Therapeutic level of functional human alpha 1 antitrypsin (hAAT) secreted from murine muscle transduced by adeno‐associated virus (rAAV1) vector

Abstract

Alpha 1 antitrypsin (AAT) is a serine proteinase inhibitor (serpin). One well-known function of this protein is to inactivate neutrophil elastase and other neutrophil-derived proteinases, and prevent the destruction of pulmonary extracellular matrix. Deficiency of AAT can cause emphysema due to degradation of interstitial elastin by elastase. The majority of circulating AAT is secreted from the liver. Muscle-directed gene therapy using recombinant adeno-associated virus 2 (rAAV2) vectors has been tested to increase the serum levels of AAT. However, inefficient transduction of rAAV2 vector makes it difficult to reach therapeutic levels of AAT in clinical trials and it remains unclear as to whether muscle-secreted AAT is functional. In the present study, we evaluated five serotypes (1, 2, 3, 4, and 5) of rAAV vectors for transduction efficiency in mouse muscle. Results from these studies showed that rAAV1 is the most efficient vector among these serotypes and mediated at least 100-fold higher levels of AAT secretion than the rAAV2 vector. Western blot analysis showed that this murine muscle-secreted human AAT (hAAT) formed a complex with human neutrophil elastase in a dose-dependent manner. An anti-elastase activity assay showed that murine muscle-secreted hAAT inhibited elastase with equal capacity as hAAT purified from plasma. These results provide strong support for the functionality of AAT in ongoing clinical studies of muscle-directed AAT gene therapy.

Distinct immune responses to transgene products from rAAV1 and rAAV8 vectors

“…Long-term and high levels of transgene expression have been achieved in animal models as well as in humans (9,17,18). However, the host immune response to a transgene product is one of the major hurdles to limit the successes of gene therapy in humans (19).…”

“…rAAV vectors were purified using iodixanol (IOD) gradients. The physical particle titers were determined by a dot-blot assay (9).…”

“…hAAT in sera, media, and cell lysates, as well as anti-hAAT antibodies, were detected by ELISA as described previously (9). For hAAT specific ELISA, purified hAAT (Athens Research Technology Inc.) was used as the standard.…”

“…It has been reported that the rAAV2 vector is less immunogenic compared to the adenovirus vector (5). Recent studies show some rAAV vectors including serotypes 1, 2, and 5 can transduce dendritic cells (DCs) and generate immune responses to transgene products (6)(7)(8)(9). Some studies have shown that rAAV8 vector has a low immunogenicity while it transduces liver, muscle, and many other cell types with high efficiency (10)(11)(12)(13)(14)(15).…”

Gene therapy: The first two decades and the current state‐of‐the‐art

“…It has a very limited packaging capacity, however (approximately 5 kb), and the original AAV2 is relatively inefficient at infecting a number of key cell types. Studies are now underway in the US and Europe with rAAV1 vectors, which have been shown to be nearly 1,000-fold more potent for murine muscle transduction than rAAV2 vector (Chao et al, 2000;Lu et al, 2006). Over 100 capsid variants are now known to exist (Gao et al, 2002.…”