Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein

Abstract

Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.

Figure 1

(A) pAAV-lacZ. ITR, inverted terminal repeat; CMV, CMV promoter; lacZ, bacterial β-gal; An, simian virus 40 (SV40) early polyadenylylation signal. (B) pAAV-Epo. Epo, human Epo coding sequence; An, SV40 polyadenylylation signal; noncoding, 2.2-kb noncoding fragment from the lacZ gene. See text for details.

Figure 2

Histochemical detection of β-gal expression in situ following injection of AAV-lacZ. The tibialis anterior of adult male BALB/c mice was injected with 8 × 10⁹ vector particles. The animals were sacrificed at (A) 2, (B) 4, (C) 8, (D) 12, (E) 24, or (F) 32 weeks after the injection. The tibialis anterior was excised, and 10-mm sections were processed for β-gal histochemistry. (×25.)

Figure 3

High-powered section of skeletal muscle 2 months after injection with AAV-lacZ. Tibialis anterior muscle was processed for in situ detection of β-gal and photographed with diffraction-interference contrast optics at ×400.

Figure 4

Secretion of human Epo by C2C12 myotubes transduced with AAV-Epo. Confluent C2C12 myoblasts were differentiated into myotubes and transduced with 3 × 10⁸ (□), 3 × 10⁹ (▨), or 3 × 10¹⁰ (▪) vector particles of AAV-Epo. Twenty-four hours following a complete change of media, the secretion of Epo was measured (3, 8, and 14 days after transduction). AAV-lacZ-transduced myotubes secreted <2.5 milliunits/ml Epo. The bar graph depicts mean concentration of Epo per well per 24 hr determined in triplicate cultures ± SEM.

Figure 5

Dose-response and time course of Epo secretion in BALB/c mice after i.m. injection of AAV-Epo. Adult BALB/c mice were injected i.m. with 1 × 10¹⁰ (▾), 3 × 10¹⁰ (▴), 1 × 10¹¹ (▪), or 3 × 10¹¹ (•) vector particles at day 0, and serum Epo levels were measured at time points postinjection. Points represent means (n = 4) ± SEM.

Figure 6

Epo secretion by primary human myotubes transduced with AAV-Epo. Confluent human myoblasts were differentiated into myotubes by culture for 14 days in differentiation media, then transduced with 3 × 10⁸ (□:), 3 × 10⁹ (▨), or 3 × 10¹⁰ (▪) vector particles of AAV-Epo. Twenty-four hour secretion of Epo was measured 3, 8, and 14 days after transduction. AAV-lacZ-transduced myotubes secreted <2.5 milliunits/ml Epo. Bar graph depicts mean concentration of Epo per well per 24 hr determined in triplicate cultures ± SEM.