene products are also abrogated in mice by liver-specific gene expression (Breous et al., 2009; Dobrzynski et al., 2006; Martino et al., 2009; Cooper et al., 2009). In contrast to skeletal muscle expression of these genes, challenge with a highly-immunogenic adenoviral vector following earlier AAV-mediated liver-specific expression of the same transgene does not result in the destruction of transduced hepatocytes. Furthermore, in some studies, secondary gene transfer with an adenoviral vector resulted in an increase in the proportion of transduced hepatocytes and an increased frequency of transgene-specific regulatory T cells (Tregs) (Martino et al., 2009). Expansion of regulatory T cells after liver-directed transgene delivery is consistently described (Breous et al., 2009; Cooper et al., 2009; Dobrzynski et al., 2006; Martino et al., 2009). Adoptive transfer of Tregs confers tolerance to naïve recipients (Dobrzynski et al., 2006; Martino et al., 2009; Cooper et al., 2009), and conversely, tolerance can be broken by the depletion of Tregs (Breous et al., 2009; Cao et al., 2007). Administration of AAV vector alone did not lead to Treg induction (Breous et al., 2009), and whilst antigen-specific Tregs were able to suppress responses against adenoviral capsid and the identical transgene product after secondary gene transfer, they could not suppress responses to other gene products nor to super-antigens (Martino et al., 2009). The type and properties of the antigen-presenting cells responsible for Treg induction remain to be determined, but a recent report indicates that an interaction between Tregs and Kupffer cells is critical for the generation of a suppressive intrahepatic microenvironment following AAV-8-mediated gene transfer (Breous et al., 2009).

Requirements for tolerance induction by the hepatic route include restriction of transgene expression to hepatocytes, use of a vector with minimal ability to trigger innate immune responses, and absence of transgene expression in professional APC (Martino et al., 2009). Tolerance induction after hepatic gene transfer is promoted by higher levels of gene expression. Minimum levels of systemic F.IX expression required to achieve tolerance were 0.5 to 2 nM, while for OVA, tolerance occurred with systemic concentrations of 2-4 nM (Dobrzynski et al., 2004). In the case of the cytosolic antigen β-gal, expression of the transgene in as few as 1-3% of hepatocytes resulted in tolerance to the transgene product (Martino et al., 2009). Titres of neutralizing antibodies against F.IX resulting from AAV-2 mediated hepatic gene transfer are inversely correlated with F.IX expression levels (Cooper et al., 2009). The combination of AAV-8 capsid with the efficient liver-specific ApoE/hAAT promoter results in substantially higher levels of transgene expression at equivalent vector doses, when compared with AAV-2-ApoE2/hAAT (Cooper et al., 2009). Correspondingly, AAV-8-ApoE2/hAAT-driven gene transfer was superior to that mediated by AAV-2-ApoE2/hAAT in inducing tolerance to Human F.IX across a range of F.IX-deficient mouse strains, including C3H/HeJ F9-/- mice which had previously been resistant to tolerance induction (Cooper et al., 2009). Expression of OVA mediated by the analogous AAV-8 vector resulted in a 15-fold increase in OVA-specific Tregs, whereas this cell population increased by 4-fold when an AAV-2 vector was used (Cooper et al., 2009). Although hepatic gene expression levels may be the single most important factor accounting for the difference in ability of different vectors to induce tolerance, there are other differences in the biology of these two vectors which may also influence their tolerogenic potency. Such differences include more rapid expression of the gene product, and more homogeneous distribution of transgene-expressing hepatocytes within the transduced liver after AAV-8 than after AAV-2 administration (Thomas et al., 2004; Cooper et al., 2009).

Of Mice and Men

rAAV-mediated liver-directed gene expression holds great promise for the amelioration of a number of human diseases, including inborn errors of metabolism, and blood coagulation disorders. For gene transfer to mediate a useful therapeutic effect in these conditions, expression of the transgene must be sufficiently high to permit correction of the disease phenotype, and of sufficient duration that only infrequent re-administration would be required. Anti-capsid antibody responses that reduce efficacy of re-administration should be minimized, and neutralizing antibody responses against the transgene product which would interfere with its action and that of the conventionally-administered protein must be avoided.

Robust rAAV-mediated liver-specific transgene expression has been obtained in mice, and indeed in larger experimental animals, including non-human primates, but this has not been replicated in clinical studies (Herzog et al., 2010). Based on convincing preclinical data from several groups, an open-label dose-escalation study of gene therapy for Hemophilia B was undertaken, using an rAAV-2-ApoE2/hAAT vector to express human F.IX in liver (Manno et al., 2006). No adverse events were associated with vector administration. Although gene expression was relatively modest, therapeutic levels of F.IX were achieved in both patients who received the highest dose of vector. There was an inverse relationship between pre-existing titres of neutralizing antibodies against AAV-2 capsid and the level of circulating F.IX. Expression of the transgene was not sustained, and the decline in F.IX levels coincided with mild-moderate liver inflammation, suggesting immune-mediated destruction of the transduced hepatocytes. CD8+ T cell responses to viral capsid, but not to F.IX, were subsequently demonstrated (Manno et al., 2006).

Rates of previous natural exposure to AAV vary significantly between serotypes, with AAV-2 having the greatest seroprevalence worldwide (Calcedo et al., 2009). Vector design could potentially be tailored to the recipient’s exposure profile to avoid neutralizing antibodies and memory T cell responses against capsid antigens, provided that the biology of the vector was otherwise favorable. For instance, although seroprevalence to AAV rh32.33 is almost negligible (Calcedo et al., 2009), the immunogenicity of this vector reduces its suitability for gene therapy (Mays et al., 2009). Increased hepatocyte transduction efficiency would favor tolerance induction, as well as improving therapeutic efficacy. In murine studies, AAV-8 has shown clear superiority in this regard (Cooper et al., 2009), but this advantage has been less apparent in dogs (Sarkar et al., 2006) and non-human primates (Nathwani et al., 2007), and may not be evident in humans. Advances in vector design, such as the combination of AAV-7, -8, or -9 with a self-complementary genome format, have further enhanced the rapidity and extent of gene expression in the liver of both small and large animals (Sun et al., 2010) and have the potential to do so in humans also, thus promoting tolerance. Nonetheless, it may be that vector design and selection will never be sufficient to overcome immune responses in all human recipients or, as suggested by Manno et al. (2006), a brief period of immunosuppression will be required to allow clearance of viral capsid proteins and permit stable gene expression and emergence of tolerance to transgene products.

Conclusions

rAAV 2/8 vectors mediate high level expression of transferred genes in the livers of both small and large animal recipients. The combination of favorable vector biology with the tolerogenic effects of intrahepatic antigen expression results in a gene transfer strategy which can generate true immunological tolerance to transgene products, an outcome which would be of immense benefit to human gene therapy. Results in animal studies are very encouraging. However, given the additional complexity introduced by genetically-diverse human populations with frequent prior AAV exposure, it remains to be seen whether these results will be able to be translated into the clinic.

References

Alexander IE, Cunningham SC, Logan GJ, Christodoulou J. Potential of AAV vectors in the treatment of metabolic disease. Gene Ther 15(11):831-9, 2008.

Atchison RW. The role of herpesviruses in adenovirus-associated virus replication in vitro. Virology 42(1):155-62, 1970.

Atchison RW, Casto BC, Hammon WM. Adenovirus-associated defective virus particles. Science 149:754-6, 1965.

Benseler V, McCaughan GW, Schlitt HJ, Bishop GA, Bowen DG, Bertolino P. The liver: A special case in transplantation tolerance. Semin Liver Dis 27(2):194-213, 2007.

Bertolino P, McCaughan GW, Bowen DG. Role of primary intrahepatic T-cell activation in the ‘liver tolerance effect’. Immunol Cell Biol 80(1):84-92, 2002.

Bishop GA, McCaughan GW. Immune activation is required for the induction of liver allograft tolerance: implications for immunosuppressive therapy. Liver Transpl 7:161-72, 2001.

Bishop GA, Wang C, Sharland AF, McCaughan GW. Spontaneous acceptance of liver transplants in rodents: Evidence that liver leucocytes induce recipient T-cell death by neglect. Immunol Cell Biol 80:93-100, 2002.

Bowen DG, Zen M, Holz L, Davis T, McCaughan GW, Bertolino P. The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity. J Clin Invest 114 (5):701-712, 2004.

Breous E, Somanathan S, Vandenberghe LH, Wilson JM. Hepatic regulatory T cells and Kupffer cells are crucial mediators of systemic T cell tolerance to antigens targeting murine liver. Hepatology 50(2):612-21, 2009.

Callery MP, Kamei T, Flye MW. The effect of portacaval shunt on delayed-hypersensitivity responses following antigen feeding. J Surg Res 46:391-4, 1989.

Cantor HM, Dumont AE. Hepatic suppression of sensitization to antigen absorbed into the portal system. Nature 215:744- 5, 1967.

Cao O, Dobrzynski E, Wang L, Nayak S, Mingle B, Terhorst C, Herzog RW. Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer. Blood 110 (4):1132-40, 2007.

Chase MW. Inhibition of experimental drug allergy by prior feeding of the sensitizing agent. Proc Soc Exp Biol Med 61(3):257-9, 1946.

Cooper M, Nayak S, Hoffman BE, Terhorst C, Cao O, Herzog RW. Improved induction of immune tolerance to factor IX by hepatic AAV-8 gene transfer. Hum Gene Ther 20(7):767-76, 2009.

Cunningham SC, Dane AP, Spinoulas A, Logan GJ, Alexander IE. Gene delivery to the juvenile mouse liver using AAV2/8 vectors. Mol Ther 16(6):1081-8, 2008.

Cunningham SC, Spinoulas A, Carpenter KH, Wilcken B, Kuchel PW, Alexander IE. AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice. Mol Ther 17(8):1340-6, 2009.

Dobrzynski E, Fitzgerald JC, Cao O, Mingozzi F, Wang L, Herzog RW. Prevention of cytotoxic T lymphocyte responses to factor IX-expressing hepatocytes by gene transfer-induced regulatory T cells. Proc Natl Acad Sci U S A 103(12):4592-7, 2006.

Dobrzynski E, Mingozzi F, Liu YL, Bendo E, Cao O, Wang L, Herzog RW. Induction of antigen-specific CD4+ T-cell anergy and deletion by in vivo viral gene transfer. Blood 104(4):969-77, 2004.

Fields PA, Arruda VR, Armstrong E, Chu K, Mingozzi F, Hagstrom JN, Herzog RW, High KA. Risk and prevention of anti-factor IX formation in AAV-mediated gene transfer in the context of a large deletion of F9. Mol Ther 4(3):201-10, 2001.

Gao G, Vandenberghe LH, Wilson JM. New recombinant serotypes of AAV vectors. Curr Gene Ther 5(3):285-97, 2005.

Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A 99(18):11854-9, 2002.

Gregorevic P, Blankinship MJ, Allen JM, Crawford RW, Meuse L, Miller DG, Russell DW, Chamberlain JS. Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nat Med 10(8):828-34, 2004.

Halbert CL, Standaert TA, Aitken ML, Alexander IE, Russell DW, Miller AD. Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration. J Virol 71(8):5932-41, 1997.

Halbert CL, Standaert TA, Wilson CB, Miller AD. Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure. J Virol 72(12):9795-805, 1998.

Hermonat PL, Muzyczka N. Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci U S A 81(20):6466-70, 1984.

Herzog RW, Cao O, Srivastava A. Two decades of clinical gene therapy — success is finally mounting. Discov Med 9(45):105-11, 2010.

Hoggan MD, Blacklow NR, Rowe WP. Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics. Proc Natl Acad Sci U S A 55(6):1467-74, 1966.

Jooss K, Yang Y, Fisher KJ, Wilson JM. Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J Virol 72(5):4212-23, 1998.

Kamada N, Davies H, Roser B. Reversal of transplantation immunity by liver grafting. Nature 292:840-2, 1981.

Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci U S A 93(24):14082-7, 1996.

Khaliq S, Khaliq SA, Zahur M, Ijaz B, Jahan S, Ansar M, Riazuddin S, Hassan S. RNAi as a new therapeutic strategy against HCV. Biotechnol Adv 28(1):27-34, 2010.

Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, Berns KI. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A 87(6):2211-5, 1990.

Laurence JM, Allen RD, McCaughan GW, Logan GJ, Alexander IE, Bishop GA, Sharland AF. Gene therapy in transplantation. Transplant Rev (Orlando) 23(3):159-70, 2009a.

Laurence JM, Wang C, Zheng M, Cunningham S, Earl J, Tay SS, Allen RD, McCaughan GW, Alexander IE, Bishop GA, Sharland AF. Overexpression of indoleamine dioxygenase in rat liver allografts using a high-efficiency adeno-associated virus vector does not prevent acute rejection. Liver Transpl 15(2):233-41, 2009b.

Lerut J, Sanchez-Fueyo A. An appraisal of tolerance in liver transplantation. Am J Transpl 6(8):1774-80, 2006.

Lin J, Zhi Y, Mays L, Wilson JM. Vaccines based on novel adeno-associated virus vectors elicit aberrant CD8+ T-cell responses in mice. J Virol 81(21):11840-9, 2007.

Lusby E, Fife KH, Berns KI. Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA. J Virol 34(2):402-9, 1980.

Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ, Ozelo MC, Hoots K, Blatt P, Konkle B, Dake M, Kaye R, Razavi M, Zajko A, Zehnder J, Rustagi PK, Nakai H, Chew A, Leonard D, Wright JF, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 12(3):342-7, 2006.

Martino AT, Nayak S, Hoffman BE, Cooper M, Liao G, Markusic DM, Byrne BJ, Terhorst C, Herzog RW. Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity. PLoS One 4(8):e6376, 2009.

Mays LE, Vandenberghe LH, Xiao R, Bell P, Nam HJ, Agbandje-McKenna M, Wilson JM. Adeno-associated virus capsid structure drives CD4-dependent CD8+ T cell response to vector encoded proteins. J Immunol 182(10):6051-60, 2009.

Mercier S, Gahery-Segard H, Monteil M, Lengagne R, Guillet JG, Eloit M, Denesvre C. Distinct roles of adenovirus vector-transduced dendritic cells, myoblasts, and endothelial cells in mediating an immune response against a transgene product. J Virol 76 (6): 2899-911, 2002

Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol 158:97-129, 1992.

Nakano Y, Monden M, Valdivia LA, Gotoh M, Tono T, Mori T. Permanent acceptance of liver allografts by intraportal injection of donor spleen cells in rats. Surgery 111:668-76, 1992.

Nathwani AC, Gray JT, McIntosh J, Ng CY, Zhou J, Spence Y, Cochrane M, Gray E, Tuddenham EG, Davidoff AM. Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates. Blood 109(4):1414-21, 2007.

Nathwani AC, Nienhuis AW, Davidoff AM. Current status of gene therapy for hemophilia. Curr Hematol Rep 2(4):319-27, 2003.

Qian J, Hashimoto T, Fujiwara H, Hamaoka T. Studies on the induction of tolerance to alloantigens. I. The abrogation of potentials for delayed-type-hypersensitivity response to alloantigens by portal venous inoculation with allogeneic cells. J Immunol 134(6):3656-61, 1985.

Rose JA, Berns KI, Hoggan MD, Koczot FJ. Evidence for a single-stranded adenovirus-associated virus genome: formation of a DNA density hybrid on release of viral DNA. Proc Natl Acad Sci U S A 64(3):863-9, 1969.

Samulski RJ, Srivastava A, Berns KI, Muzyczka N. Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell 33(1):135-43, 1983.

Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J 10(12):3941-50, 1991.

Srivastava A, Lusby EW, Berns KI. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol 45(2):555-64, 1983.

Sun J, Sheil AGR, Wang C, Wang L, Rokahr KL, Sharland AF, Jung S-E, McCaughan GW, Bishop GA. Tolerance to rat liver allografts IV. Acceptance depends on the quantity of donor tissue and on donor leukocytes. Transplantation 62:1725-30, 1996.

Sun X, Lu Y, Bish LT, Calcedo R, Wilson JM, Gao G. Molecular analysis of vector genome structures after liver transduction by conventional and self-complementary adeno-associated viral serotype vectors in murine and nonhuman primate models. Hum Gene Ther, epub ahead of print, May 5, 2010.

Thomas CE, Storm TA, Huang Z, Kay MA. Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors. J Virol 78(6):3110-22, 2004.

Thomas HC, Ryan CJ, Benj IS, Blumgart LH, MacSween RNM. The immune response in cirrhotic rats. The induction of tolerance to orally administered protein antigens. Gastroenterology 71:114-7, 1976.

Tratschin JD, West MH, Sandbank T, Carter BJ. A human parvovirus, adeno-associated virus, as a eukaryotic vector: transient expression and encapsidation of the prokaryotic gene for chloramphenicol acetyltransferase. Mol Cell Biol 4(10):2072-81, 1984.

Triger DR, Cynamon MH, Wright R. Studies on hepatic uptake of antigen. I. Comparison of inferior vena-cava and portal vein routes of immunization. Immunology 25(6):941-50, 1973.

Vandenberghe LH, Wang L, Somanathan S, Zhi Y, Figueredo J, Calcedo R, Sanmiguel J, Desai RA, Chen CS, Johnston J, Grant RL, Gao G, Wilson JM. Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid. Nat Med 12(8):967-71, 2006.

Wang C, Sheil AG, Sun J. Outcome of different models of multiorgan transplantation in rats. Microsurgery 19(7):318-23, 1999a.

Wang L, Calcedo R, Wang H, Bell P, Grant R, Vandenberghe LH, Sanmiguel J, Morizono H, Batshaw ML, Wilson JM. The pleiotropic effects of natural AAV infections on liver-directed gene transfer in macaques. Mol Ther 18(1):126-34, 2010.

Wang L, Takabe K, Bidlingmaier SM, Ill CR, Verma IM. Sustained correction of bleeding disorder in hemophilia B mice by gene therapy. Proc Natl Acad Sci U S A 96(7):3906-10, 1999b.

Wang Z, Zhu T, Qiao C, Zhou L, Wang B, Zhang J, Chen C, Li J, Xiao X. Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol 23(3):321-8, 2005.

Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 70(11):8098-108, 1996.

Xiao X, Li J, Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72(3):2224-32, 1998.

Yang R, Liu Q, Grosfeld JL, Pescovitz MD. Intestinal venous drainage through the liver is a prerequisite for oral tolerance induction. J Paed Surg 29:1145-8, 1994.

Zimmermann FA, Butcher GW, Davies HS, Brons G, Kamada N, Turel O. Techniques for orthotopic liver transplantation in the rat and some studies of the immunologic responses to fully allogeneic liver grafts. Transpl Proc 11(1):571-7, 1979.

[Discovery Medicine, 9(49):519-527, June 2010.]

Access This PDF as a Subscriber |
Close
Close
E-mail It
Close