used to design the vector sequence has a strong influence on the gene expression rate.
For example, DNA vectors harbouring NLS sequences, DNA binding proteins such
as histones and HMG proteins, Simian Virus 40 (SV40) promoter and origin of
replication have been shown to increase the intranuclear delivery and expression
of genes. Furthermore, the presence of impurities (e.g. endotoxins) in the DNA can
also lower the transfection efficiency.
The optimal quantity of DNA used for transfection also needs to be titrated to
regulate the gene copy number in the transfected cells to obtain optimal gene
expression and to avoid post-integrative gene silencing due to high copy number
or overexpression of exogenous genes. In certain situations, the non-viral vector
does not integrate into the nuclear genome and remains episomal. The tendency of
non-viral vectors to stay episomal can be considered beneficial for cellular
reprogramming of somatic cells into stem cells (iPS cells) [111, 140]. However,
when used for transgenesis and stable expression of genes, the episomal form of
non-viral vectors is not desirable because they are not passed on to daughter cells.
The development of self-replicating vectors, vectors without regions prone to
epigenetic silencing and vectors containing scaffold or nuclear matrix attachment
regions (S/MARs) to keep them in transcriptionally active regions are some of the
approaches that have shown promise in increasing the persistence of expression for
episomal vectors.
Apart from mosaic and variable expression of genes, NP-mediated gene delivery
methods also suffer from integration-mediated activation or inhibition of other
nearby genes. Random genomic integration may also lead to insertional mutagenesis and/or trans-activation of cellular proto-oncogenes, resulting in cellular transformation to cancerous cells. This can be addressed by proper design of DNA
vector to include homologous sequences for locus-specific gene targeting, Sleeping
Beauty transposon-transposase or piggyBac transposition for non-random preferential integration at microsatellite repeats [141–143] or by using a fC31 integrase
system [144, 145].
Post-integrative gene silencing can occur for a variety of reasons, such as high
copy number or overexpression of exogenous genes, random integration into
heterochromatin regions and episomal silencing due to heterochromatin spreading
[146]. Although optimizing the DNA concentration can reduce the chances of high
gene copy number, random integration into heterochromatin regions can be overcome by incorporating insulator sequences into the vectors [140]. Several regulatory sequences that have insulating properties have been described [147],
including S/MARs [148]. Evidence suggests that inclusion of an S/MAR region
can provides an insulating effect by inhibiting promoter region methylation and
silencing, as seen for CMV and HAAT promoters [140]. S/MAR-containing vectors
have been used to drive transgene expression in hematopoietic stem cells [149].
6.6 NP Size
The size of the NP–DNA complexes is of crucial importance for their cellular
uptake through endocytosis and/or pinocytosis and subsequent transfer into the
Nanoparticles for Gene Delivery into Stem Cells and Embryos
75
For example, DNA vectors harbouring NLS sequences, DNA binding proteins such
as histones and HMG proteins, Simian Virus 40 (SV40) promoter and origin of
replication have been shown to increase the intranuclear delivery and expression
of genes. Furthermore, the presence of impurities (e.g. endotoxins) in the DNA can
also lower the transfection efficiency.
The optimal quantity of DNA used for transfection also needs to be titrated to
regulate the gene copy number in the transfected cells to obtain optimal gene
expression and to avoid post-integrative gene silencing due to high copy number
or overexpression of exogenous genes. In certain situations, the non-viral vector
does not integrate into the nuclear genome and remains episomal. The tendency of
non-viral vectors to stay episomal can be considered beneficial for cellular
reprogramming of somatic cells into stem cells (iPS cells) [111, 140]. However,
when used for transgenesis and stable expression of genes, the episomal form of
non-viral vectors is not desirable because they are not passed on to daughter cells.
The development of self-replicating vectors, vectors without regions prone to
epigenetic silencing and vectors containing scaffold or nuclear matrix attachment
regions (S/MARs) to keep them in transcriptionally active regions are some of the
approaches that have shown promise in increasing the persistence of expression for
episomal vectors.
Apart from mosaic and variable expression of genes, NP-mediated gene delivery
methods also suffer from integration-mediated activation or inhibition of other
nearby genes. Random genomic integration may also lead to insertional mutagenesis and/or trans-activation of cellular proto-oncogenes, resulting in cellular transformation to cancerous cells. This can be addressed by proper design of DNA
vector to include homologous sequences for locus-specific gene targeting, Sleeping
Beauty transposon-transposase or piggyBac transposition for non-random preferential integration at microsatellite repeats [141–143] or by using a fC31 integrase
system [144, 145].
Post-integrative gene silencing can occur for a variety of reasons, such as high
copy number or overexpression of exogenous genes, random integration into
heterochromatin regions and episomal silencing due to heterochromatin spreading
[146]. Although optimizing the DNA concentration can reduce the chances of high
gene copy number, random integration into heterochromatin regions can be overcome by incorporating insulator sequences into the vectors [140]. Several regulatory sequences that have insulating properties have been described [147],
including S/MARs [148]. Evidence suggests that inclusion of an S/MAR region
can provides an insulating effect by inhibiting promoter region methylation and
silencing, as seen for CMV and HAAT promoters [140]. S/MAR-containing vectors
have been used to drive transgene expression in hematopoietic stem cells [149].
6.6 NP Size
The size of the NP–DNA complexes is of crucial importance for their cellular
uptake through endocytosis and/or pinocytosis and subsequent transfer into the
Nanoparticles for Gene Delivery into Stem Cells and Embryos
75