Hyperactive Sleeping Beauty Transposase Enables Persistent Phenotypic Correction in Mice and a Canine Model for Hemophilia B

Abstract

Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases.

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…Liu et al, 2006b;Kren et al, 2009;Hausl et al, 2010) (Aronovich et al, , 2009, cancer (Ohlfest et al, 2005a;Peng et al, 2009;Jin et al, 2011), and type 1 diabetes (He et al, 2004). In addition, important steps have been made toward SB-mediated gene transfer in the lung for potential therapy of a 1 -antitrypsin deficiency, cystic fibrosis, and a variety of cardiovascular diseases (Belur et al, 2003;Liu et al, 2004).…”

“…Indeed, components of the SB transposon have been incorporated into integrase-defective lentiviral particles that showed efficient gene transfer in a range of human cell types and an insertion profile favorable to conventional lentiviral vectors (Staunstrup et al, 2009;Vink et al, 2009;Moldt et al, 2011). Hybrid adenovirus-SB vectors (Yant et al, 2002) have been used to efficiently deliver SB transposon vectors expressing FIX into the liver in a hemophilic dog model (Hausl et al, 2010). Retroviral vectors disabled in generating a cDNA copy of the retroviral vector have been shown to deliver the SB transposase mRNA into target cells with impressive efficiency (Galla et al, 2011).…”

Cell-Free Transmission of Human Adenovirus by Passive Mass Transfer in Cell Culture Simulated in a Computer Model

“…Ad5 was incubated with an Ad2/5 neutralizing antiserum (kindly supplied by Anja Ehrhardt [43]) in 96-well dishes containing 50 l of DMEM and 1% BSA. HLC-A549 cells were added to the wells, incubated at 37°C for 7 days, fixed, and stained with the cell dye crystal violet (0.25 mg/ml crystal violet in H 2 O containing 5% ethanol).…”

Adenoviral Vectors for Hemophilia Gene Therapy

“…An alternative approach to achieve stable transgene levels in the presence of hepatocyte turn-over is based on delivery of hyperactive Sleeping Beauty transposase system by HDAd that results in somatic integration of FIX into the hepatocyte genome [50]. By this approach, stable canine FIX expression levels from integrated vector have been observed long term both in mice and hemophilia B dogs [50].…”

“…Should transgene expression fall below therapeutic levels over time, a HDAd of a different serotype may be re-administered to overcome the neutralizing anti-Ad antibody elicited with the first administration [48,49]. An alternative approach to achieve stable transgene levels in the presence of hepatocyte turn-over is based on delivery of hyperactive Sleeping Beauty transposase system by HDAd that results in somatic integration of FIX into the hepatocyte genome [50]. By this approach, stable canine FIX expression levels from integrated vector have been observed long term both in mice and hemophilia B dogs [50].…”