Search

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications. INSET: Synopsis. [ABSTRACT FROM AUTHOR]

An advantage of plasmid-based vector systems for gene therapy over virus-based is their low immunogenicity. The Sleeping Beauty (SB) transposon system, which has the ability to integrate genes into human chromosomes, is a candidate for gene therapy of mucopolysaccharidoses (MPS), genetic disorders of lysosomal metabolism that require sustained expression of the therapeutic transgene. However, our attempts to achieve long-term expression of human alpha -L- iduronidase (hIDUA) or beta-glucuronidase (hGUSB) without immune suppression resulted in almost complete loss of transgene expression and maintenance by 4 weeks postinjection in both MPS and wild type (WT ) mice. In cyclophosphamide-immune-suppressed MPS I mice, the initial supranormal, 1-day plasma activity levels dropped approximately 150-fold by 2 weeks, but then persisted at detectable levels in some mice (see abstract by Aronovich et al). This suggested that cells that expressed the human therapeutic gene induced immune response and were cleared from the liver of some treated mice.The purpose of this study was to analyze contribution of cell- mediated immune response to the clearance of liver cells expressing foreign transgenes. We used high-pressure (hydrodynamic), tail-vein injection to deliver the transposon vectors pT2/mCAGGS-(hIDUA/hGUSB/Luc)//Ub-SB11 to the liver of WT C57Bl/6 mice. The efficiency of injection was evaluated 24 hr postinjection for transgene expression by either enzyme assay for IDUA or GUSB in plasma or in vivo imaging of luciferase (Luc). Mice were euthanized at 7, 10, 14, 17, 21 and 28 days and livers were extracted. Adjacent 5-μm sections from each liver were analyzed for transgene expression (histochemical staining for GUSB), for the presence of cellular infiltrates (H&E staining), and for the presence of CD4+ and CD8+ lymphocytes (immunohistochemical staining). In addition, the maintenance of the hIDUA, hGUSB and Luc transgene, as well as persistence of the transposon plasmid, were determined by PCR. Our results show the temporal relationship between expression of the therapeutic transgene and induction of immune response in the livers of transgene-treated mice.Molecular Therapy (2006) 13, S384–S384; doi: 10.1016/j.ymthe.2006.08.1090 [ABSTRACT FROM AUTHOR]

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 α-L-iduronidase (IDUA) and β-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 (GdCl3) 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. GdCl3 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 GdCl3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 μg/mL). It is concluded that GdCl3 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Retroviruses and transposons are mobile genetic elements with the ability to integrate genetic information into chromosomes for human gene therapy. However, integration-site preferences for mobile elements are poorly understood. We analyzed the insertion sites of several mobile elements to detect patterns of integration that can lead to the prediction of preferred integration sites. Based on our findings, we developed an automated method, ProTIS©, capable of faithfully predicting which base pairs in chromosomal DNA are preferred sites for Sleeping Beauty (SB) transposons. The preference for integration into certain sites is based upon structural characteristics of sequences flanking the dinucleotide insertion site. For any gene, chromosome, or whole genome, ProTIS© can generate a profile of predicted integration events. We have described an in vitro assay of transposition that uses a mathematical description of DNA deformability, Vstep, which allowed us to identify shared structural patterns among several preferred integration sites for SB transposons. For the SB transposon system we established the following equation that allows prediction of the relative likelihood of integration of an SB transposon into defined lengths of sequence:Likelihood of Integration = [10(# preferred sites) + 5(# semi-preferred sites) + (# basal sites)]ProTIS is valid for integration of SB transposons into limited and extended regions of genomic DNA up to 3.2 Mbp. We used a similar approach to analyze DNA integration patterns of other mobile genetic elements including retroviruses, lentiviruses and insect transposons. We analyzed 11,791 piggyBac integration events into its target TTAA sites and 5,070 integrations of P-elements, which do not have defined target sites. We were unable to find consistent Vstep pattern shared among either the piggyBac or P-element integration sites. We examined 695 MLV, 1371 HIV-1, 148 SIV and 551 ASLV integration sites for Vstep patterns that would aid in predicting integration preferences. Although we found symmetric patterns that overlapwith the base pairs involved in the target site duplication for most viral vectors, their patterns are based on the same compilations used to identify unique, weak consensus sequences for the various viruses and therefore cannot be used to generate algorithms for predicting integration sites based on structure rather than sequence. Our data suggest that as the density of integrations into a defined sequence increases our approach using ProTIS will have utility for human gene therapy applications for 'randomly' integrating vectors.Molecular Therapy (2006) 13, S200–S200; doi: 10.1016/j.ymthe.2006.08.591 [ABSTRACT FROM AUTHOR]

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70–80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy. [Copyright &y& Elsevier]

To expand the repertoire of transposable elements available for gene transfer in higher vertebrates, we investigated the properties of the Tol2 transposon in mammalian cells and in vivo. Tol2 is a member of the hAT family of DNA transposons and is derived from an active element found in the tyrosinase gene of medaka fish (Oryzias latipes). Tol2 can transpose in mouse ES cells, suggesting its potential use as a gene transfer reagent in higher vertebrates.We first tested the gene transfer activity of Tol2 in cultured human HeLa and HT1080 cell lines by G418 resistant colony forming assay and found that it is comparable to that of Sleeping Beauty. Furthermore, inceasing the amount of transposase-expressing plasmid resulted in increased transposition activity until a plateau was reached. In contrast to Sleeping Beauty, overexpression inhibition was not observed for Tol2. We also tested the effect of transposon size on Tol2 transposition and found that elements of up to 10 kilobases in size transpose as efficiently as smaller 2 kilobase transposons.The Tol2 transposable element routinely used to date contains a significant portion of the transposase open reading frame complete with multiple splice sites, promoter and polyadenylation cassettes. As these DNA elements pose a potential insertional mutagenesis risk in gene therapy applications, we sought to identify minimal sequences required for Tol2 transposition. In contrast to a previously published report, we found that internal sequences are not necessary for transposition, and we were able to reduce the size of the element by about 3 kilobases, creating Tol2mini.To investigate the activity of Tol2 in vivo, we coinjected a luciferase-expressing Tol2 transposon along with transposase-expressing plasmid rapidly and in high volume into the tail vein of wild type mice. We observed long term expression of the luciferase transgene in mouse liver, suggesting genomic integration of the transgene. Increasing the dose transposase led to increase in transposition efficiency without overexpression inhibition.Finally, we used both full length Tol2 and Tol2mini to correct the genetic defect in a mouse model of hereditary tyrosinemia type 1 by rapid, high volume co-injection with a transposase-encoding plasmid and transposon encoding fumaryl acetoacetate hydrolase and firefly luciferase. Liver repopulation was quantitatively monitored over time by increasing luciferase activity, measured as light emitted from the liver (please see abstract by Wilber et al), and required co-administration of transposase-encoding plasmid.Our results suggest that the Tol2 transposable element has excellent potential as a gene therapy tool and may be particularly suitable for applications where large cDNAs or cell type-specific promoters are required for full therapeutic potential. In addition, the apparent lack of overexpression inhibition suggests that Tol2 can be readily optimized for a variety of gene therapy applications.Molecular Therapy (2006) 13, S147–S147; doi: 10.1016/j.ymthe.2006.08.446 [ABSTRACT FROM AUTHOR]