No abstract

The Sleeping Beauty transposon system has been extensively tested for integration of reporter and therapeutic genes in vitro and in vivo in mice. Dogs were used as a large animal model for human therapy and minimally invasive infusion of DNA solutions. DNA solutions were delivered into the entire liver or the left side of the liver using balloon catheters for temporary occlusion of venous outflow. A peak intravascular pressure between 80 and 140 mmHg supported sufficient DNA delivery in dog liver for detection of secretable reporter proteins. Secretable reporters allowed monitoring of the time course of gene products detectable in the circulation postinfusion. Canine secreted alkaline phosphatase reporter protein levels were measured in plasma, with expression detectable for up to 6 weeks, while expression of canine erythropoietin was detectable for 7-10 days. All animals exhibited a transient increase in blood transaminases that normalized within 10 days; otherwise the treated animals were clinically normal. These results demonstrate the utility of a secreted reporter protein for real-time monitoring of gene expression in the liver in a large animal model but highlight the need for improved delivery in target tissues to support integration and long-term expression of Sleeping Beauty transposons.

The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by a-L-iduronidase (IDUA) and b-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl 3 ) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl 3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl 3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 lg/mL). It is concluded that GdCl 3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative regimens will be required to achieve therapeutic levels of transgene products.

S174the nasal and intrapulmonary airways of IFITM locus knockout mice are more efficiently transduced by GP64-FIV than their heterozygous littermates. In anticipation of transitioning our studies into pig models of airway disease and clinical trials in humans, we investigated the ability of pig and human IFITMs to restrict lentiviral gene transfer. We observed that both human and pig IFITMs partially restricted both VSV-G-FIV and GP64-FIV transduction in vitro. These results implicate the IFITM proteins as restriction factors that can limit lentiviral gene transfer to airway epithelia. The findings are relevant to future pre-clinical and clinical airway gene therapy trials using lentiviral-based vectors.

Gene TarGeTinG and Gene CorreCTion i junction. Recently, we provided proof of principle that ZFNmediated, AAVS1-targeted GFP addition can be achieved in human keratinocytes and in long-term repopulating epithelial stem cells in a validated preclinical model of xenotransplantation of human skin equivalents on immunodeficient mice.This project aims at the demonstration of a successful in situ correction of the LAMB3 gene in primary keratinocytes from Herlitz JEB patients. Recently TALEN-based gene correction for dystrophic EB has been reported. Similarly, we have developed a genome editing approach for JEB. In particular we have designed TALENs specific for the second intron of LAMB3 gene and a HR cassette including a splicible LAMB3 cDNA (from exon 3 to the end of the gene). In particular immortalized JEB keratinocytes were transfected with TALEN mRNAs and infected with an IDLV vector carrying the HR cassette. The in situ gene correction has been evaluated by site-specific PCR and knock-in expression of the corrected LAMB3 gene on bulk population. We then assessed targeting efficiency and specificity by extensive molecular analyses of single-cell clones isolated by limiting dilution from the TALENs/IDLV-treated immortalized JEB population. We isolated 256 clones and expanded 69 of them. Sixteen out of 69 clones showed an in vitro adhesion advantage, hosted the HR cassette correctly integrated into the predetermined locus, expressed the corrected LAMB3 gene and produced the laminin-332 protein. In parallel, CRISPR-Cas9 nuclease has been designed on the same locus to compare the transduction efficiency and cleavage activity and to translate the knock-in targeting platform to primary JEB keratinocytes.