Sleeping Beauty: a candidate for safer hybrid vectors in HIV therapy Essay

Submitted By geekygreeky
Words: 6581
Pages: 27

Contents

Introduction 2
The HIV life cycle 5
Sizing up the methods 7
Altered viral vectors can help deliver 9
Gene therapy for HIV 11
System potential in HIV treatment 13
Conclusions 15
Referenced websites/sources: 18
Bibliography 18

Introduction

The Sleeping Beauty (SB) transposon system was developed as a synthetic construct based on a fish gene which was gradually inactivated due to accumulated mutations (Ivics et al, 1997). It was creatively termed due to the transposon having been ‘awakened’ from its evolutionarily developed inactive state. Transposons are naturally occurring mobile elements which excise from one position in the genome to another. The SB transposon is constructed from an ancient salmonid Tc1/mariner transposon, undergoing a series of site directed mutagenesis to remove the accumulated mutations which had led to its inactivation (Z Ivics et al, 1997). By modifying the transposons natural functions they were able make the SB system a randomly integrating vector system for genetic engineering.
Specifically the enzyme transposase binds the Terminal Inverted Repeats (TIRs) which are on each end of the transgene. It then catalyses excision and subsequent reinsertion into target DNA in a TA dinucleotide and leaves behind a double-strand DNA break to be repaired by cellular machinery. Once excised the insertion region must include TA sites at each end for successful transposition. However the transposase may also be designed to be encoded on the same plasmid as the transgene, or introduced in vitro directly into the cell in the form of coding mRNA. Insertion of the transposon is largely considered to be random since it only requires a TA site at the target sequence for integration. However a study has shown half the transpositions have preference for 10% of the total TA sites in euchromatin, i.e. in lightly packed chromatin (Geurts et al. 2006). In addition, a study by Liu et al. (2005) showed that particular deformations in the double helix can present TA sites with about 16 fold preference for transposon integration. In this sense the integration of the transgene is not truly random, but cannot be considered directed as any TA site in the genome is technically a potential target.
Since it was first engineered in 1997 alterations have been made to produce the hyperactive forms of the transposase enzyme such as the SB100X developed by Mátés et al (2009) and HSB5 transposase (de Silva et al., 2010). In the case of SB100X a 100-fold efficiency increase was achieved compared to the original transposase as well as 35–50% stable gene transfer in human CD34+ cells. As will be discussed, efficacy of integration plays a major role in the maintenance of therapeutic transcription, so these enhancements are significant in developing viable in vivo therapies.

Two plasmids can be used within a functional transposon system: recombinant and helper. Recombinant plasmid contains the transgene, i.e. the sequence which is to be introduced into the target’s genome. At each end of the transgene are terminal inverted repeats (TIRs). A helper plasmid codes for a transposase which is the enzyme necessary to perform the ‘cut’ function from the recombinant plasmid. Four of these enzymes bind to the TIRs of the transgene, removing the bonds that hold it to the plasmid.

Figure 1: Stages in transposition and different ways of delivering transposase. Transposase can be encoded by the recombinant plasmid (A) or by a helper plasmid (left B) or by insertion of mRNA encoding the transposase (right B). First the transposon is bound to by transposase enzymes which form a complex with each other bringing the ends of the transposon close to each other. This is followed by the ‘cutting’ action when then the enzymes guide the transposon to the TATA site and insert, or ‘paste’, it there. Sourced from Izsvák et al. (2010)

The Journal of Gene medicine web database as of February 2012 estimates approximately 1786 gene therapy clinical trials…