Strategies to insulate lentiviral vector-expressed transgenes

Abstract

Lentiviruses are capable of infecting many cells irrespective of their cycling status, stably inserting DNA copies of the viral RNA genomes into host chromosomes. This property has led to the development of lentiviral vectors for high-efficiency gene transfer to a wide variety of cell types, from slowly proliferating hematopoietic stem cells to terminally differentiated neurons. Regardless of their advantage over gammaretroviral vectors, which can only introduce transgenes into target cells that are actively dividing, lentiviral vectors are still susceptible to chromosomal position effects that result in transgene silencing or variegated expression. In this chapter, various genetic regulatory elements are described that can be incorporated within lentiviral vector backbones to minimize the influences of neighboring chromatin on single-copy transgene expression. The modifications include utilization of strong internal enhancer-promoter sequences, addition of scaffold/matrix attachment regions, and flanking the transcriptional unit with chromatin domain insulators. Protocols are provided to evaluate the performance as well as the relative biosafety of lentiviral vectors containing these elements.

Fig. 5.1

Schematic diagram illustrating the two main and separate properties of insulators. (A) An insulator element (I) can protect against chromosomal position effects by blocking the advancement of adjacent repressive chromatin. (B) Many insulators also provide enhancer blocking function by preventing the activity of an enhancer (E), but only when placed between the enhancer and a promoter (P).

Fig. 5.2

Schematic diagram showing the reporter plasmid pRL-TKGFP-CMVE used in the flow cytometry-based barrier function assay (A) and the reporter plasmid pRL-TKGFP-CMVE-TKRFP used in the flow cytometry-based enhancer blocking assay (B). Abbreviations: 1.2 kb 5′HS4, 1.2-kb fragment of the chicken β-globin 5′HS4 insulator; GFP, enhanced GFP gene; RFP, DsRed.T4 gene; E, human CMV immediate early region enhancer; P, herpes simplex virus TK promoter.