A transposon and transposase system for human application

Abstract

The stable introduction of therapeutic transgenes into human cells can be accomplished using viral and nonviral approaches. Transduction with clinical-grade recombinant viruses offers the potential of efficient gene transfer into primary cells and has a record of therapeutic successes. However, widespread application for gene therapy using viruses can be limited by their initially high cost of manufacture at a limited number of production facilities as well as a propensity for nonrandom patterns of integration. The ex vivo application of transposon-mediated gene transfer now offers an alternative to the use of viral vectors. Clinical-grade DNA plasmids can be prepared at much reduced cost and with lower immunogenicity, and the integration efficiency can be improved by the transient coexpression of a hyperactive transposase. This has facilitated the design of human trials using the Sleeping Beauty (SB) transposon system to introduce a chimeric antigen receptor (CAR) to redirect the specificity of human T cells. This review examines the rationale and safety implications of application of the SB system to genetically modify T cells to be manufactured in compliance with current good manufacturing practice (cGMP) for phase I/II trials.

Figure 1

SB transposon-mediated gene transfer into chromosomal DNA for long-term expression of a therapeutic gene. A SB transposon, with flanking inverted terminal repeats (ITRs, blue arrowheads), in a plasmid (blue line) provides only transient expression of a transgene (yellow) from a promoter (green) unless the transposon vector transposes into a host genome (green line) by SB transposase (red expression cassette). The lightning bolt represents any of several methods for delivery of the transposon system into a cell and its nucleus. The structure of SB transposase, which is composed of three functional domains, is diagrammed in the box. The transposase's a-helical rich N-terminal domain binds to the ITRs of the transposon, the nuclear localization sequence (NLS) directs import of the transposase into the nucleus, and the catalytic domain catalyzes the cut-and-paste transposition reaction and recognizes TA base pairs into which the transposon will be inserted. The N-terminal domain is further divided into two subdomains, PAI and RED, each of which has helix-turn-helix regions (green and blue boxes, respectively) that are separated by an arginine-rich sequence GRRR. Three signature amino acids (D, D, and E) in the catalytic domain are indicated by blue boxes.

Figure 2

The T2/onc transposon vector to introduce mutations. This DNA vector contains elements designed to elicit either transcriptional activation (MSCV 5′ LTR and splice donor, SD) or inactivation [splice acceptor (SA) and polyadenylation signal, pA] when landing within/near an endogenous gene. IRL and IRR denote the left and right inverted terminal repeats of the SB transposon, respectively. MSCV, murine stem cell virus.

Figure 3

Schematic for the electroporation of SB system and subsequent selective propagation of CAR⁺ T cells on aAPC. The culturing of T cells on aAPC leads to their expansion as well as provides a window in which pharmacologic agents can be added to reprogram T cells to exhibit desired properties (e.g., those associated with a central memory or naive phenotypes). The peripheral blood mononuclear cells can be collected by steady-state apheresis or a simple blood draw. The release testing before human application is performed on the cryopreserved product manufactured in compliance with current good manufacturing practice for phase I/II trials. aAPC, artificial antigen-presenting cells; EGFP, enhanced green fluorescent protein; SB, Sleeping Beauty.