Evaluating risks of insertional mutagenesis by DNA transposons in gene therapy

Abstract

Investigational therapy can be successfully undertaken using viral- and nonviral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently, the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR(+) T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease.

Copyright © 2013 Mosby, Inc. All rights reserved.

Figures

Fig. 1. Genetic consequences of integration of therapeutic vectors into genomes

In this instance a transposon vector is illustrated, but the considerations apply to any vector system. A therapeutic transposon can integrate into four general categories of chromatin. Heterochromatin will suppress expression of the transgene – no gain-of-function (GOF) or loss-of-function (LOF) will occur. The most desirable integration events will be into intergenic regions where the Therapeutic Gene (TG) will be expressed. Integration into or proximal to a transcriptional regulatory region can have several outcomes including GOF of the transgenic cassette, as well as either enhancement or loss of expression of the neighboring gene (Gene X). As reviewed in the text, in some cases the transcriptional regulatory elements of the transgene can activate quiescent chromosomal genes. Integration of the transposon into a transcriptional unit may allow expression of the transgene but block expression of the host gene leading to a phenotypic loss of function due to blockage of the gene or alterations in splicing. Integration within some genes can also lead to a dominant gain-of-function (DGF) and/or production of a dominant-negative form (DNF) of the original Gene X.

Fig. 2. DNA transposition

DNA transposition, as exemplified by the Sleeping Beauty (SB) transposon system, is a cut-and-paste reaction in which a transposon containing an expression cassette with a therapeutic gene (TG) and its promoter (pentagon) is delivered to target cells wherein the transposon is cut out of the plasmid and inserted into a chromosome. The inverted terminal repeats (inverted set of double arrowheads) define a transposon. The second part of the SB system is SB transposase, which in this example is carried in a separate expression cassette that is on the same plasmid, but not in the transposon. The SB transposon will only integrate into TA-dinucleotide basepairs (about 200 million in mammalian genomes). 1) The plasmid is delivered into a cell by any of several means and proceeds through the nuclear membrane (dotted oval) by a poorly understood process. 2) The SB gene is expressed. 3) The transposase molecules enter the nucleus and bind to the transposon. 4) Four transposases work in concert to cut the transposon out of the plasmid and paste it (dotted lines) into an AT sequence in chromatin (tangled line). A plasmid excision product is left behind in this reaction (the transposon site is marked by an X). Integration into a chromosome can confer sustained expression of the gene of interest that is contained within the transposon.

Fig. 3. Potential consequences of remobilization of SB transposons

The schematic illustrates the initial transposon integration site (orange) and subsequent hopping to other TA sites in chromatin, one of which might induce an adverse transforming event.

Fig. 4. The T2/onc transposon vector

This SB vector contains elements designed to elicit either transcriptional activation (Mouse Stem Cell Virus 5'-LTR and splice donor [SD]) or inactivation (splice acceptors [SA] and polyadenylation signals [pA]). The inverted terminal repeats are indicated by the arrows labeled ITR

Figure 5

Using SB for cancer-gene screens in mice. Step 1. Breed SB transposon and transposase transgenes together. In some cases, arrangements for tissue-specific expression of the transposase will be made or specific cancer-predisposed backgrounds could be used. Step 2. Transposition in somatic cells causes random insertion mutations. A correctly designed transposon vector can cause gain or loss-of-function mutations. Step 3. Mice are aged for tumor development. Step 4. Tumors develop as a result of transposon-induced mutations. Step 5. Transposon insertions are cloned from tumor genomic DNA. Step 6. Clones are sequenced. Step 7. Insertion sites are mapped and annotated with respect to nearest genes. Those genes repeatedly mutated in multiple, independent tumors are designated as common insertion sites or CIS. Step 8. CIS can be analyzed to determine what genes and genetic pathways contribute to cancer. Cancer genetic fingerprints are obtained that can be characterized by networks of interacting cancer-gene mutations. These genes can be queried in relevant human cancers.