Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery ofSleeping BeautyTransposons: Implications for Non-Viral Gene Therapy of Systemic Disease

Abstract

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.

Mucopolysaccharidosis Type I: Current Treatments, Limitations, and Prospects for Improvement

“…Hydrodynamic co-delivery of SB transposase and the SB transposon encoding IDUA into MPS I mice or MPS I dogs resulted in successful expression of the enzyme in the liver. However, the animals’ immune response resulted in the subsequent decline of IDUA levels [ 203 , 204 ]. Immunosuppression or the use of immunodeficient NOD/SCID-MPS I mice allowed prolonged expression of IDUA in the liver, reduction of GAG accumulation, and correction of some skeletal manifestations [ 205 ].…”

Molecular Mechanisms in Pathophysiology of Mucopolysaccharidosis and Prospects for Innovative Therapy

“…This system has shown promise in preclinical trials for various diseases, including cancer and genetic disorders [314][315][316][317][318]. In vivo MPS I and VII animal model experiments demonstrated positive results with this method [319][320][321][322]. On the other hand, for ex vivo gene therapy, this method has been studied mainly for the generation of chimeric antigen receptor T (CAR-T) cells [323,324].…”

Preclinical and clinical advances in transposon-based gene therapy

“…Hydrodynamic (HD) injection is considered one of the most common methods for in vivo transposon delivery through high-speed injection of large volume of DNA solution. This approach can be assisted by catheter and has been successfully demonstrated in a broad range of animal models including mouse [ 43 , 70 ], rat [ 71 , 72 ], rabbit [ 73 ], pig [ 74 ], dog [ 75 , 76 ], and monkey [ 44 ]. HD injection primarily targets gene transfer in liver; however, in vivo kidney and muscle transfection can also be attained using this method.…”

“…These SB100X-modified MSCs contributed to sustained expression and activity of α- L -iduronidase in vitro , over a 1-year time-course [ 101 ]. Direct in vivo administration of transposons has also been explored for MPS gene therapy in several preclinical animal models [ 76 , 102 – 104 ]. Usually this method is used in conjunction with immunomodulatory molecules, such as gadolinium chloride (GdCl 3 ), to suppress immune responses in the hope to prolong expression of therapeutic enzymes.…”