Therapeutic gene delivery with Sleeping Beauty transposon vectors: Assessment of preclinical efficacy and safety in a mouse model of Gaucher disease
Final Report Abstract
Gaucher disease (GD) is an inherited autosomal recessive lysosomal storage disorder caused by mutations in the GBA gene that lead to a deficiency in the lysosomal enzyme GCase. This results in a reduction in glucosylceramide degradation, which accumulates in excess, particularly in mononuclear phagocytes. These disease-defining Gaucher cells infiltrate the visceral organs, and lead to hepatosplenomegaly, anemia and thrombocytopenia. Enzyme replacement therapies are expensive and do not offer permanent cure. On the other hand, infusion of genetically corrected autologous hematopoietic stem and progenitor cells (HSPCs) into patients represents a reasonable therapeutic option potentially offering life-long clinical benefit. However, insertional mutagenesis by integrating retroviral vector systems can increase the risk of cellular transformation and tumorigenesis, thereby presenting severe adverse events arising from HSPC gene therapy. Since enzyme replacement and substrate reduction therapy do not offer long-term treatment, new and innovative gene therapy methods for the treatment of GD are needed. Such methods should ideally avoid the toxicity and immunogenicity that occur as side effects in viral transfer systems. The SB transposon system represents a promising alternative to viral vectors. The hyperactive SB system (SB100X) is the first non-viral vector that is able to mediate both stable gene transfer and long-term expression of therapeutic genes with an efficiency comparable to viral vectors. In this project, we set out to investigate the feasibility of using the SB transposon system for gene therapy of GD. We have demonstrated the effectiveness as well as safety of the SB system vectorized as minicircle (MC) DNA for the transposon carrying a gene-of-interest and a specially formulated, stabilized mRNA encoding the SB100X transposase in human CD34+ HSPCs, and showed that these genetically engineered cells are capable of long-term engraftment and hematopoietic reconstitution in immunodeficient mice. Our in vitro studies with murine Lin- cells nucleofected with MC-Venus and SB100X transposase supplied as mRNA demonstrate very efficient stable Venus expression reaching more than 70% of the cells in liquid culture as well as in colony-forming assays. Vector integration site analysis in both human CD34+ and mouse Lin- HSPCs following ex vivo delivery of MC-Venus and SB100X by nucleofection demonstrates random genomic distribution of the vector without profound preferences for integration into genes and their transcriptional regulatory genes, implying a relative safety advantage of the SB system over conventional retroviral and lentiviral vectors. Our preliminary in vivo data indicate that Venus-expressing Lin- cells engraft and differentiate into all types of mature blood cells if kept in culture for less than 4 days. Similarly, MC-huGBA/SB100X based gene delivery in primary human GCase-deficient fibroblasts revealed stable expression of HA-tagged functional active GCase enzyme in about 30-40% of the cells. We will continue to investigate the feasibility of using the SB transposon system for effective and stable GBA transgene expression in HSPCs with the final aim to establish an efficient and safe non-viral gene therapy protocol for correction of autologous HSPCs in a mouse model of GD.
Publications
- (2017) Gene Therapy with the Sleeping Beauty Transposon System. Trends Genet. 2017 Nov;33(11):852-870
Kebriaei P, Izsvák Z, Narayanavari SA, Singh H, Ivics Z
(See online at https://doi.org/10.1016/j.tig.2017.08.008) - (2017) Wide Awake and Ready to Move: 20 Years of Non-Viral Therapeutic Genome Engineering with the Sleeping Beauty Transposon System. Hum Gene Ther. 2017 Oct;28(10):842-855
Hodge R, Narayanavari SA, Izsvák Z, Ivics Z.
(See online at https://doi.org/10.1089/hum.2017.130) - Improved Transposon System For Gene Delivery. EP3350335A1; EP3350335B1; WO2017046259A1
Aneja Manish; Geiger Johannes; Holstein Marta; Ivics Zoltan; Rudolph Carsten
- (2018) Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors. Mol Ther. 2018 Apr 4;26(4):1137-1153
Holstein M, Mesa-Nuñez C, Miskey C, Almarza E, Poletti V, Schmeer M, Grueso E, Ordóñez Flores JC, Kobelt D, Walther W, Aneja MK, Geiger J, Bonig HB, Izsvák Z, Schleef M, Rudolph C, Mavilio F, Bueren JA, Guenechea G, Ivics Z.
(See online at https://doi.org/10.1016/j.ymthe.2018.01.012) - (2018) Non-viral therapeutic cell engineering with the Sleeping Beauty transposon system. Curr Opin Genet Dev. 2018 Oct;52:100-108
Hudecek M, Ivics Z.
(See online at https://doi.org/10.1016/j.gde.2018.06.003) - (2019) A highly soluble Sleeping Beauty transposase improves control of gene insertion. Nat Biotechnol. 2019 Dec;37(12):1502-1512
Querques I, Mades A, Zuliani C, Miskey C, Alb M, Grueso E, Machwirth M, Rausch T, Einsele H, Ivics Z, Hudecek M, Barabas O
(See online at https://doi.org/10.1038/s41587-019-0291-z)
