Expression of the Primary Coxsackie and Adenovirus Receptor Is Downregulated during Skeletal Muscle Maturation and Limits the Efficacy of Adenovirus-Mediated Gene Delivery to Muscle Cells

Abstract

Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

CAR is a cell–cell adhesion protein in human cancer cells and is expressionally modulated by dexamethasone, TNFα, and TGFβ

“…The expression level of CAR in human and murine tissues can vary tremendously 3,23 and was found to be highly regulated during murine development. 24,25 In the developing mouse brain, CAR expression peaked in the brains of newborn mice and transfection of murine CAR cDNA increased cell-cell adhesion of murine glioma cells. 25 Previous immunofluorescence studies on lung epithelial tissue cells revealed restriction of CAR to basolateral membranes, with immunostaining most prominently at cell-cell adhesive structures 6 and tight junctions.…”

Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA

“…The latter is caused by a maturation-dependent loss of the myofiber's susceptibility to rAd uptake which is due to structural and biochemical changes during muscle maturation. 5,[12][13][14] When an adenovirus particle enters a target cell, two types of receptor-mediated process are involved. First, the fiber knob of the adenovirus capsid binds to the attachment receptor on the cell surface, such as a Coxsackie B and adenovirus receptor (CAR) [15][16][17][18] and a MHC class I ␣2 domain.…”

“…Gene Therapy (2001) 8, 20-27. suggests a high-affinity attachment receptor. 14 This is followed by binding of the penton base to low-affinity internalization receptors, such as ␣ v ␤ 3 , ␣ v ␤ 5 and ␣ 5 ␤ 1 integrins, [20][21][22] which results in internalization of virus capsids. 23 Thus, the densities of attachment and internalization receptors on target cells influence their susceptibility to adenoviral infection.…”

Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin

“…For Ad vectors, the major viral attachment receptors, coxsackie adenovirus receptor and the integrins a v b3 and a v b5, are downregulated during myoblast differentiation and myofiber maturation. 37,38 Thus, Ad vectors were found to primarily target satellite cells, myoblasts and immature myofibers, and the highest transduction of skeletal muscle mediated by Ad is achieved in neonatal or regenerating muscle tissue. 5,[37][38][39] Effective transduction by VSV-G-pseudotyped lentiviral vectors also requires appropriate expression of several proteins such as heparan sulfate proteoglycans and glycosaminoglycans (GAGs) on target cell surfaces.…”

“…37,38 Thus, Ad vectors were found to primarily target satellite cells, myoblasts and immature myofibers, and the highest transduction of skeletal muscle mediated by Ad is achieved in neonatal or regenerating muscle tissue. 5,[37][38][39] Effective transduction by VSV-G-pseudotyped lentiviral vectors also requires appropriate expression of several proteins such as heparan sulfate proteoglycans and glycosaminoglycans (GAGs) on target cell surfaces. 40 However, the seeming inefficiency of lentiviral transduction of muscle in vivo relative to cultured cell lines may not be due primarily to differences in receptor levels, since we efficiently transduced freshly isolated single myofiber fragments and primary myoblasts from mice (Figures 4a, b, and 5b).…”