work reasonably well in vitro and overcome some of the disadvantages of viral-based
gene delivery systems such as immunological response, fatal infections, etc.
The genetic material for treatment of a variety of genetic disorders can be of
three types: (1) pDNA, to express a gene of interest under the control of a suitable
promoter (has to reach the nucleus), which will result in the increased production of
a protein [94, 95]; (2) oligomeric genetic material such as antisense OligoDeoxyNucleotides (ODN), short RNA molecules such as small interfering RNA (siRNA)
micro-RNA (miRNA) or short hairpin RNA (shRNA), or a DNAzyme in order to
silence a specific gene by reducing the target/protein activity. The short RNA
molecules as well as DNAzyme have to reach the cytoplasm and more precisely
the RNA-induced silencing complex (RISC) without being destroyed in the late
endosomes or lysosomes [96–98].
1.4.2 Requirements for Efficient Gene Therapy
Complexation and Compaction/Condensation
For most cell types, the size requirement for particle uptake via endocytosis is of the
order of 200 nm or less. As DNA has a R h of a few hundred nanometers when its
molecular weight is a few thousand base pairs, the polymers should not only
complex DNA but also condense or compact it into smaller particles. The polymer
remains unable to condense DNA until the neutralization of a critical amount of
negative charges on the DNA. For instance, Wilson and Bloomfield have calculated
that in order to condense DNA, 90% of the phosphonate moieties have to be
neutralized when the condensing agent is spermine or spermidine [99].
For condensation, strong (quaternary ammonium, etc.) as well as weak polyelectrolytes (containing amino acids such as Arg, Lys, etc. or poly[(2-dimethylamino)
ethyl methacrylate], PDMAEMA [100]) can be used, which should possess a minimum number of cationic charges at physiological pH. For instance, as a weak
polyelectrolyte, linear PEI (LPEI, 22 kDa) has 75% of its amino groups protonated
at physiological pH [101]. At pH 8, with a degree of polymerization (DP) of
32, PDMAEMA has approximately 24% of its amino groups protonated [102].
Extracellular Barriers and Physico-chemical Aspects
A major drawback of current transfection vectors is that they have poor in vivo
transfection efficiency and only confer transient gene expression. Indeed, poor
transfection efficiency is due, in part, to the lack of stability of the non-viral
vector–DNA complex under physiological conditions and its ability to interact
with blood plasma proteins after intravenous injection, the extracellular matrix,
and undesirable cells, and its possible degradation by enzymes, even before
reaching the intracellular compartment (Scheme 12). In order to overcome these
problems and to enable the carrier to translocate across cellular membranes (thus
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
127
gene delivery systems such as immunological response, fatal infections, etc.
The genetic material for treatment of a variety of genetic disorders can be of
three types: (1) pDNA, to express a gene of interest under the control of a suitable
promoter (has to reach the nucleus), which will result in the increased production of
a protein [94, 95]; (2) oligomeric genetic material such as antisense OligoDeoxyNucleotides (ODN), short RNA molecules such as small interfering RNA (siRNA)
micro-RNA (miRNA) or short hairpin RNA (shRNA), or a DNAzyme in order to
silence a specific gene by reducing the target/protein activity. The short RNA
molecules as well as DNAzyme have to reach the cytoplasm and more precisely
the RNA-induced silencing complex (RISC) without being destroyed in the late
endosomes or lysosomes [96–98].
1.4.2 Requirements for Efficient Gene Therapy
Complexation and Compaction/Condensation
For most cell types, the size requirement for particle uptake via endocytosis is of the
order of 200 nm or less. As DNA has a R h of a few hundred nanometers when its
molecular weight is a few thousand base pairs, the polymers should not only
complex DNA but also condense or compact it into smaller particles. The polymer
remains unable to condense DNA until the neutralization of a critical amount of
negative charges on the DNA. For instance, Wilson and Bloomfield have calculated
that in order to condense DNA, 90% of the phosphonate moieties have to be
neutralized when the condensing agent is spermine or spermidine [99].
For condensation, strong (quaternary ammonium, etc.) as well as weak polyelectrolytes (containing amino acids such as Arg, Lys, etc. or poly[(2-dimethylamino)
ethyl methacrylate], PDMAEMA [100]) can be used, which should possess a minimum number of cationic charges at physiological pH. For instance, as a weak
polyelectrolyte, linear PEI (LPEI, 22 kDa) has 75% of its amino groups protonated
at physiological pH [101]. At pH 8, with a degree of polymerization (DP) of
32, PDMAEMA has approximately 24% of its amino groups protonated [102].
Extracellular Barriers and Physico-chemical Aspects
A major drawback of current transfection vectors is that they have poor in vivo
transfection efficiency and only confer transient gene expression. Indeed, poor
transfection efficiency is due, in part, to the lack of stability of the non-viral
vector–DNA complex under physiological conditions and its ability to interact
with blood plasma proteins after intravenous injection, the extracellular matrix,
and undesirable cells, and its possible degradation by enzymes, even before
reaching the intracellular compartment (Scheme 12). In order to overcome these
problems and to enable the carrier to translocate across cellular membranes (thus
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
127