Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System

Nafiseh Nafissi,Roderick Slavcev

doi:10.1186/1475-2859-11-154

Abstract

BackgroundWhile safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event.ResultsWe constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called “Super Sequence”, and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc—linear covalently closed (Tel/TelN-cell), or mini ccc—circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpart.ConclusionWe offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of “minicircle” DNA vectors and virtually eliminate the potential for undesirable vector integration events.

Highlights

While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile
R-cells exhibit temperature-regulated recombinase expression Recombinant E. coli cells (R-cells) were constructed that place tel or telN recombinase genes under control of the bacteriophage λ strong promoter, pL, that is regulated by the temperature-sensitive λ repressor, CI[Ts]857 (Figure 1A)
Linearization of pNN9 and extraction of the mini lcc vector, was much more efficient in Tel+, as compared to the TelN+ counterpart (Figure 3C). These findings indicate that Tel is active upon pal+ constructs in vivo and that the modification of pal target site through the addition of loxP, FRT, and telRL minimal target sequences into non-binding regions of the full 142 bp pal target site does not abrogate Tel-pal functionality

Summary

Introduction

While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may impart the potential for chromosomal integration, potentiating oncogenesis. Plasma nucleases digest the unprotected DNA within just a few minutes, so DNA vectors need to rapidly cross the plasma membrane of target cells This is further complicated by the fact that the plasma membrane is composed of dense lipoprotein barriers that intrinsically inhibit efficient DNA translocation. This difference may be attributed to charge per unit ratio of linear versus supercoiled circular DNA and provides yet another intriguing opportunity for lcc vectors [7]

Methods

Results

Discussion

Conclusion