Quantitative Analysis of α-L-Iduronidase Expression in Immunocompetent Mice Treated with the Sleeping Beauty Transposon System

Abstract

The Sleeping Beauty transposon system, a non-viral, integrating vector that can deliver the alpha-L-iduronidase-encoding gene, is efficient in correcting mucopolysaccharidosis type I in NOD/SCID mice. However, in previous studies we failed to attain reliable long-term alpha-L-iduronidase expression in immunocompetent mice. Here, we focused on achieving sustained high-level expression in immunocompetent C57BL/6 mice. In our standard liver-directed treatment we hydrodynamically infuse mice with plasmids containing a SB transposon-encoding human alpha-L-iduronidase, along with a source of SB transposase. We sought to 1) minimize expression of the therapeutic enzyme in antigen-presenting cells, while avoiding promoter shutdown and gender bias, 2) increase transposition efficiency and 3) improve immunosuppression. By using a liver-specific promoter to drive IDUA expression, the SB100X hyperactive transposase and transient cyclophosphamide immunosuppression we achieved therapeutic-level (>100 wild-type) stabilized expression for 1 year in 50% of C57BL/6 mice. To gain insights into the causes of variability in transgene expression, we quantified the rates of alpha-L-iduronidase activity decay vis-a-vis transposition and transgene maintenance using the data obtained in this and previous studies. Our analyses showed that immune responses are the most important variable to control in order to prevent loss of transgene expression. Cumulatively, our results allow transition to pre-clinical studies of SB-mediated alpha-L-iduronidase expression and correction of mucopolysaccharidosis type I in animal models.

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

“…2. SB transposons for cSEAP expression and human (h)GUSB and hIDUA have been described previously, 13,18,19,41,45 as have plasmids for co-delivery of expression cassettes for SB transposases, pCMV-SB11, mini(m)Ub-SB11, 19,46 and pCMV-SB100X. 47 Liver-specific promoter (LSP) ApoEHCRhAAT 40 was a gift from Dr. Mark Kay (Stanford University, CA).…”

“…The SB transposon pKT2/ApoEHCRhAAT-cGUSB was engineered by inserting the full-length canine (c)GUSB into a unique EcoRI site of the pKT2-ApoE-hAAT-BGintron plasmid. 13 The plasmid containing cGUSB was a gift from Dr. Kathy Ponder (Washington University, St. Louis, MO). Prior to injection into dogs, plasmids were hydrodynamically infused into C57BL/6 mice to ensure transgene expression and transposition.…”

“…[11][12][13] Importantly, a nearly random integration profile makes this vector safer than its viral counterparts. 1,2,4,10,14 The SB transposon system has proved effective in mouse models of several monogenic diseases, including hemophilia A 15 and B 11,16,17 and mucopolysaccharidoses (MPS) types I and VII.…”

“…23 Preclinical studies using SB transposons in MPS I mice have demonstrated lifelong IDUA expression in the liver at about 100fold wild type (WT), which results in therapeutic effects comparable to those attained with retroviral liver-directed gene therapy. 13,19 However, mouse data are insufficient for translation to human clinical trials due to many differences, including organ size, metabolism, and life-span. 24 Dog models exist for both MPS I 25,26 and MPS VII.…”

Nanotechnology applied to treatment of mucopolysaccharidoses

“…The two approaches can even be combined[87].Recent progresses in genome editing technology using engineered zinc finger nucleases (ZFN), transcriptional activator-like effector nucleases (TALEN), and the clustered regularly interspaced short palindromic repeat (CRISPR) system have enabled the possibility of precisely modifying target sites in the genome without the use of viruses[88][89][90]. A recent study demonstrated that a transposable element (transposons) was able to integrate exogenous DNA into host cells, and showed benefits in MPS I mice[91,92]. Sangamo BioSciences Inc.(Richmond, CA, USA), is evaluating through a clinical trial (NCT02702115) the safety, the tolerability and the effect on plasma IDUA enzyme activity of ascending doses of SB-318.…”