In Vivo Applications Liver Transduction

The liver is an attractive gene therapy target because the fenestrated endothelium permits exposure to intravenously delivered vector, hepatocytes are well suited for secretion of therapeutic proteins into the circulation for systemic delivery, and it is the affected organ in many genetic disorders. Helper-dependent adenoviral vectors are particularly attractive vectors for liver-directed gene therapy because of their ability to efficiency transduce hepatocytes following intravenous injection.

To evaluate the utility of liver-directed, HDAd-mediated gene therapy in a large animal model, three baboons were intravenously injected with 3.3 to 3.9 x 1011 vector particles per kilogram of a HDAd expressing human aj-antitrypsin (hAAT).[5] Human aj-antitrypsin antagonizes neutrophilic elastase and is abundantly expressed in hepatocytes and at a lower level in macrophages and aj-antitrypsin-deficient patients have shortened life expectancies because of emphysema. Expression of hAAT persisted for more than j year in two of the three animals (Fig. 2). Maximum levels of serum hAAT of 3 to 4 mg/mL were reached 3 to 4 weeks postinjection in these two 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 had significantly lower levels of serum hAAT which rapidly declined to undetectable levels after 2 months. 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 three baboons at any time, starting 3 days postinjection. In contrast, hAAT expression lasted only 3 to 5 months in all four baboons

Fig. 2 Serum levels of hAAT in baboons following intravenous administration of the HDAd AdSTK109 or the FGAd AdhAATDEl. Baboons 12402 and 12486 were injected with

6.2 x 1011 particles/kg of AdhAATDEl. Baboons 12490 and 12497 were injected with 1.4 x 1012 particles/kg of AdhAATDE1. Baboons 13250, 13729, and 13277 were injected with

3.3 x 1011, 3.9 x 1011 and 3.6 x 1011 particles/kg, respectively, of AdSTK109.

Fig. 2 Serum levels of hAAT in baboons following intravenous administration of the HDAd AdSTK109 or the FGAd AdhAATDEl. Baboons 12402 and 12486 were injected with

6.2 x 1011 particles/kg of AdhAATDEl. Baboons 12490 and 12497 were injected with 1.4 x 1012 particles/kg of AdhAATDE1. Baboons 13250, 13729, and 13277 were injected with

3.3 x 1011, 3.9 x 1011 and 3.6 x 1011 particles/kg, respectively, of AdSTK109.

injected with an FGAd expressing hAAT (Fig. 2). This was shown to be due to the generation of a cellular immune response against viral proteins expressed from the vector backbone of the FGAd resulting in the elimination of transduced hepatocytes. These experiments convincingly demonstrated that HDAds were superior to FGAd with respect to duration of transgene expression and hepatotoxicity in a nonhuman primate.

The potential of liver-directed, HDAd-mediated gene therapy was investigated for the phenotypic correction of hypercholesterolemia in the apolipoprotein E-deficient (apoE_/_) mouse model.[6] Apolipoprotein E, a 34-kDa plasma glycoprotein, is a component of all plasma lipoproteins except low-density lipoprotein (LDL) and plays a major role in lipoprotein catabolism by acting as a ligand for the LDL-receptor (LDLR) and the LDLR-related protein for transport of excess cholesterol from the peripheral tissues to the liver for excretion. The apoE_/_ mouse is an excellent model for cardiovascular disease because they develop severe hypercholesterolemia and atherosclerotic lesions similar to those found in humans. Chan and coworkers investigated correction of hypercholesterolemia in apoE_/_ mice with either a FGAd or a HDAd expressing apoE.[6] Injection of apoE_/_ mice with FGAd resulted in an immediate rise in plasma apoE levels and a concomitant drop in plasma cholesterol levels to within normal range. However, this effect was transient, as apoE levels rapidly declined to pretreatment levels by day 28 and plasma cholesterol levels increased after 28 days, returning to pretreatment levels by 112 days. In contrast, a single injection of HDAd resulted in immediate lowering of plasma cholesterol to subnormal or normal levels for the rest of the natural lifespan of the animal (2.5

years). Plasma apoE levels reached ~ 200% wild-type and remained at supraphysiological levels for >4 months, only to decline slowly to wild-type levels at 1 year and remained at 60-90% physiological concentrations for the lifetime of the animals (2.5 years). Analysis of total plasma cholesterol revealed normalization of the plasma lipoproteins back to a predominately HDL pattern seen in wild-type mice. Aortas in all mice, examined at 2.5 years after treatment with HDAd, were essentially free of atherosclerotic lesions in contrast to saline-injected mice whose aortas were completely covered with lesions. Significantly, this study demonstrated that a single injection of HDAd encoding apoE could confer lifetime protection against aortic atherosclerosis. Toxicity studies revealed that whereas injection of FGAd resulted in significant chronic hepatotoxicity, no such evidence of damage was observed following injection of HDAd. In summary, this study demonstrated the tremendous potential of HDAds for gene therapy; a single injection of HDAd resulted in lifelong expression of a therapeutic transgene and permanent phenotypic correction in a mouse model of a genetic disease without long-term toxicity.

Muscle Transduction

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. Dystrophin is an essential structural component of the skeletal muscle cell membrane, linking intracellular actin filaments with the dystrophin-associated proteins (DAPs) in the sarcolemma. 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 but following the development of HDAds with large cloning capacity, gene transfer of the full-length dystrophin cDNA became feasible. Gilbert et al.[7] demonstrated that a single injection of a HDAd carrying two copies of the full-length human dystrophin cDNA resulted in transduction of 34% of the fibers of the total tibialis anterior (TA) muscle in neonatal mdx mice, a genetic and biochemical mouse model for DMD. The amount of dystrophin produced in these muscles was five times that in normal human muscle. 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. Indeed, injection of a HDAd expressing the full-length murine dystrophin cDNA into the tibialis anterior muscle of neonatal mdx mice resulted in sustained expression for at least 1 year in 52% of the muscle fibers. The treated muscle showed restored dystrophin-glycoprotein complex to the sarcolemma, significant improvement in isometric force production, resistance to high stress muscle contraction damage, and improved muscle histopathology.[8] This study demonstrates the tremendous potential of HDAd-mediated DMD gene therapy.

in negligible inflammation, with transgene expression lasting for up to 15 weeks.[10] Moreover, intranasal administration of a HDAd bearing the CFTR gene resulted in expression of CFTR in the airway epithelial cells of CFTR-knockout mice and could protect the lung from bacterial challenge.[11] These studies suggest that HDAd-mediated CF gene therapy would benefit CF patients by reducing susceptibility to opportunistic pathogens.

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