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-␣ 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 ؋ 10 13 particles into skeletal muscles of several mouse strains (C57BL͞6, BALB͞c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 g͞ml in SCID and over 400 g͞ml in C57BL͞6 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. Alpha-1-antitrypsin (AAT) deficiency is the second most common monogenic lung disease, accounting for 3% of all early deaths due to obstructive pulmonary disease. AAT is produced in the liver, secreted into the serum, and circulated to the lung where it protects elastin fibers and other connective tissue components of the alveolar wall from degradation by neutrophil elastase. Current therapy for AAT deficiency includes avoidance of cigarette smoke exposure and weekly i.v. infusions of recombinant human AAT (hAAT) protein (1). Attempts at gene augmentation have been limited by the short duration of expression and by the high circulating levels of AAT, which are required for therapeutic effect (800 g͞ml) (2).Several groups have demonstrated that adeno-associated virus (AAV) vectors are capable of stable in vivo expression (3-5) and are less immunogenic than other viral vectors (6). AAV is a nonpathogenic human parvovirus whose life cycle includes a mechanism for long-term latency. In the case of wild-type AAV (wtAAV), this persistence is due to sitespecific integration into a site on human chromosome 19 (AAVS1) (7), whereas with recombinant AAV (rAAV) vectors, persistence occurs by both episomal persistence and integration into non-chromosome 19 locations (8-9). rAAV latency also differs from that of wtAAV in that wtAAV is rapidly converted to double-stranded DNA in the absence of helper virus (e.g., Ad) infection, whereas rAAV-leading st...

A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

“…We used iodixanol as the ultracentrifugation gradient medium because this agent has been successfully used to isolate cells [12,13], organelles [14,15], macromolecules [16,17], and viruses [18][19][20][21][22]. Using iodixanol discontinuous density ultracentrifugation followed by sizeexclusion chromatography, we successfully isolated and purified both RGD-modified and non-RGD-modified adenovectors with a degree of purity and infectivity comparable to that of adenovectors purified by traditional 2× CsCl ultracentrifugation.…”

Identification of the immunodominant cytotoxic T-cell epitope of human -1 antitrypsin

“…Administration of vectors such as adeno-associated virus (AAV), adenovirus and helper-dependent adenovirus to the liver or muscle achieved stable therapeutic expression of human AAT (hAAT) in small and large animal models. [1][2][3][4][5] Interestingly, no B cell response to the hAAT transgene product was detected in these studies. However, there is a considerable lack of information on the cytotoxic T lymphocyte (CTL) response to hAAT.…”

“…AAT deficiency is the secondmost common lung disease and accounts for 3% of all early deaths due to obstructive pulmonary disease. 1 Given the limited treatment options for AAT deficiency, gene therapy represents a promising treatment for long-term correction of this metabolic disorder. Administration of vectors such as adeno-associated virus (AAV), adenovirus and helper-dependent adenovirus to the liver or muscle achieved stable therapeutic expression of human AAT (hAAT) in small and large animal models.…”

Adeno-associated virus-mediated human IL-10 gene transfer suppresses the development of experimental autoimmune orchitis

“…17,18 Another is recognized as having some superiority to other vectors with regard to their safety and efficiency, without known pathology (see Appendix A). 17,18 Since humoral substances such as cytokine have been considered suitable for use in AAV-mediated gene therapy, 19,20 we attempt prevention of EAO onset by the intramuscular (i.m.) human IL-10 (hIL-10) gene administration.…”