الفهرس 
Abstract
Graphical AbstractOnly 14 pages are availabe for public view
 |  | from | 188 |  |  |
| | | from | 188 | | |
Abstract
Gene therapy is an experimental technique that uses genetic
materials to treat or prevent disease.
Although gene therapy is a
promising treatment option for a number of diseases (including inherited
disorders and cancer), the technique remains risky and is still under study
to make sure that it will be safe and effective.
One of the major challenges in gene therapy is the development of
suitable vectors that protect therapeutic genetic material from degradation
and facilitate effective transfection of host cells with stable genetic
payload. A gene vector is needed to deliver this precious material to the
target site. Viral vectors are highly efficient for gene therapy, but their
use is associated with high toxicity and immunogenicity. However,
nonviral vectors are considered attractive alternatives because of their
inherent low toxicity and minimum potential to induce immune
responses.
This thesis is structured as three chapters. Each chapter has a
specific aim as described below.
Chapter 1: Formulation and delivery of reporter gene using
methacrylate-based cationic polymers as non-viral gene delivery
system.
This chapter represented a preliminary study using non-specific
genetic material such as plasmid encodes for Green Fluorescent Protein
(pAcGFP1-C1, reporter gene) to compare safety and efficacy of
quaternary amine-containing methacrylate polymer Eudragit® RL PO
(ERL) and poly[N-(2-hydroxypropyl)methacrylamide]-poly(N,NAbstract
II
dimethylaminoethylmethacrylate) copolymer (pHPMA-b-pDMAEMA),
which contains secondary and tertiary amines, as effective gene vectors.
Ethidium bromide (EtBr) intercalation and gel retardation assays
demonstrated that pHPMA-b-pDMAEMA most effectively condensated
pAcGFP1-C1 plasmid into electrostatically stabilized nanoassemblies at a
mass ratio of 1:0.513 using phosphate-buffered saline (PBS), pH 7.4, as
vehicle. Polyplexes of pDNA with ERL, pHPMA-b-pDMAEMA, and
various pHPMA-b-pDMAEMA/ERL blends were fabricated at different
pDNA/polymer ratios. Particle size and zeta potential were determined
using dynamic laser light scattering. These cationic polymers up to
concentrations of 560 μg/mL did not significantly compromise HeLa
cells viability. pHPMA-b-pDMAEMA had a good buffering capacity,
which is considered as a good indication for the endosomal escape.
Consequently, pHPMA-b-pDMAEMA had higher ability to deliver the
genetic material than ERL-containing nanoassemblies. Inclusion of serum
significantly decreased transfection efficiency of pHPMA-bpDMAEMA-
containing polyplexes by ~30% at N/P=4 and ~50% at
N/P=2, respectively. The transfection efficiency in presence of serum was
comparable to the positive control “TurboFect™ transfection reagent”.
N.B/ N/P ratio is a stoichiometric ratio of positively charged amine
groups in a cationic polymer and negatively-charged phosphate groups in
DNA moieties.
These results imply that polyplexes fabricated with the secondary
and tertiary amine-containing pHPMA-b-pDMAEMA copolymer
represent more effective gene delivery systems than nanoassemblies
composed of the quaternary amine-containing ERL and should be further
explored as promising nonviral gene vector.
Abstract
III
Chapter 2:
Formulation and delivery of therapeutic genetic materials
using pHPMA-b-pDMAEMA as a non-viral vector to up-regulate or
to down-regulate gene expression
Gene therapy can be used to up-regulate the expression of genes,
which is defective or to down-regulate the expression of unwanted genes.
Multidrug-resistance (MDR), which represents a major cause for
chemotherapy failure can be modulated by P-glycoprotein (P-gp) downregulation
via inhibiting Nuclear Factor-κB (Nuclear Factor kappa-lightchain-
enhancer of activated B cells, NF-κB) transcriptional activity using
NF-κB decoy.
Placental insufficiency accounts for 75% of all cases of intrauterine
growth restriction (IUGR). There is no treatment at the present
time for placental insufficiency except premature delivery. IUGR is
recognized as the failure of fetus to achieve its growth potential in utero
and is the second leading cause of perinatal morbidity and mortality.
Insulin-like growth factor (IGF-1) plays an important role in childhood
growth and continues to have anabolic effects in adults. Up-regulation of
this gene may be used to alleviate placental insufficiency and its resulting
IUGR.
The objectives of this chapter were to encapsulate the genetic
materials NF-κB decoy oligodeoxynucleotide (ODN) or the plasmid
encodes for IGF-1 within the previously tested pHPMA-b-pDMAEMA
polymer to decrease P-gp-associated MDR via inhibition of NF-κB
transcription activity or to alleviate the fetal growth restriction via upregulation
of IGF-1 expression, respectively.
Ethidium bromide intercalation and agarose gel retardation
demonstrated that pHPMA-b-pDMAEMA has high DNA condensation
Abstract
IV
capacity. Particle size and zeta potential were determined using dynamic
laser light scattering. Nanoparticles prepared with and without the genetic
materials did not significantly compromise cellular safety at all tested
N/P ratios. Transfection efficiency of NF-κB decoy polyplexes was
visually assessed by confocal microscopy. Gene target effects were
quantified using P-gp immunoblots and intracellular uptake of the P-pg
substrate Rhodamine123 (Rho-123). It was found that pHPMA-bpDMAEMA/
NF-κB ODN polyplexes significantly reduced P-gp levels
and significantly increased intracellular Rho-123 concentration to twofolds
in absence of serum.
In presence of 10% (v/v) serum, the
intracellular Rho-123 was increased to 1.9-fold. The intracellular Rho-
123 was non-significantly decreased in presence of serum (p=0.1).
Trophoblast-selective transgene expression was induced in vitro
using pHPMA-b-pDMAEMA and PLAC1-modified expression plasmids.
In vivo transfection and functionality was assessed by direct placentalinjection
of PLAC1-huIGF-1 polyplexes into a mouse model of IUGR
and proved that these polyplexes were able to restore the normal fetal
growth. Placental histology showed no abnormal morphology in
polyplexes-treated placentas.
It was concluded that polyplexes fabricated with pHPMA-bpDMAEMA
effectively deliver the therapeutic genetic materials into the
cells and regulate the gene expression. NF-κB decoy ODN polyplexes
effectively modulate P-gp expression and function in Caco-2 cells
suggesting interference with NF-κB binding sites in the promoter region
of the ABCB1 gene encoding for human P-gp. PLAC1-huIGF-1
polyplexes alleviated IUGR and consequently, representing innovative
building blocks towards human placental gene therapies.
Abstract
V
Chapter 3: Optimized cationic lipid–polycation–DNA formulations to
obtain serum resistant lipopolyplexes as non-viral gene delivery
system
This chapter aimed to improve the transfection efficiency of
pHPMA-b-pDMAEMA/pAcGFP1-C1 polyplexes in presence of serum.
This aim was achieved through coating of the polyplexes with 1,2-
dioleoyl-3-trimethylammonium-propane, chloride salt (DOTAP)/1,2-
dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipid systems
forming lipopolyplexes.
The effect of different lipopolyplex fabrication methods on particle
size and zeta potential in both presence and absence of serum was
studied. The best fabrication method was used for further characterization
of lipopolyplexes at different lipid/DNA weight ratios (10, 20 and 40)
and compared to the ‘parent’ lipid-free polyplexes. Under serum
conditions, transfection activity of lipopolyplexes at lipid/DNA weight
ratio of 20 was maximal and significantly higher than lipid-free
polyplexes. This higher transfection efficiency was due to the serum
resistance effect of the lipopolyplexes, which was clear from
physicochemical properties. In 10% (v/v) FBS, lipopolyplexes showed
homogenous size distribution (300- 500 nm, PDI of approx. 0.2)
and a
reversed zeta potential (about -10 mV), while the corresponding lipid-free
polyplexes showed significant dissociation and aggregation under serum
conditions (PDI of approx. 1).
Lipopolyplex formulations of pHPMA-b-pDMAEMA in
combination with DOTAP/DOPE are promising tools for gene delivery
under serum conditions.
General