A. Liver Gene Transfer

The liver possesses a variety of characteristics that make this organ very attractive for gene therapy. Because of the fenestrated structure of its endothelium, the liver parenchymal cells are readily accessible to large particles such as viruses present in the blood. With respect to blood circulation, the liver can serve as a secretory organ for the systemic delivery of many therapeutic proteins. In addition, in many inborn errors of metabolism the liver is the mainly affected organ. Adenoviral vectors gained considerable interest for liver gene therapy owing to their capacity to very efficiently transduce quiescent hepatocytes in vivo. In fact, upon intravenous injection into the tail vein of mice, a large proportion of adenovirus particles preferentially localizes to the liver. However, in immunocompetent animals and with first-generation adenoviral vectors, transgene expression in general is transient both due to the loss of transduced hepatocytes and to promoter inactivation. Immunological and toxic effects in transduced cells due to viral gene expression significantly limit the use of first-generation vectors for hepatic gene transfer in vivo.

An HC-Ad vector expressing the human al-antitrypsin gene was used in several instructive experiments. Using the loxP helper virus production system, an HC-Ad vector was generated containing the 19-kb genomic human al-antitrypsin locus that included both the macrophage and liver-specific promoters, all exons and introns, and the natural polyadenylation signal [2]. al-Antitrypsin antagonizes neutrophilic elastase and is abundantly expressed in hepatocytes and at a lower level in macrophages. Expression in the two cell types is regulated by different tissue-specific promoters. Currently, al-antitrypsin-deficient patients have a shortened life expectancy due to emphysema. Patients are treated with weekly injections of human al-antitrypsin purified from human plasma.

Gene transfer of 2 x 1010 particles of this vector in immunocompetent C57BL/6J mice resulted in tissue-specific and stable gene expression for longer than 1 year. Transcription of the human al-antitrypsin RNA in the liver of transduced animals was initiated from the liver-specific promoter, but not from the macrophage-specific promoter. Gene transfer with increasing vector doses resulted in high and stable al-antitrypsin levels in serum. Significantly, with increasing vector doses, serum levels of al-antitrypsin were obtained that would be considered supraphysiological in humans. Even these very high vector doses were not accompanied by liver toxicity. Mice that received the same dose of a first-generation vector carrying the human al-antitrypsin cDNA under the control of the murine phosphogylcerate kinase (PGK) promoter experienced liver damage as documented by histological abnormalities and elevated liver enzymes detected in the serum of transduced mice [50]. Gene transfer of this vector in baboons resulted in relatively stable transgene expression for longer than 16 months in two of three baboons [39], In these animals only a slow decline was observed to 19% and 8% of peak levels at 16 and 24 months, respectively. This was not surprising for two reasons. First, hepatocytes are not postmitotic and there is a regular, albeit slow, turnover in this cell type. Second, the animals were young and still growing when they were injected. Therefore, a decline of al-antitrypsin levels correlated with animal growth. In a third baboon, the generation of anti-al-antitrypsin antibodies was associated with a short duration of expression of only 2 months. Transgene expression in all three animals injected with a first-generation vector was limited to 3 to 6 months. The lack of anti-al-antitrypsin antibodies in these animals and further immunological studies suggested that cellular immune responses against viral proteins might have resulted in the elimination of vector-transduced hepatocytes. In summary, these studies demonstrated the main advantages of HC-Ad vectors: increased capacity allowing the incorporation of large DNA fragments and even some genes in the genomic context, improved levels and persistence of transgene expression, and significantly reduced toxicity.

Improved expression and decreased liver toxicity has also been observed following gene transfer with an HC-Ad vector expressing the murine leptin cDNA from the human cytomegalovirus (HCMV) promoter [29]. Leptin is a potent modulator of weight and food intake. In leptin-deficient ob/ob mice, daily delivery of recombinant leptin protein suppresses appetite, induces weight reduction, and decreases blood insulin and glucose levels. Results from gene transfer experiments with a first-generation vector suggested that delivery of the leptin cDNA might provide therapeutic benefit equivalent to daily leptin protein treatment. However, the effects were only transient in both lean and ob/ob mice due to the loss of DNA and due to significant inflammatory changes in liver. Using an HC-Ad vector carrying the same expression cassette, leptin expression and physiological consequences were analyzed following gene transfer. In lean mice, tail vein injection of 1-2 x 1011 particles of the HC-Ad vector resulted in long-term leptin expression. Gene expression in ob/ob mice (which are leptin-deficient and therefore not tolerant to leptin) following gene transfer with the same dose of an HC-Ad vector was improved, prolonged, and associated with increased weight loss. However, even in HC-Ad vector transduced ob/ob mice leptin serum levels declined and finally disappeared due to the generation of anti-leptin antibodies.

Relatively realistic disease targets for HC-Ad vectors are the clotting disorders hemophilias A and B. The hemophilias are characterized by spontaneous and prolonged bleeding into joints, muscle, and internal organs. Current treatment of the hemophilias, which are often life-threatening and frequently associated with disabling arthropathy due to recurring joint bleeding, consists of protein-replacement therapy with infusion of plasma-derived or recombinant factor VIII (FVIII) or factor IX (FIX). The hemophilias are attractive candidates for gene therapy since they are due to single gene defects. A significant advantage is the fact that the therapeutic window is relatively broad. In addition, tissue-specific expression and precise control of the transgene expression is probably not required. Importantly, even moderate increases of FVIII or FIX levels would be sufficient to convert a severe hemophilia to a milder form. Intravenous injection of first-generation adenoviral vectors expressing the human or canine B-domain-deleted FVIIII cDNA in normal or hemophilic mice and dogs resulted in therapeutic and physiological levels of biologically active FVIII that was accompanied by a correction of bleeding tendency. However, both in hemophilic mice and dogs FVIII levels gradually declined, resulting in only short-term phenotypic correction. In mice transduced with a first-generation adenoviral vector expressing the human FVIII gene, anti-FVIII antibodies were not detectable. However, in hemophilic dogs, neutralizing FVIII antibodies were generated upon gene transfer of first generation vectors expressing either the human or canine FVIII cDNA [for review see 51].

Recently, an HC-Ad vector that carries the full-length human FVIII cDNA under the control of the 12.5-kb albumin promoter was injected into hemophilic mice, resulting in efficient hepatic gene transfer and therapeutic FVIII expression which led to the correction of the phenotype. However, FVIII levels declined, possibly due to the generation of inhibitory antibodies to the human FVIII protein. Histopathological findings of vector-induced toxicity were not observed [52]. Therapeutic expression levels could only be observed with relatively high vector doses (2 x 1011 viral particles per mouse). With a 10-fold lower vector dose FVIII could not be detected in the serum. These results suggested a nonlinear "threshold" effect which also has been observed with first-generation vectors [53].

Two further examples of liver gene transfer by HC-Ad vectors are mentioned since they point to additional advantages of this new vector type. In one instance an HC-Ad vector was generated to express murine erythro-poetin (mEPO), a glycoprotein regulating erythropoiesis [35]. EPO is mainly secreted by kidney peritubular cells in response to hypoxia and promotes late erythroid precursor proliferation and terminal differentiation of erythrocytes. Patients suffering from chronic renal failure show anemia as a major complication resulting from the destruction of EPO-secreting cells. These patients are treated with administration of recombinant human EPO protein. As an alternative treatment, delivery of the human EPO gene via an HC-Ad vector was tested and compared to a first-generation adenovirus vector with the same expression cassette. Relatively low amounts of an HC-Ad vector (3 x 105 infectious units or 3 x 107 particles per mouse) were sufficient to elevate hematocrit levels significantly, although with varying efficiencies, in different immunocompetent mouse strains. In this system the HC-Ad vector was at least 100-fold more efficient than a first generation vector. Because the low vector doses did not initiate any detectable neutralizing antibody response, intravenous readministration of the vector was possible without a need for immunosuppression. In contrast, a second injection of a first-generation virus into mice that had been previously transduced with the same vector induced a much smaller and only transient hematocrit increase.

A second example concerns the use of the mifepristone inducible gene expression system within the HC-Ad vector context [34]. In this system a chimeric trans-activator was used consisting of a mutated progesterone receptor ligand-binding domain, part of the activation domain of the human p65 subunit of the NF-kB complex, and a GAL4 DNA-binding domain. Expression of the fraws-activator was under the transcriptional control of the liver-specific transthyretin (TTR) promoter. A second expression cassette was located on the same vector and consisted of a 17-mer GAL4-binding site just upstream of a minimal TATA box containing the promoter and cDNA of human growth hormone (hGH). In the presence of the progesterone antagonist mifepristone the transactivator dimerizes, binds to the Gal4 DNA binding site and induces hGH expression. In vitro studies in HepG2 cells and in vivo experiments in mice demonstrated extremely tight control of gene expression and very strong induction of hGH expression upon administration of mifepristone. Following liver gene transfer, repetitive induction was possible for longer than 1 year [34, and unpublished data].

B. Gene Transfer into Skeletal Muscle

The first in vivo application of HC-Ad vectors was for gene transfer studies toward a treatment for Duchenne muscular dystrophy (DMD), an inherited muscular dystrophy caused by mutations in the dystrophin gene. The dystrophin cDNA is 14-kb in length; thus, only shortened versions of this cDNA could be accommodated by first-generation or second-generation adenoviral vectors. Therefore, HC-Ad vectors provided the potential to deliver the full-length dystrophin cDNA with an adenoviral vector. DMD is the most common form of muscular dystrophy with an incidence of 1:3500 male births. Mutations in the dystrophin gene result in the absence of the cytoskeletal dystrophin protein that is normally located at the cytoplasmic face of the cell membrane in skeletal and cardiac muscle. In normal muscle, dystrophin serves as a link in a network of proteins that span from actin within the muscle cell to laminin in the extracellular matrix. The absence of dystrophin results in a secondary loss of dystrophin-associated proteins, increased fragility of the muscle membrane, and cycles of degeneration followed by regeneration. Ultimately, the regenerative process fails and muscle fibers are replaced with fibrosis.

HC-Ad vectors encoding the dystrophin cDNA were developed by several groups [5, 8, 14]. Two groups incorporated a muscle-specific muscle creatine kinase (MCK) promoter [5, 8], allowing demonstration of striated muscle-specific expression of dystrophin from the vector. Direct intramuscular injection of these dystrophin-encoding HC-Ad vectors in the dystrophin-deficient mdx mouse model resulted in expression of recombinant dystrophin that properly localized to the muscle sarcolemma [7, 14]. Furthermore, dystrophin-associated proteins, which are lost in DMD and mdx muscle secondary to the primary absence of dystrophin, were restored in muscle fibers expressing HC-Ad vector-delivered dystrophin [54]. The prevention of dystrophic morphologic changes in muscle of mdx mice receiving an intramuscular injection of dystrophin-encoding HC-Ad vector was a second indicator of normal function provided by the recombinant dystrophin that was expressed from the HC-Ad vector [7].

One HC-Ad vector encoding a MCK-driven murine dystrophin cDNA and an HCMV-controlled lacZ gene, called AdDYSpgal, resulted in a profound cellular infiltrate composed primarily of CD4+ and CD8+ T cells when injected intramuscularly in nondystrophic, normal mice, even when gene delivery was performed during the neonatal period [54], Expression of ยก3-galactosidase was identified as the principal cause of the observed cellular immune response by performing parallel intramuscular injections of AdDYS^gal in neonatal mice with a germline lacZ transgene on the same genetic background. LacZ-transgenic mice did not develop a cellular infiltrate in skeletal muscle at any time point after intramuscular AdDYSfigal delivery [54]. Further studies demonstrated that dystrophin expression from AdDYSPgal in skeletal muscle of mdx mice also could induce at least an antibody-mediated immune response to dystrophin antigens (P.R.C., unpublished observations). When immunity to the vector was largely eliminated in direct muscle gene transfer studies, the AdDYSPgal vector DNA was stably maintained in skeletal muscle for at least 5 months [33]. Furthermore, the integrity of vector DNA remained intact [33]. This provided assurance that HC-Ad vector DNA could remain as a stable episome in transduced muscle cells.

These studies clearly show the utility of HC-Ad vectors for muscle gene transfer. An important issue to address in future studies is the nature of immunity induced by transgene proteins and adenoviral capsid antigens in the context of specific disease applications. It is likely that the underlying pathology of a muscle disorder will influence immunity induced or augmented by HC-Ad vector-mediated gene delivery. The low efficiency and extent of gene delivery to muscle is a second issue that currently prevents clinical applications of HC-Ad vectors. Targeting of HC-Ad vectors for muscle gene delivery may permit systemic administration that could result in transduction of muscle tissue widespread throughout the body.

C. Gene Transfer into the Eye and into the CNS

Adenoviral vectors have successfully been used for transgene delivery to different anatomic compartments and cell types of the eye, in vitro and in vivo. Several groups have demonstrated efficient transduction of retinal cells with first-generation adenoviral vectors expressing reporter or therapeutic genes [see for example 55-61]. The eye is considered a site of immune privilege, which is immunologically tolerant to foreign antigens similar to the testis, ovary, and uterus [62]. However, following adenoviral-mediated gene transfer into different ocular cell types, gene expression has always been transient. The short duration of gene expression obtained, together with the limited insertion capacity of first-generation Ad vectors, recently prompted studies that aimed at developing HC-Ad vectors for somatic gene therapy of human retinal degenerative diseases. R. Kumar-Singh et al. constructed an "encapsi-dated adenovirus mini-chromosome" containing a full-length murine cDNA encoding the P-subunit of the guanosine 3',5'-monophosphate (cyclic GMP) phosphodiesterase (P-PDE) under control of a human P-PDE promoter which is transcriptionally active in photoreceptor cells of the neuronal retina [28, 63]. This vector was prepared by cotransfection of 293 cells with helper virus DNA and a circular plasmid with head-to-head-oriented adenoviral ITRs generating linear adenoviral "mini-chromosomes" following rescue in 293 cells. The vector particles contained either monomers of the 13-kb starting material, or dimers in a head-to-head, head-to-tail, or tail-to-tail configuration [28, 63]. The P-PDE HC-Ad vector was delivered to the subretinal space of homozygous rd mice. These mice, which show a similar retinal phenotype as retinitis pigmentosa patients, suffer from an early-age onset of degeneration of retinal photoreceptors due to a loss-of-function mutation in the (3-PDE gene. Expression of (3-PDE in transgenic rd mice is known to rescue photoreceptor degeneration in this model [64]. In the P-PDE HC-Ad vector-treated animals, expression of the transgene in the neuronal retina was demonstrated by RT-PCR, Western blot analysis and functional enzymatic assays [28, 63]. When the thickness of the outer nuclear layer, as a marker of photoreceptor cell rescue, was evaluated at 2-week intervals, significant differences were observed between mice injected with the (3-PDE HC-Ad vector and control vector up to 12 weeks postinfection [28, 63]. Despite these encouraging results the expression of the P-PDE Ad vector was transient and loss of expression was complete at 120 days following subretinal injection. Whether the loss of expression was due to an immune response directed against contaminating first-generation helper virus or against the transgenic protein, to promoter shutdown, or simply to instability of the vector DNA is not clear at the time of this writing. Since quiescent cells of the CNS allow efficient gene transfer by adenoviral vectors, glial and neuronal cells are very interesting target cells for HC-Ad vectors. In an in vitro study primary neuronal cells isolated from the cerebellum of 8- to 9-day-old mice were transduced with either a first-generation or an HC-Ad vector expressing E. coli p-galactosidase [65]. Compared to gene transfer with a first-generation vector, transduction of these primary cells with the HC-Ad vector resulted in a marked decline in vector-mediated toxicity as assessed by morphological and metabolic studies. In particular, this was evident at moderate vector doses, corresponding to up to 50 multiplicities of infection (m.o.i.), a vector dose that resulted in an 85% transduction rate. However, at very high doses, the HC-Ad vector exhibited cytotoxicity, though not as severe as could be observed with a first-generation vector control.

A problem of clinical significance that has been rarely addressed concerns the fate of a viral vector following the superinfection by a virus of the same or a closely related serotype. Stereotactic injection in rats into the striatum of the brain of both a first-generation and an HC-Ad vector expressing lacZ resulted in stable gene expression over at least 60 days with both vectors [36]. However, challenge by peripheral subcutanous injection of a first-generation vector expressing an immunologically unrelated transgene resulted in a strong inflammatory response in the brain of rats that had received the first-generation vector but not the HC-Ad vector. Gene expression was completely abolished in rats that were injected with the first-generation vector while expression from the HC-Ad vector was stable. This experimental setup is mirrored by a clinical situation in which therapeutic gene transfer is followed at a later time by infection with a virus of the same or a closely related serotype.

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