E3

Promoter Transgeno

Figure 3 Schematic of adenovirus genome. The bar indicates the DNA genome of 36 kb. The black arrows indicate the transcription units: El A immediate-early transcription unit and the delayed-early El B, E2, E3, and E4 transcription units. The expression cassette replacing the El region is depicted. The cassette contains a promoter with transcriptional elements used to drive expression of the transgene. The transgene is the open reading frame in the recombinant transcript that translates the desired protein. First-generation adenoviruses are made by substituting an expression cassette for the El and/E3 regions. Second-generation adenovirus vectors are generated by the additional deletion of genes necessary for viral replication, e.g., E2a DNA-binding protein [114, 115]; E4 region [113]; the E2b-encoded terminal protein and viral DNA polymerase [120], Third-generation vectors are vectors deleted for all viral genes, but retain the cis-acting sequences necessary for viral replication and packaging (see Chapter 15).

Promoter Transgeno

Figure 3 Schematic of adenovirus genome. The bar indicates the DNA genome of 36 kb. The black arrows indicate the transcription units: El A immediate-early transcription unit and the delayed-early El B, E2, E3, and E4 transcription units. The expression cassette replacing the El region is depicted. The cassette contains a promoter with transcriptional elements used to drive expression of the transgene. The transgene is the open reading frame in the recombinant transcript that translates the desired protein. First-generation adenoviruses are made by substituting an expression cassette for the El and/E3 regions. Second-generation adenovirus vectors are generated by the additional deletion of genes necessary for viral replication, e.g., E2a DNA-binding protein [114, 115]; E4 region [113]; the E2b-encoded terminal protein and viral DNA polymerase [120], Third-generation vectors are vectors deleted for all viral genes, but retain the cis-acting sequences necessary for viral replication and packaging (see Chapter 15).

proteins that are presented in the context of MHC classl molecules to elicit a cytotoxic T-cell response [63]. For these reasons additional changes have been introduced into the backbone of adenovirus vectors to render them more replication-defective and thus further reduce their potential for viral gene expression.

2. Second-Generation Adenoviral Vectors

Two regions of adenovirus, E2 and E4, which play critical roles in viral DNA synthesis and late gene expression have been targeted for deletion [110-113]. A temperature-sensitive mutation, tsl25, and a deletion have been introduced into the E2a region [111, 114, 115], vectors containing this mutation showed prolonged transgene expression in CBA mice, Cotton rats, and nonhuman primates. However, contrary to these reports, a recent study using BALB/C mice and hemophilia B dogs demonstrates that this E2a mutation is insufficient for achieving persistent expression [116].In the case of E4-deleted vectors long-term gene expression is dependent on both the promoter used to control expression and the context of the E4 region [110]. Interpretation of the earlier work on deletions in the E2 and E4 regions were complicated by the immunogenic transgenes which were used in these studies [117, 118]. Another version of a second-generation vector was generated using a nonimmunogenic protein the hCFTR [119]. The vector contained wild-type E2 and E4 with a partial deletion in the E3 region and when instilled into the lungs of various strains of immunocompetent mice persistent transgene expression was measured in lung tissue up to 70 days. In this vector the persistence of transgene expression was attributed in part to the CMV enhancer-promoter used for transgene expression in conjunction with a wild-type E4 region [110, 119].

Other groups have also reported on the optimization of vectors deleted for El and DNA polymerase. This significantly modified vector expressing the highly immunogenic P-gal transgene was shown to persist in the livers of immunocompetent mice for up to 2 months [120], Such a vector could have broad benefits for use in human gene therapy in which the encoded transgene may be seen as a neoantigen by the human immune system.

3. Helper-Dependent Vectors

More recently, vectors deleted for all viral coding sequences (helper-dependent or "gutless" Ad vectors) have been developed, so that leaky expression of viral protein is eliminated [121-124] (see Chapter 15). Such a helper-dependent vector has been generated which contains the entire human alpha 1-anti-trypsin gene under the control of a tissue (liver)-specific promoter. This vector results in more than 1 year of stable expression, provides supraphysiological levels of hAAT in the mouse, and demonstrates less hepato-toxicity compared with first-generation vectors [125, 126]. Although many of the advantages of this gutless vector can be attributed to elimination of leaky viral gene expression it was later shown that the inclusion of a tissue-specific promoter also helped reduce the development of a host immune response to the transgene [127].

4. Tissue-Specific Promoters

The use of tissue-specific promoters may be helpful in avoiding host immune responses to transgenic proteins in human gene therapy, the rationale behind this being that expression in antigen-presenting cells would be greatly reduced. Thus the transcriptional unit responsible for expression of the transgene in an adenoviral vector could have a substantial effect on the nature of the ensuing immune response. Most experiments have used constitutively active promoters that may express efficiently in dendritic cells. A reduced immune response is seen with vectors that contain more specific promoters, which may not express efficiently in antigen presenting cells [127]. For example, when a helper-dependent Ad vector expressing the hAAT cDNA from a liver-specific promoter was used to express hAAT in C3H/HeJ mice, anti-hAAT antibodies did not develop and long-term expression of hAAT resulted [128]. In contrast, use of a non-liver-specific promoter to drive expression of the same transgene resulted in antibody production to hAAT in the same mouse strain. Thus, vector-specific differences in transgene expression within APCs due to choice of promoter could explain some of the variation in immune responses that have characterized in vivo applications of gene therapy vectors.

5. E3 Region

In addition to making progressive deletions of the adenoviral backbone other groups have coexpressed the Ad early region (E3) with the transgene of interest [128], Injection of Ad-overexpressing E3-encoded gene products leads to inhibition of cytotoxic activity toward Ad-infected cells in addition to marked down regulation of antibody formation to structural viral proteins. Genes encoded by the E3 region downregulate surface MHC class I expression, which in turn interferes with presentation of viral peptides and reduced CD8+ cytotoxic T lymphocytes. Thus the absence of Ad-specific CTLs in animals injected with this vector was expected. What was unexpected was the inhibition of a humoral antibody response to this E3 overexpressing vector. The authors suggest that transduction of an E3 containing Ad vector into liver cells in the absence of CTL or TNFa-induced cytolysis may result in poor antigen release, consequently there is little antigen presentation by APCs to initiate an antibody response. TNFa is one of the cytokines that controls dendritic cell maturation and migration; thus, early antigen presentation may be downregulated by inhibition of this cytokine by E3 proteins [128].

However, other studies using an Ad vector overexpressing the herpes simplex ICP47 gene suggest that use of any vector that downregulates MHC class I presentation should be assessed carefully [129]. Coexpression of ICP47 has a similar result to expression of E3 in that there is downregulation of MHC class I presentation. Administration of this vector to the lungs of rhesus monkeys inhibited the generation of Ad-specific CTLs. However, natural killer cell activity was enhanced, suggesting that strategies to protect the Ad-transduced cell without interfering with MHC class 1 expression should also be explored.

B. Species and Strain

To date the majority of animal studies evaluating the efficacy of adenovirus vectors have been performed with vectors expressing bacterial or human proteins. Clearly these proteins also constitute potential antigens recognized as foreign by the host immune response. Administration of vectors encoding the murine erythropoietin resulted in long-lasting elevated hematocrit levels in mice [130]. In contrast, injection of adenoviruses carrying the human erythropoietin induced a strong immune response directed against the human protein, which resulted in transient expression of the transgene.

Most in vivo studies are performed in inbred mouse strains of various MHC haplotypes whose immune systems might react differently to a given antigen. Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation Factor IX from viral vectors. This is in part because systemic expression of the secreted protein is not limited by antibody responses following intravenous (iv) injection of vector. Importantly iv injection of an Ad vector results in sustained expression of human FIX in normal or hemophilic C57B1/6 mice, while antibodies against FIX develop in other strains [131, 132]. A similar observation was seen with an Ad vector encoding human Factor VIII under the control of a liver-specific promoter following treatment of hemophilic C57B1/6 mice. High-level human FVIII expression was detected in the serum of the mice for over 5 months with no antibody production against the transgene [133]. In contrast treatment of FVIII-deficient hemophilic dogs with an Ad vector encoding human FVIII resulted in a strong antibody response directed to the human protein [133].

A reason for the difference in antibody response between different mouse strains was thought to be due to the MHC haplotype. C57BL/6 mice fhaplotype H-2b) lack MHC class II allele IE, and may therefore have some deficiency in humoral immune responses. However, mice of another strain with the same haplotype, and therefore the same lack of the IE allele, did mount an immune response to human FIX following systemic administration of a similar adenoviral vector. This suggests that the data produced in studies based on C57BL/6 mice often cannot be extrapolated to other species. The mechanism of tolerance to FIX or FVIII by iv injection of adenoviral vector in C57BL/6 mice remains elusive, but illustrates the difficulty of extrapolation of results obtained in inbred strains of mice and highlights the importance of studies in other animal models [132].

Similarly, others have reported [109, 134] that intravenous administration of an El-deleted adenovirus vector carrying the human alpha-antitrypsin (hAAT) cDNA leads to a strain-related variation in persistence of expression of transgene. Transient expression of hAAT was seen in C3H/HeJ and Balb C mice with longer persistence of expression seen in C57B1/6 mice [109, 134], Persistence was shown to correlate with poor anti-hAAT antibody formation in these mouse strains while Balb C and C3 H mice developed significant levels of anti-hAAT antibodies which resulted in a corresponding disappearance of hAAT in the serum [134]. In contrast, when a helper-dependent Ad vector expressing the hAAT cDNA from a liver-specific promoter was used C3H/HeJ mice failed to develop antibodies and demonstrated long-term expression of hAAT [127],

Careful identification and characterization of the host factors involved in the formation of anti-transgene antibody responses should provide insight into the development of useful gene therapy systems for the treatment of patients with genetic diseases involving null mutations. A good understanding of these immune responses is critical to the appropriate interpretation of many previous gene therapy studies and to the design of future studies. Although new generations of adenoviral vectors may offer many advantages compared to first-generation vectors in terms of persistence of transgene expression, it is important to establish an improved experimental paradigm to evaluate the effect of vector modifications on transgene expression, especially in the context of a highly immunogenic transgene.

C. Route of Delivery

1. Intravenous versus Intraperitoneal

Studies by Gahery-Segard et al. [135] investigating the humoral immune response to Ad capsid components demonstrated that routes of immunization modulate virus-induced Ig subclass shifts. Two routes of immunization, intravenous (iv) and intraperitoneal (ip) were compared for the response induced against the adenovirus particle in particular the three major components of the viral capsid, hexon, penton base, and fiber. The molecular components of the viral capsid are differentially recognized depending on the route of administration. The sera from mice immunized ip recognized only the hexon protein and a preferential switch to the IgG2a subclass was obtained. The sera from mice immunized iv had a more complex response. At the beginning of the response an isotype bias toward the IgG2a subclass was observed, but the isotype distribution changed during the period of the response. Neutralizing activity was maximum 45 days after immunization by both routes. However, iv serum displayed a higher neutralizing activity than ip serum, while the two routes of immunization did not induce the same IgG isotypes to neutralize viral infectivity.

2. Lung Instillation

The delivery of adenoviral vectors to the lung has received much attention due to the concerted efforts of many groups to develop gene therapy vectors for the treatment of lung disorders such as cystic fibrosis. To correct the CFTR defect, CFTR cDNA needs to be delivered to the respiratory epithelium in situ and must direct gene expression independent of cell division. Many groups have shown that Ad vectors can deliver CFTR cDNA to airway epithelial cells, leading to protein expression [105, 108, 136] and correction of the CF phenotype in vivo and in vitro [137-139], although the efficiency of repeated Ad administrations is diminished by the development of serum and mucosal neutralizing antibodies to the Ad vector [140]. In a study with repeat administrations to the nonhuman primate lung both IgG and slgA antibodies against Ad2 were detected and it was shown that slgA alone can contribute to neutralization of the infectivity of Ad particles entering the lung [105, 140, 141],

3. Delivery to the Brain

Interestingly, injection of an El-deleted adenovirus into the brain triggered a humoral immune response to the adenovirus and its gene products but no neutralizing antibody was detected thus repeat administration of the adenovirus was possible [142]. The authors claim that one reason for the lack of neutralizing activity may be due to the relative immunological privilege in the brain. Similarly, when adenovirus vectors are injected into the subretinal space, which is also considered to be immunologically privileged, they do not elicit humoral immune responses and repeated administration of adenovirus vectors is possible [143]. Thus there are regions within the body that may be more resistant to immune clearance on the basis of their anatomic structure. The retina for example, which is a derivative of the central nervous system, has the equivalent of a blood-brain barrier. In another report intranasal immunization of mice with wild-type adenovirus 1 month before intratumoral administration with an Ad vector of the same serotype did not efficiently inhibit repeat administration to the tumor [144]. It is likely that the structural integrity of the tumor or the extracellular matrix around the tumor presented a barrier to the neutralizing antibodies.

4. Intramuscular Delivery

More recently it was shown that effective repeat administration of adenovirus vectors to muscle was not hindered by the presence of neutralizing antibodies in the serum [145]. The authors reasoned that the concentration of adenovirus-specific neutralizing antibodies in the muscle may be considerably lower than in the serum, thus permitting effective multiple dosing to the muscle. The ability to repeat dose to the muscle has significant implications for cardiovascular gene therapy where it has been shown that intramuscular administration of adenovirus vectors expressing vascular endothelial growth factor (VEGF)-stimulated angiogenesis in hind-limb ischemia in rats [146]. Thus, the ability to repeat dose to the muscle for both peripheral and coronary vascular disease could significantly improve the efficacy of gene therapies for cardiovascular disorders.

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