phago-lysosome after bacterial uptake [2]. For the last two decades,
clinical trials have advanced various types of bacterial vectors for the
delivery of genes to certain cells for antiangiogenic, immunotherapy, and other therapeutic purpose [3–6]. Gene delivery using such
nonviral bacterial vectors has been especially highlighted targeting
antigen-presenting cells (APCs). In such a context, the bacterial
vector possesses natural adjuvant features capable of triggering
activation features of the APC through the production of nitric
oxide to attract additional APCs to the site of infection and accelerate digestion of the bacteria [7]. To enhance endosomal escape after
bacterial vector phagocytosis in APCs, LLO has been naturally
leveraged or recombinantly introduced during bactofection [8].
While bactofection offers potential advantages in mammalian
gene delivery, the approach still depends on a foreign bacterial cell,
which can cause unwanted side effects and potential excesses in
immune reactivity. Common bacterial agent attenuation
approaches include chemical and biological-genetic cellular weakening [9]. This chapter will feature an alternative attenuation
method used previously in vaccine-based bactofection strategies,
namely introduction of the bacteriophage ϕX174 lethal lysis gene E
(LyE) into Escherichia coli (E. coli) to promote safer and improved
delivery of antigenic content to APCs [10]. The inclusion of LyE
expression within the vector serves to both enhance bacterial degradation, thus, minimizing possible negative side effects caused by
the bacterial cells, and improve release of DNA/RNA in APCs (in a
similar function as LLO). Resulting bacterial vectors with the LyE
gene have shown enhanced gene and protein release and inducible
attenuation [11].
Figures 1 and 2 describe the mechanism of LyE activity affecting the cell wall and compares traditional bactofection with LyE
integrated bactofection, respectively. The new LyE integrated
strains exhibited improved gene delivery and reduced cytotoxicity
profiles when tested with murine RAW 264.7 macrophage APCs.
This chapter will describe the protocol used to engineer E. coli
strains with the LyE gene and steps to assess membrane disruption,
protein/DNA release, and cytotoxicity of the new bacterial vectors
to serve as antigen delivery vehicles.
2 Materials
2.1 Molecular
Biology Reagents
and Equipment
1. Bacteriophage ΦX174, (ATCC).
2. Restriction enzymes: EcoRI, HindIII.
3. Primers.
Forward: 5
0 -GGGAATTCGATGGTACGCTGGACTTTGTGG-3
0
Reverse: 5
0 -AGGAAGCTTTCACTCCTTCCGCACGTAATT-3
0
4
Dongwon Park et al.
clinical trials have advanced various types of bacterial vectors for the
delivery of genes to certain cells for antiangiogenic, immunotherapy, and other therapeutic purpose [3–6]. Gene delivery using such
nonviral bacterial vectors has been especially highlighted targeting
antigen-presenting cells (APCs). In such a context, the bacterial
vector possesses natural adjuvant features capable of triggering
activation features of the APC through the production of nitric
oxide to attract additional APCs to the site of infection and accelerate digestion of the bacteria [7]. To enhance endosomal escape after
bacterial vector phagocytosis in APCs, LLO has been naturally
leveraged or recombinantly introduced during bactofection [8].
While bactofection offers potential advantages in mammalian
gene delivery, the approach still depends on a foreign bacterial cell,
which can cause unwanted side effects and potential excesses in
immune reactivity. Common bacterial agent attenuation
approaches include chemical and biological-genetic cellular weakening [9]. This chapter will feature an alternative attenuation
method used previously in vaccine-based bactofection strategies,
namely introduction of the bacteriophage ϕX174 lethal lysis gene E
(LyE) into Escherichia coli (E. coli) to promote safer and improved
delivery of antigenic content to APCs [10]. The inclusion of LyE
expression within the vector serves to both enhance bacterial degradation, thus, minimizing possible negative side effects caused by
the bacterial cells, and improve release of DNA/RNA in APCs (in a
similar function as LLO). Resulting bacterial vectors with the LyE
gene have shown enhanced gene and protein release and inducible
attenuation [11].
Figures 1 and 2 describe the mechanism of LyE activity affecting the cell wall and compares traditional bactofection with LyE
integrated bactofection, respectively. The new LyE integrated
strains exhibited improved gene delivery and reduced cytotoxicity
profiles when tested with murine RAW 264.7 macrophage APCs.
This chapter will describe the protocol used to engineer E. coli
strains with the LyE gene and steps to assess membrane disruption,
protein/DNA release, and cytotoxicity of the new bacterial vectors
to serve as antigen delivery vehicles.
2 Materials
2.1 Molecular
Biology Reagents
and Equipment
1. Bacteriophage ΦX174, (ATCC).
2. Restriction enzymes: EcoRI, HindIII.
3. Primers.
Forward: 5
0 -GGGAATTCGATGGTACGCTGGACTTTGTGG-3
0
Reverse: 5
0 -AGGAAGCTTTCACTCCTTCCGCACGTAATT-3
0
4
Dongwon Park et al.