Transduction of the Liver

The liver is very attractive target for gene therapy. The fenes-trated structure of its endothelium permits exposure of the parenchymal cells to intravenously delivered vector and secretion of vector-encoded therapeutic proteins into the circulation for systemic delivery. Ads are particularly attractive vectors for liver-directed gene therapy because of their efficiency at transducing hepatocytes following intravenous injection.

^-Antitrypsin antagonizes neutrophilic elastase and is abundantly expressed in hepatocytes and at a lower level in macrophages. a1-Antitrypsin-deficient patients have shortened life expectancies due to emphysema. The utility of HDAds for liver-directed gene transfer was demonstrated in a series of studies using a HDAd containing the 19-kb genomic human ^-antitrypsin (hAAT) locus (AdSTK109). In the first of these studies, immunocompetent C57BL/6J mice, which do not generate anti-hAAT antibodies, were intravenously injected with 2 X 1010 particles of AdSTK109 (67). Serum hAAT levels reached a plateau of ~50 ^g/mL 3 weeks postinjection and were sustained for the duration of the 10-month experiment. In contrast, in C57BL/6J mice injected with 2 X 1010 particles AdhAATAE1, a FGAd vector bearing the hAAT cDNA, hAAT concentrations reached a peak of 2 ^g/mL 3 days postinjection, followed by a slow decline over 10 months to less than 10% of the peak levels. The superior expression levels induced by AdSTK109 compared with AdhAATAE1 were attributed to the more favorable genomic context of the hAAT in the HDAd compared with the cDNA inserted in the FGAd. The duration of hAAT expression correlated with persistence of vector genome in the liver. Specifically, a total of 65% of the FGAd genome was lost between 3 days and 12 weeks compared with a loss of only 30% in the case of AdSTK109, with only a 6% loss at 6 and 12 weeks. Histopath-ological examination of livers from C57BL/6J mice injected with AdSTK109 revealed normal morphology between 3 days and 12 weeks postinjection, whereas significant hepatotoxic-ity was observed from 6 to 12 weeks in the livers of mice injected with AdhAATAEl. To further examine the issue of hepatotoxicity, Ragl-immunodeficient mice were injected with AdSTK109 or AdhAATAEl. As with C57BL/6J mice, no evidence of liver toxicity was associated with AdSTK109. In contrast, hepatotoxicity was observed in AdhAATAE1-in-jected Ragl mice, similar to that observed in C57BL/6J mice. These results suggested that the observed hepatotoxicity resulted from direct cytotoxic affect of the viral proteins expressed from the AdhAATAE1 backbone, instead of toxicity due to induction of a cytotoxic T lymphocyte (CTL) response because Ragl mice do not produce CTLs (67).

Increasing the dose of AdSTK109 to 1.1 X 1011 particles resulted in 1 mg/mL hAAT in the serum, which is close to the normal human range (1.3 mg/mL) (78). Further increases in vector dose to 3.2 X 1011 particles resulted in supraphysio-logical levels of 5-6 mg/mL, which were sustained for the duration of the experiment (8 weeks). In contrast, injection of identical doses of AdhAATAE1 resulted in only transient hAAT expression. Clinically relevant markers of liver toxicity (ALT, alanine aminotransferase; AST, aspartate aminotransferase; and AP, alkaline) remained within the normal range in mice injected with AdSTK109 throughout the observation period, beginning 3 days to 8 weeks postinjection. In contrast, these markers were significantly elevated in mice injected with AdhAATAE1 for at least 6 weeks. Histopathological studies performed 5 days postinjection with the highest dose of AdSTK109 (3.2 X 1011 particles) revealed only mild signs of liver injury, including minor evidence of inflammation as quantitated by neutrophil infiltration. In contrast, extensive liver injury was observed at the highest dose of AdhAATAE1 (3.2 X 1011 particles), including cell degeneration, cell necrosis, and abundant inflammation. Morphological changes were also observed in the spleen at the highest dose, including large, expanded lymphoid follicles with active germinal centers and increased extramedullary hematopoiesis. These splenic changes were more pronounced in mice injected with Ad-hAATAE1. Southern blot analyses on liver DNA revealed that AdSTK109 DNA did not decline significantly for the duration of the experiment. In contrast, the amount of AdhAATAE1 DNA in the liver at 8 weeks was significantly reduced compared with 5 days in mice injected with high dose, consistent with the observed decline in hAAT expression.

To evaluate the utility of HDAds in a large animal model, three baboons were intravenously injected with 3.3-3.9 X 1011 particles/kg of AdSTK109 (79). hAAT expression persisted for more than 1 year in 2 of the 3 animals (Fig. 4). Maximum levels of serum hAAT of 3-4 mg/mL were reached 3 to 4 weeks postinjection in these 2 baboons and slowly declined to 8% and 19% of the highest levels after 24 and 16 months, respectively. The slow decline in hAAT expression was attributed to the fact that the baboons were young (7.5 and 9 months old) when injected and that the decrease in hAAT concentrations was correlative to the growth of the animals. The third baboon injected with AdSTK109 had significantly lower levels of serum hAAT, which rapidly declined

weeks

Figure 4 Serum levels of hAAT in baboons following intravenous administration of the HDAd AdSTK109 or the FGAd Ad5-hAATAE1. Baboons 12402 and 12486 were injected with 6.2 X 1011 particles/kg of Ad5hAATAE1. Baboons 12490 and 12497 were injected with 1.4 X 1012 particles/kg of Ad5hAATAE1. Baboons 13250,13729, and 13277 were injected with 3.3 X 1011 particles/kg, 3.9 X 1011 particles/kg, and 3.6 X 1011particles/ kg, respectively, of AdSTK109. (From Ref. 79, copyright 1999 National Academy of Sciences, USA.)

weeks

Figure 4 Serum levels of hAAT in baboons following intravenous administration of the HDAd AdSTK109 or the FGAd Ad5-hAATAE1. Baboons 12402 and 12486 were injected with 6.2 X 1011 particles/kg of Ad5hAATAE1. Baboons 12490 and 12497 were injected with 1.4 X 1012 particles/kg of Ad5hAATAE1. Baboons 13250,13729, and 13277 were injected with 3.3 X 1011 particles/kg, 3.9 X 1011 particles/kg, and 3.6 X 1011particles/ kg, respectively, of AdSTK109. (From Ref. 79, copyright 1999 National Academy of Sciences, USA.)

to undetectable levels after 2 months (Fig. 4). This baboon had generated anti-hAAT antibodies, thus accounting for the low level and rapid loss of serum hAAT. No abnormalities in blood cell counts and chemistries were observed in these

3 baboons at any time, starting 3 days postinjection. In contrast to baboons injected with AdSTK109, hAAT expression lasted only 3 to 5 months in all 4 baboons injected with AdhAATAE1 (Fig. 4). This was shown not to be due to the generation of anti-hAAT antibodies but was attributed to the generation of a cellular immune response against viral proteins expressed from the vector backbone, resulting in the elimination of vector-transduced hepatocytes. These early experiments convincingly demonstrated that HDAd were superior to FGAd with respect to duration of transgene expression and hepatotoxicity in mice and, significantly, in a nonhuman primate.

Leptin is a potent modulator of weight and food intake. Leptin deficient ob/ob mice gain considerable weight (~70 g) compared with lean littermates (~28 g) at 8 to 12 weeks of age. Morsy et al. (62) compared HDAd with FGAd, with respect to safety and efficacy, for leptin gene therapy in ob/ ob mice. Intravenous injection of 1-2 X 1011 particles of a FG vector encoding murine leptin (Ad-leptin) into ob/ob mice resulted in an increase in serum leptin levels for only the first

4 days, returning to baseline levels 10 days postinjection (Fig. 5A). Increased leptin levels were associated with transient weight loss of ~25% followed by weight gain 2 weeks after treatment (Fig. 5B). In contrast, ob/ob mice injected with an HDAd-encoding leptin (HD-leptin) resulted in about 2-fold higher serum levels of leptin up to ~15 days postinjection (Fig. 5A). However, expression was transient and gradually returned to baseline levels 40 days postinjection. Rapid weight loss to levels approaching that of normal lean mice (>60%

Figure 5 Phenotypic correction of leptin-deficient ob/ob mice with HDAd-mediated gene therapy. (A) Serum leptin levels and (B) weights of ob/ob mice intravenously injected with HD-leptin (black circle), Ad-leptin (crossed circle), or uninjected controls (white triangles). Weights of normal lean mice are shown in (B) as *. (C) Phenotypic correction of ob/ob mice. On the left is an ob/ob mouse treated with HD-leptin, next to an ob/ob littermate treated with Ad-leptin at 54 days postinjection. Control normal lean mouse and untreated ob/ob mouse are shown to the right for comparison. See the color insert for acolor version of this figure. (Modified from Ref. 62, copyright 1998 National Academy of Sciences, USA.)

Figure 5 Phenotypic correction of leptin-deficient ob/ob mice with HDAd-mediated gene therapy. (A) Serum leptin levels and (B) weights of ob/ob mice intravenously injected with HD-leptin (black circle), Ad-leptin (crossed circle), or uninjected controls (white triangles). Weights of normal lean mice are shown in (B) as *. (C) Phenotypic correction of ob/ob mice. On the left is an ob/ob mouse treated with HD-leptin, next to an ob/ob littermate treated with Ad-leptin at 54 days postinjection. Control normal lean mouse and untreated ob/ob mouse are shown to the right for comparison. See the color insert for acolor version of this figure. (Modified from Ref. 62, copyright 1998 National Academy of Sciences, USA.)

weight reduction) was observed in the HD-leptin-treated animals by 1 month, but the loss was only maintained for 6 to 7 weeks postinjection (Figs. 5B and 5C). Analogous to the case of hAAT in 1 baboon, loss of leptin in both Ad-leptin and HD-leptin mice correlated with the development of antileptin antibodies. This is undoubtedly due to the fact that ob/ob mice are naive to leptin. Hepatotoxicity was compared in mice treated with HD-leptin and Ad-leptin 1, 2, and 4 weeks postin-

jection. Pronounced liver toxicity, as measured by —10-fold increase in AST and —5-fold increase in ALT, was observed in Ad-leptin, but not HD-leptin treated mice. Liver histopa-thology at these time points revealed evidence of inflammation and cellular infiltration in livers of Ad-leptin-treated mice, whereas livers from HD-leptin-treated mice were histologi-cally indistinguishable from untreated control livers. These results indicated that HDAd were significantly less toxic than their FG counterparts. Importantly, in contrast to ob/ob mice, sustained high levels of serum leptin were observed in lean mice, for which leptin would not be a foreign antigen, following injection of HD-leptin but not Ad-leptin (62). Therefore, this study, together with the hAAT baboon study (79), suggested that in the absence of an immune response to the transgene product HDAds can provide prolonged transgene expression to achieve phenotypic correction of a genetic disease with negligible toxicity.

Kim et al. (66) investigated correction of hypercholesterol-emia in apolipoprotein E (apoE)-deficient mice by using either an FGAd-encoding mouse apoE cDNA (FG-Ad5-cE), an HDAd-encoding mouse apoE cDNA (HD-Ad5-cE), or an HDAd-bearing mouse genomic apoE locus (HD-Ad5-gE). Intravenous injection of ApoE-deficient mice with 5 X 1012 particles/kg of FG-Ad5-cE resulted in an immediate fall in plasma cholesterol levels to within normal range (Fig. 6A). However, this effect was transient and plasma cholesterol levels increased after 28 days, returning to pretreatment levels by 112 days. Correlative with the plasma cholesterol levels, the levels of plasma apoE immediately increased shortly after injection but rapidly declined to pretreatment levels by day 28 (Fig. 6B). Similarly, intravenous injection of 7.5 X 1012 particles/kg of HD-Ad5-cE produced a complete and immediate lowering of plasma cholesterol to normal levels, but in contrast to FG-Ad5-cE, the reduced levels lasted about 1 year before gradually increasing (Fig. 6A). ApoE appeared in plasma within 1 week and remained at a level —25% of wildtype (<10% of normal plasma levels of apoE is sufficient to maintain normal plasma cholesterol) but slowly declined to <10% of wild-type after about 1.5 years (Fig. 6B), at which time plasma cholesterol levels rose to —50% of untreated mice (Fig. 6A). Intravenous injection of 7.5 X 1012 particles/kg of HD-Ad5-gE also resulted in a complete and immediate lowering of plasma cholesterol to subnormal levels for about 9 months, with levels subsequently staying within the normal range for the rest of the natural lifespan of the animal (2.5 years) (Fig. 6A). In this case, plasma apoE reached —200% wild-type levels within 4 weeks and remained at supraphysio-logical levels for >4 months, at which time it slowly declined to about wild-type levels at 1 year and remained at 60% to 90% physiological concentrations for the lifetime of the animals (2.5 years) (Fig. 6B).

Because the duration of ApoE expression from HD-Ad5-gE was superior to HD-Ad5-cE, it would appear that genomic-based transgenes may be more effective than cDNA-based transgenes. Although the duration of expression from HD-Ad5-gE was impressive, lasting the lifetime of the mice, it would likely not be sustained in animals with significantly

Figure 6 HDAd-mediated phenotypic correction of atherosclerosis in ApoE-deficient mice. (A) Plasma cholesterol and (B) plasma ApoE levels in ApoE-deficent mice injected with dialysis buffer (white triangle), FG-Ad5-cE (black triangle), HD-Ad5-gE (black circle), or HD-Ad5-cE followed (indicated by the bold arrow) by HD-Ad2-gE (white circle). (C) Aortas from HDAd-treated and control dialysis buffer (DB)-treated mice stained with Oil Red at 2.3 years postinjection. Atherosclerotic lesion areas, stained red, determined by quantitative morphometry were 91.45 mm2 for DB-treated animals; 0.81 mm2 and 0.31 mm2 for HD-Ad5-gE-treated animals; and 5.89 mm2 and 1.74 mm2 for HD-Ad5-cE followed by HD-Ad2-gE-treated animals. See the color insert for a color version of this figure. (Modified from Ref. 66, copyright 2001 National Academy of Sciences, USA.)

Figure 6 HDAd-mediated phenotypic correction of atherosclerosis in ApoE-deficient mice. (A) Plasma cholesterol and (B) plasma ApoE levels in ApoE-deficent mice injected with dialysis buffer (white triangle), FG-Ad5-cE (black triangle), HD-Ad5-gE (black circle), or HD-Ad5-cE followed (indicated by the bold arrow) by HD-Ad2-gE (white circle). (C) Aortas from HDAd-treated and control dialysis buffer (DB)-treated mice stained with Oil Red at 2.3 years postinjection. Atherosclerotic lesion areas, stained red, determined by quantitative morphometry were 91.45 mm2 for DB-treated animals; 0.81 mm2 and 0.31 mm2 for HD-Ad5-gE-treated animals; and 5.89 mm2 and 1.74 mm2 for HD-Ad5-cE followed by HD-Ad2-gE-treated animals. See the color insert for a color version of this figure. (Modified from Ref. 66, copyright 2001 National Academy of Sciences, USA.)

longer lifespans, such as humans. Simply readministering the vector when transgene expression wanes is not possible due to the potent neutralizing anti-Ad antibody response that is elicited by the first administration. Indeed, mice previously treated with HD-Ad5-cE could not be successfully retreated again with the same vector. One solution to overcoming this problem is to administer a vector of a different serotype (72) (see Section V.F). To evaluate this strategy, Kim et al. generated a serotype 2 version of the genomic ApoE vector (HD-Ad2-gE) (66), using the serotype 2 helper virus described by Parks et al., (72) and showed that it could be successfully administered to mice previously treated with the serotype 5 HD-Ad5-cE to lower plasma cholesterol levels and raise plasma ApoE levels for the remainder of the animals' lives (Figs. 6A and 6B).

Aortas in all mice, examined at 2.3 years after treatment with HDAds, were essentially free of atherosclerotic lesions as determined by quantitative morphometry (Fig. 6C), demonstrating that a single injection of HDAd-encoding ApoE could confer lifetime protection against aortic atherosclerosis. Kim et al. also investigated the associated toxicities and found that, whereas injection of FG-Ad5-cE resulted in significant hepa-totoxicity as indicated by significant elevation of AST and ALT (>10- to 20-fold), no such evidence of damage was observed following injection of any of the various HDAd constructs, even after a second administration with the serotype 2 HDAd (66).

In summary, this study elegantly demonstrated many of the advantages of HDAds for gene therapy. First, the large cloning capacity of the vector permits delivery of transgenes in their native chromosomal context, resulting in superior kinetics and duration of expression. Indeed, a single intravenous injection of HDAd, in this case, resulted in lifelong expression of the therapeutic transgene and permanent phenotypic correction of a genetic disease. Second, if transgene expression diminishes over time, administration of an alternative serotype HDAd is effective at circumventing the humoral immune response generated by the initial treatment. Third, negligible hepatotoxicity was associated with HDAd administrations.

Hemophilia A is the most common inherited severe bleeding disorder; it is caused by a deficiency in coagulation factor VIII (FVIII) and affects about 1 in 10,000 males. Patients with <1% normal plasma FVIII activity suffer from spontaneous and prolonged bleeding into joints, muscle, and internal organs. Hemophilia A is an attractive target for gene therapy because it is caused by a single gene defect, and even moderate increases of FVIII levels can convert a severe phenotype to a milder form. Reddy et al. (41) compared the efficacy and safety of hemophilia A gene therapy using a multiply deleted (E1, E2a, E3 deleted) Ad vector (Av3H8101) and an HDAd (AGV15huFVIII), both containing the identical B-domain-deleted human FVIII expression cassette. In this study, hemophiliac A mice were intravenously injected with 6 X 1010 particles of AGV15huFVIII or Av3H8101. Plasma hFVIII levels in mice treated with AGV15huFVIII peaked at 2 weeks postinjection and were 10-fold higher than levels achieved using Av3H8101 (Fig. 7A). Expression of hFVIII in AGV15-

huFVIII-treated mice was sustained for at least 40 weeks although a ~ 10-fold decrease in plasma levels was observed between weeks 2 and 40 (Fig. 7A). In contrast, plasma hFVIII levels in Av3H8101-treated mice rapidly decreased to below the limit of detection (<25 mU/mL) by 12 weeks (Fig. 7A). It is interesting to note that the decrease in plasma hFVIII levels in both AGV15huFVIII and Av3H8101 mice was not due to the development of anti-hFVIII antibodies. At a dose of 3 X 1011 particles (1.5 X 1013 particles/kg), both AGV15-huFVIII and Av3H8101 induced hepatotoxicity as evident by ~ 10-fold increase in AST and ALT levels measured 1 day after vector administration (Fig. 7B). These levels returned to baseline by day 3. However, by day 7, animals treated with Av3H8101 showed a 10-fold elevation in AST and ALT levels, whereas those treated with AGV15huFVIII remained at baseline levels (Fig. 7B). AST and ALT levels did not return to baseline levels until day 28 in the Av3H8101-treated animals. These results suggested that the initial increase in liver trans-aminases observed at day 1 was caused by direct toxicity of the virion capsid protein from both AGV15huFVIII and Av3H8101. The toxicity observed at day 7 and beyond for Av3H8101, but not AGV15huFVIII, may have been due to viral gene expression from the Av3H8101 backbone. This study, unlike most others (see above), investigated vector-mediated toxicity shortly after administration and showed that HDAd can cause acute hepatoxicity by day 1. This toxicity was resolved by day 3, explaining why most others (who measured hepatotoxicity >3 days postinjection) have not observed hepatoxicity with HDAds.

Reddy et al. (41) also investigated the affect of helper virus contamination on the level and duration of hFVIII expression, vector-mediated toxicity, and development of anti-hFVIII antibodies, and found that these parameters were unaffected following intravenous injection of AGV15huFVIII preparations with 0.5%, 1%, 2.5%, 5%, and 10% helper virus contamination.

B. Transduction of Muscle

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, degenerative muscle disease with a frequency of 1 in 3500 male births caused by mutations in the dystrophin gene. Dys-trophin is an essential structural component of the skeletal muscle cell membrane, linking intracellular actin filaments with the dystrophin-associated proteins (DAPs) in the sarco-lemma. Dystrophin deficiency results in instability of the muscle cell membrane causing muscle fiber degeneration. The length of the dystrophin cDNA (14 kb) precluded its inclusion into most gene therapy viral vectors. Following the development of HDAds with large cloning capacity, gene transfer of the full-length dystrophin cDNA became feasible. Indeed, the first in vivo application of HDAds was transduction of skeletal muscle for DMD gene therapy (50). In that study, an HDAd, AdDYSpgal, was constructed bearing the dystrophin cDNA under the control of a muscle-specific murine creatin kinase promoter and the LacZ reporter gene under the control of the CMV promoter (50). Direct intramuscular injection of 2 X 107 particles of AdDYSpgal into the gastrocnemius muscle

Figure 7 (A) Comparison of serum hFVIII levels in hemophiliac mice injected with 6 X 1010 particles of the HDAd AGV15huFVIII (squares) or the multiply deleted Ad Av3H8101 (circles). (B) Comparison of hepatotoxicity, as determined by measuring the serum levels of AST and ALT, in hemophiliac mice injected with 3 X 1011 particles of AGV15huFVIII (squares) or Av3H8101 (circles). (Modified from Ref. 41.)

Figure 7 (A) Comparison of serum hFVIII levels in hemophiliac mice injected with 6 X 1010 particles of the HDAd AGV15huFVIII (squares) or the multiply deleted Ad Av3H8101 (circles). (B) Comparison of hepatotoxicity, as determined by measuring the serum levels of AST and ALT, in hemophiliac mice injected with 3 X 1011 particles of AGV15huFVIII (squares) or Av3H8101 (circles). (Modified from Ref. 41.)

of mdx mice, a genetic and biochemical model for DMD, resulted in expression of the 400-kDa full-length dystrophin protein, which was correctly localized to the sarcolemma membrane, restoring the DAPs to the muscle membrane and resulting in significant improvement of the histological pheno-type (80). Most muscle fibers that expressed dystrophin also expressed p-galactosidase. However, by 6 weeks postinjection, the proportion of muscle fibers expressing dystrophin and p-galactosidase decreased. Expression of p-galactosidase from AdDYSpgal was identified as the principle cause of the loss of dystrophin expression (81). When immune response to p-galactosidase was eliminated, transgene expression persisted for at least 84 days with no significant loss of the Ad-DYSpgal vector DNA (81). Of course, for clinical use, therapeutic vectors would not contain an immunogenic reporter transgene such as LacZ.

More recently, Gilbert et al. (82) demonstrated that a single injection of an HDAd carrying two copies of the full-length human dystrophin cDNA under the control of a powerful hybrid CMV-enhancer/p-actin promoter, resulted in transduc-tion of 34% of the fibers of the total tibialis anterior (TA) muscle in neonatal mdx mice. The amount of dystrophin produced in these muscles was 5 times that in normal human muscle, as determined by Western blot analyses. However, only 7% transduction was achieved following injection into the TA muscle of adult mdx mice. In these transduced adult fibers, the amount of dystrophin produced was only 10% of the amount in normal humans. However, the high levels of transduction were transient and a humoral immune response was mounted against the foreign human dystrophin protein in the mdx mice. Importantly, such a response was not observed in immunodeficient SCID mice suggesting that sustained expression could be achieved in the absence of an immune response to the transgene product. Interestingly, a 6-fold increase in transduction was observed in TA muscles by the HDAd in the presence of an FGAd vector. The authors proposed that gene products synthesized by the cotransducing FGAd acted in trans to increase transgene expression from the HDAd. The exact mechanism responsible for this intriguing result remains to be fully elucidated, although the Ad E4 region has been implicated to play a role (83).

Despite these encouraging results, effective gene therapy for DMD would likely require transduction of a critical number of muscle fibers in numerous major muscle groups throughout the body, including the limb, trunk, and respiratory muscles, a feat that is not currently feasible. However, muscle remains an attractive tissue for transduction because, like the liver, it may serve a more general purpose in gene therapy: (1) muscle constitutes as much as 40% of the total body mass and much of it is readily accessible, (2) skeletal myocytes can be transduced in vivo, (3) skeletal myofibers have a relatively long half-life and therefore represent a stable platform for transgene expression, (4) muscle is highly vascularized and skeletal muscle can efficiently secrete recombinant proteins into the circulation for systemic delivery, and (5) the high seroprevalence in the adult human population of preexisting anti-Ad neutralizing antibodies, an obstacle for intravenous vector delivery, may be minimized through localized delivery into the muscle. This strategy was investigated in a study by Maione et al. (84) in which mice were intramuscularly injected with C4AFO-mEPO, an HDAd-expressing mouse erythropoi-etin, as a marker gene. All mice intramuscularly injected with a dose of 1 X 106 infecious units (i.u.) or 3 X 105 i.u. retained transgene expression for at least 4 months. At a dose of 3 X 106 i.u., 30% of the mice slowly lost transgene expression. In contrast, all mice injected intramuscularly with FGAd lost transgene expression by day 21. The effect of preexisting anti-Ad antibody on the effectiveness of intramuscular administration was also investigated. Mice were first immunized with 1 X 1010 particles of a FGAd and, following generation of anti-Ad antibodies, the mice were challenged with intramuscular injections of HDAd at various doses. The authors found that successful intramuscular gene transfer could be accom plished in preimmunized mice, although a 30- to 100-fold higher dose was required to achieve 87% and 100%, respectively, the levels of transgene expression in naive mice. This is in sharp contrast to intravenous gene transfer in which 60% of preimmunized mice were completely refractory to trans-duction, whereas the remaining 40% were transduced but exhibited transgene expression levels comparable to naive mice intravenously injected with 1000-fold lower dose.

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