Translating Sleeping Beauty transposition into cellular therapies: victories and challenges

Abstract

Recent results confirm that long-term expression of therapeutic transgenes can be achieved by using a transposon-based system in primary stem cells and in vivo. Transposable elements are natural DNA transfer vehicles that are capable of efficient genomic insertion. The latest generation, Sleeping Beauty transposon-based hyperactive vector (SB100X), is able to address the basic problem of non-viral approaches - that is, low efficiency of stable gene transfer. The combination of transposon-based non-viral gene transfer with the latest improvements of non-viral delivery techniques could provide a long-term therapeutic effect without compromising biosafety. The new challenges of pre-clinical research will focus on further refinement of the technology in large animal models and improving the safety profile of SB vectors by target-selected transgene integration into genomic "safe harbors." The first clinical application of the SB system will help to validate the safety of this approach.

Figure 1

The plasmid-based SB system. The SB plasmid-based transposon system combines the advantages of viral vectors with those of naked DNA molecules. The advantage of transposon-based gene delivery as compared to classical, plasmid-based, and non-viral delivery approaches is that, due to stable genomic insertion of expression cassettes, it can lead to both long-term and efficient transgene expression. However, in contrast to viral vectors, transposon vectors can be maintained and propagated as plasmid DNA. Making them simple and inexpensive to manufacture – an important issue regarding the implementation of future clinical trials. The SB transposon system has two functional components: a specific piece of DNA that frames a gene to be moved into the cell’s genome and a protein (the transposase) that mobilizes the transposon. Following DNA delivery to the cells, the transposase (purple) binds the terminal inverted repeats (arrows) flanking the gene of interest (GOI) and catalyzes the excision and subsequent genomic integration of the transposon. The transposase can be provided A: in cis (from the same plasmid molecule) or B: in trans in the form of a second plasmid. Notably, the transposase can be provided as mRNA as well, thereby reducing the risk of “re-hopping” of the transposon-based vector [109]. Integration occurs at TA dinucleotides, which are duplicated to flank the transposon at the site of insertion. Although the transposition efficiency decreases with increasing insert size [110], inserts of up to ~8 kb can be efficiently transferred by the SB-based vector. When the cargo is flanked by two transposons (two-ended arrows) in a “sandwich” configuration, transposition efficiencies of inserts over 8 kb (GOI*) can be significantly improved [24]. Since the transposition mechanism does not involve reverse transcription, DNA-based transposon vectors are not prone to incorporate mutations and can tolerate larger and more complex transgenes, including those containing repeated DNA motifs. Moreover, the use of SB-based gene delivery eliminates the risk of rearrangements of the expression cassette that, as part of a transposing unit of DNA, integrates into chromosomal DNA in an intact form [20].

Figure 2

Genomic insertion preferences of integrating vector systems. Genomic integration of viral vector systems shows considerable preference for genes [–10, 111]. In contrast, SB integration can be considered fairly random on a genomic level [37, 40]. Only about one-third of SB insertions occur in transcribed regions (the vast majority in introns), suggesting that SB might be a safer vector for therapeutic gene delivery than most viruses that are currently used. The genomic integration profile of piggyBac [33] or Tol2 resembles that of integrating viral vectors [30].

Figure 3

Experimental strategies for targeting SB transposition. The common components of the targeting systems include a transposable element that contains the terminal inverted repeats (TIRs, black arrowheads) of the transposon and a GOI equipped with a suitable promoter. The transposase (purple oval) binds to the TIRs and catalyzes transposition. A DNA-binding protein domain (yellow sphere) recognizes a specific sequence (blue box) in the target DNA (parallel lines). A: Targeting with transposase fusion proteins. Targeting is achieved by fusing a specific DNA-binding protein domain to the transposase. B: Targeting with fusion proteins that bind the transposon DNA. Targeting is achieved by fusing a target-specific DBD to another DBD (red sphere) that binds to a specific DNA sequence within the transposable element (red box). In this strategy, the transposase is not modified. C: Targeting with fusion proteins that interact with the transposase. Targeting is achieved by fusing a target-specific DBD to a protein binding domain (PBD, green sphere) that interacts with the transposase. In this strategy, neither the transposase nor the transposon is modified.

Figure 4

Balloon-catheter-based DNA delivery to liver in large animals. Catheter-mediated delivery to the liver is made via the hepatic vein with incisions into the femoral or jugular vein followed by snaking the catheter through the IVC. Inflatable balloons on the catheter(s) are used to occlude appropriate veins and arteries to restrict outflow from the liver. The most common approach (Table 2) has been to insert a one or more balloon catheters into the IVC from entry points in either a femoral or jugular vein. With a double-balloon catheter, the DNA solution can be infused into the entire liver following temporary occlusion of the IVC above and below the access sites of the hepatic veins. In addition to the catheter that is used to deliver the DNA solution, other balloon catheters may be introduced into vessels such as arteries and/or the portal vein to isolate or semi-isolate the liver by blocking outflow of the infused solution. The DNA is shown in green exiting from multiple ports in the catheter to the large hepatic veins and hence into the sinusoids. The infusion is retrograde to normal blood flow. Figure drawn by Lynn Fellman.

Figure 5

The circuit algorithm of clinical trials. The SB system lends itself to translation science, as gene therapy applications can be developed in the laboratory, assessed in humans and the results used to refine subsequent bench research for future clinical trials.

Figure 6

Kinetics of numeric expansion of genetically modified T cells. Primary human T cells from peripheral blood can be nucleofected ex vivo to express a CD19-specific CAR and the genetically modified T cells can be selectively propagated by recursive passaging (every 7 days) on γ-irradiated CD19⁺ aAPC in presence of soluble cytokines. The graph shows the kinetics (from 0 to 28 days) of numeric expansion of CD3⁺ and CAR⁺ T cells after electro-transfer of SB plasmids on day 0. This time of ex vivo propagation can lead to differentiation of T cells and potential for replicative senescence. However, this period of tissue culture also presents opportunities as the T cells can be monitored for undesired autonomous cell growth and the culturing environment can be manipulated. Within 28 days, almost all T cells express CAR at numbers suitable for adoptive transfer in clinical trials.