HMGXB4 Targets Sleeping Beauty Transposition to Germinal Stem Cells.

Transposons are parasitic genetic elements that frequently hijack vital cellular processes of their host. HMGXB4 is a known Wnt signaling-regulating HMG-box protein, previously identified as a host-encoded factor of Sleeping Beauty (SB) transposition. Here, we show that HMGXB4 is predominantly maternally expressed, and marks both germinal progenitor and somatic stem cells. SB piggybacks HMGXB4 to activate transposase expression and target transposition to germinal stem cells, thereby potentiating heritable transposon insertions. The HMGXB4 promoter is located within an active chromatin domain, offering multiple looping possibilities with neighboring genomic regions. HMGXB4 is activated by ERK2/MAPK1, ELK1 transcription factors, coordinating pluripotency and self-renewal pathways, but suppressed by the KRAB-ZNF/TRIM28 epigenetic repression machinery, also known to regulate transposable elements. At the post-translational level, SUMOylation regulates HMGXB4, which modulates binding affinity to its protein interaction partners and controls its transcriptional activator function via nucleolar compartmentalization. When expressed, HMGXB4 can participate in nuclear-remodeling protein complexes and transactivate target gene expression in vertebrates. Our study highlights HMGXB4 as an evolutionarily conserved host-encoded factor that assists Tc1/Mariner transposons to target the germline, which was necessary for their fixation and may explain their abundance in vertebrate genomes.

Gene Therapy with the Sleeping Beauty Transposon System.

The widespread clinical implementation of gene therapy requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective, and economical manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient nonviral gene delivery approaches that are prevalent in ongoing clinical trials. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here, we review the most important aspects of using SB for gene therapy, including vectorization as well as genomic integration features. We also illustrate the path to successful clinical implementation by highlighting the application of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.

Sleeping Beauty transposition: from biology to applications.

Sleeping Beauty (SB) is the first synthetic DNA transposon that was shown to be active in a wide variety of species. Here, we review studies from the last two decades addressing both basic biology and applications of this transposon. We discuss how host-transposon interaction modulates transposition at different steps of the transposition reaction. We also discuss how the transposon was translated for gene delivery and gene discovery purposes. We critically review the system in clinical, pre-clinical and non-clinical settings as a non-viral gene delivery tool in comparison with viral technologies. We also discuss emerging SB-based hybrid vectors aimed at combining the attractive safety features of the transposon with effective viral delivery. The success of the SB-based technology can be fundamentally attributed to being able to insert fairly randomly into genomic regions that allow stable long-term expression of the delivered transgene cassette. SB has emerged as an efficient and economical toolkit for safe and efficient gene delivery for medical applications.

Multiple leptospiral sphingomyelinases (or are there?).

Culture supernatants of leptospiral pathogens have long been known to haemolyse erythrocytes. This property is due, at least in part, to sphingomyelinase activity. Indeed, genome sequencing reveals that pathogenic Leptospira species are richly endowed with sphingomyelinase homologues: five genes have been annotated to encode sphingomyelinases in Leptospira interrogans. Such redundancy suggests that this class of genes is likely to benefit leptospiral pathogens in their interactions with the mammalian host. Surprisingly, sequence comparison with bacterial sphingomyelinases for which the crystal structures are known reveals that only one of the leptospiral homologues has the active site amino acid residues required for enzymic activity. Based on studies of other bacterial toxins, we propose that leptospiral sphingomyelinase homologues, irrespective of their catalytic activity, may possess additional molecular functions that benefit the spirochaete. Potential secretion pathways and roles in pathogenesis are discussed, including nutrient acquisition, dissemination, haemorrhage and immune evasion. Although leptospiral sphingomyelinase-like proteins are best known for their cytolytic properties, we believe that a better understanding of their biological role requires the examination of their sublytic properties as well.

Wide Awake and Ready to Move: 20 Years of Non-Viral Therapeutic Genome Engineering with the Sleeping Beauty Transposon System.

Gene therapies will only become a widespread tool in the clinical treatment of human diseases with the advent of gene transfer vectors that integrate genetic information stably, safely, effectively, and economically. Two decades after the discovery of the Sleeping Beauty (SB) transposon, it has been transformed into a vector system that is fulfilling these requirements. SB may well overcome some of the limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are being used in the majority of ongoing clinical trials. The SB system has achieved a high level of stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, representing crucial steps that may permit its clinical use in the near future. This article reviews the most important aspects of SB as a tool for gene therapy, including aspects of its vectorization and genomic integration. As an illustration, the clinical development of the SB system toward gene therapy of age-related macular degeneration and cancer immunotherapy is highlighted.

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans.

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity. Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested.

Structural analysis of the Leptospiral sphingomyelinases: in silico and experimental evaluation of Sph2 as an Mg-dependent sphingomyelinase.

BACKGROUND: Leptospiral sphingomyelinases are candidate virulence factors present only in pathogenic Leptospira spp. Leptospira interrogans serovar Lai encodes Sph1, Sph2, Sph3, Sph4 and SphH. Except for Sph4, they all possess the exo-endo-phosphatase domain that groups them under the DNase I superfamily. METHODS, RESULTS AND CONCLUSIONS: Modeling of exo-endo-phosphatase domains reveals high-level structural similarity of Sph2 with the crystal structure of SmcL and BC SMase sphingomyelinases from Listeria ivanovii and Bacillus cereus, respectively. A ß-hairpin loop, essential for host cell membrane interaction, is absent in leptospiral sphingomyelinases. Instead, several aromatic amino acids were oriented outward from the surface of these molecules and formed clusters of hydrophobic regions that possibly enables the anchoring of these molecules into the host cell membrane, as demonstrated in Sph2 and Sph3. Sph2 is unique and possesses the Mg(++)-binding Glu53 residue in the metal-binding site and two His residues (His151 and His286) in the catalytic site. We demonstrate experimentally the Mg(++)-dependent hemolysis of erythrocytes by rSph2 and its ability to cleave sphingomyelin to ceramide. Anti-Sph2 antibodies neutralized the hemolytic activity of Sph2. In conclusion, we provide evidence showing that Sph2 is a Mg(++)-dependent hemolysin with both sphingomyelinase and hemolytic activities.