Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors

Abstract

Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-alpha promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 x 10(13) particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 micrograms/ml in SCID and over 400 micrograms/ml in C57BL/6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions.

Figures

Figure 1

Transient transfection of C2C12 myoblasts with AAV-AAT. (Upper) Four AAV-AAT vector cassettes. The A-AT and B-AT constructs contain the promoters from the small nuclear RNA genes, U1a and U1b, respectively. The C-AT construct contains the CMV promoter, whereas the E-AT vector uses the human elongation factor 1-α (ELF in the figure) promoter. ITR, AAV inverted terminal repeat; An, polyA signal; Tk, the HSV thymidine kinase promoter; neo, the Tn5 neomycin phosphotransferase gene. (Lower) The rates of secretion from transfected cultures. C-AT does not differ significantly from E-AT, but both differ from A-AT and B-AT (P < 0.05).

Figure 2

Stable transduction of C2C12 myoblasts with AAV-AAT vectors in vitro. The mean rates of secretion from G418-resistant cultures 1 mo after transduction with either packaged E-AT vector or packaged C-AT vector are shown. In each instance, a “low” multiplicity transduction (4 × 105 particles/cell) and a high multiplicity transduction (4 × 106 particles/cell) were performed. E-AT “low” and “high” are greater than “high” multiplicity C-AT (P = 0.02) but are not significantly different from each other (n = 3).

Figure 3

Sustained secretion of therapeutic levels of hAAT from the C-AT vector in either SCID or C57BL mice. (A) The mean total serum levels of hAAT observed in groups of either SCID (squares) or C57BL (circles) mice receiving either low dose (5 × 1011 particles) (open symbols) or high dose (1.4 × 1013 particles) (filled symbols) single injections of the C-AT vector measured at time points ranging from 1 to 16 wk after injection. For each strain, the high-dose curve is significantly different from the low-dose curve (P = 0.009 for SCID, P = 0.02 for C57BL), but the strains do not differ from each other. (B) Analogous data with the E-AT vector. None of these differences were significant. (C) An immunoblot of sera taken from several of the C-AT vector-treated mice at 11 wk after vector administration. Ten microliters of a 1:100 dilution of serum was electrophoresed by 10% SDS/PAGE, blotted, and incubated with 1:1,500 dilution of goat anti-hAAT-horseradish peroxidase conjugate (Cappel/ICN). Samples from three high-dose SCID (h1–h3), one high-dose C57BL (h3), and three low-dose C57BL (lo1–lo3) were included, along with one negative control (saline-injected = sal) serum to indicate the level of reactivity with endogenous mAAT. As a standard, hAAT was added either to negative-control C57BL serum (first hAAT lane) or to PBS (second hAAT) lane to a final equivalent serum concentration of 100 μg/ml.

Figure 4

Some BALB/c mice mount humoral immune responses to hAAT, which correlate with lower serum levels but no observable toxicity. (a) Serum hAAT levels (upper graph) and serum anti-hAAT antibody levels (lower graph) as determined by ELISA performed on serum taken from mice injected with 1 × 1011 particles of the C-AT vector. Each set of symbols represents an individual animal (□, no. 1; ▵, no. 2; ○, no. 3). Note the inverse correlation between the presence of antibody and the presence of circulating hAAT. (b) hematoxylin/eosin-stained sections from the injection site in BALB/c mice (original magnification, X2000). Section A is from the hAAT-expressing animal (animal no. 1 from a); section B is from an anti-hAAT-positive mouse which did not have appreciable hAAT activity in the serum (animal no. 2 from a). Section C is from the site of injection in a BALB/c mouse injected with a similar dose of the E-AT vector.

Figure 5

Persistence of rAAV-AAT vector DNA in high molecular weight form. PCR products were amplified from DNA prepared by Hirt extraction from three SCID mice injected 16 wk earlier with 5 × 1011 resistant-particles of C-AT and analyzed by Southern blot. The high molecular weight Hirt pellet (genomic DNA lanes) and the low molecular weight supernatant (episomal DNA lanes) were analyzed separately. Control lanes include a sample in which an hAAT cDNA plasmid was the template DNA (+) and a control in which water was the template (−). In this internal PCR reaction, a 500-bp product is expected regardless of whether or not the vector genome is integrated.