Genomic DNA And Complementary DNA Libraries construction.
Receptor targeted polyplexes for pdna and sirna delivery emadi
1. Receptor-TargetedReceptor-Targeted
Polyplexes for pDNA andPolyplexes for pDNA and
siRNA DeliverysiRNA Delivery
Receptor-TargetedReceptor-Targeted
Polyplexes for pDNA andPolyplexes for pDNA and
siRNA DeliverysiRNA Delivery
2. The aim of the implementation of nucleic acid molecules as medical
agent is to rectify possible mistakes that cause inherited or acquired
disease.
The primary roadblock for the successful application of nucleic acids in
human therapy is their extracellular and intracellular delivery.
The host organisms have developed defense mechanisms to protect
themselves against exogenous genetic information.
Nonviral vectors are synthesized from diverse natural and synthetic
molecules.
Design of these “synthetic viruses” must be further optimized with
respect to efficiency.
3. Nucleic Acids in Therapy
• Research on gene therapy focuses on many different types of nucleic acids with
therapeutic potential.
• Therapeutic nucleic acids differ in molecular and biophysical properties
resulting in different effects at the genetic level.
• pDNA vectors gain of function
• microRNA or siRNA loss of function
Polymers Designed for Gene Delivery
• Extracellular and intracellular delivery of therapeutic nucleic acid molecules is
the biggest hindrance for efficient gene therapy.
• Direct delivery of “naked” nucleic acids can be applied with reasonable
efficiency only rarely (problems such as undesired interactions with blood
components, degradation, and complement activation).
• Therefore, viral and nonviral delivery systems have been developed to stabilize
nucleic acids and cellular recognition.
4. Nonviral vectors of nanostructure size are enabled by electrostatic
interaction between nucleic acid and polymers or lipids resulting into
complexes such as “lipoplexes” or “polyplexes”.
General requirements on polymers are low toxicity, biodegradability,
good complexation of nucleic acid into nanostructures and reliable
dissociation within target cells.
The most widely studied polymers for gene delivery are poly(l-lysine)
(PLL), polyethylenimine (PEI), and polyamidoamine (PAMAM)
dendrimers.
5. Gene transfer efficiency strongly varies between the different
formulations.
PEI and PAMAM dendrimers are the most effective polycations with
excellent and consistent transfection efficiency on several cell lines.
The buffering capacity of these polymers offers the opportunity to
escape from the endosome “proton sponge effect”.
Drawbacks of PEI and PAMAM dendrimers are significant toxicity and
lack of degradability.
The best transfection results for siRNA delivery were achieved by
BPEI.
PEI PLL
PAMAM
6. PLL
Advantage:
• PLL is biodegradable, which is a big advantage for in vivo applications.
Limitation:
• The ineffective in vivo transfection of PLL is due to:
• its binding to plasma proteins
• limited intracellular endosomal release
Solutions:
• coating with polyethylene glycol (PEG)
• inclusion of a targeting ligand
• introduction of histidine residue to cause a proton sponge effect
• cotransfection with endosomal disruptive agents
PLL
7. Chitosan
Advantages:
•biocompatibility
•biodegradability
•mucoadhesive and permeability-enhancing properties
Limitation:
•low transfection efficiency which is strongly influenced by several factors:
•molecular weight of chitosan
•its degree of deacetylation
•the charge ratio of chitosan to DNA/siRNA (N/P ratio)
•chitosan salt form
•the preparation techniques of chitosan/nucleic acid particles
8. histones and protamine
Protein-mediated delivery systems such as histones and protamine
have recently emerged as an alternative gene transfer method.
Advantages:
•nuclear localization signals (NLSs)
•simplicity in preparation and application
•no restriction to the type or size of nucleic acid
•ability to target nucleic acid to specific cell types
The combination of oligo (ethylene amino) acids with hydrophobic
modifications and bioreversible disulfide cross-linking sites leads to
transfection polymers with tailor-made endosomolytic and DNA-
binding properties.
9. siRNA versus pDNA: Similarities and Differences in Delivery
structural similarities between pDNA and siRNA:
•both are double-stranded nucleic acids
•anionic phosphodiester backbones
structural differences between pDNA and siRNA:
•in size (pDNA > siRNA)
•in structure
•chemistry of pDNA and siRNA (siRNA is more degradable)
•both types of nucleic acids vary in their site of action
For the delivery of siRNA and pDNA, polycations with different
charge densities are necessary to achieve the same complexation and
extracellular stability.
10.
11.
12. Physical Restrictions of Polyplexes
Nanoparticle (NP) size seems to be a general critical factor for drug
targeting.
Factors that strongly influence the in vitro and in vivo gene transfer
efficiency:
•size and modification of the nucleic acid–binding element
•size and sequence of the nucleic acid
•procedure of complex formation
•nitrogen/phosphate (N/P) ratio
large particle sizes and a high aggregation tendency were observed at a low N/P ratio
high N/P ratio led to formation of smaller particles
An increase in the aggregation tendency was observed with higher
DNA concentrations and depended on buffer ionic strength.
13. Undesired Interactions with Plasma Proteins, Enzymes, and Nontarget
Tissue
Once in vivo, polyplexes are surrounded by a variety of compounds present in
blood plasma and the physicochemical properties of the polyplexes change.
Opsonins may coat the polyplex causing aggregation, dissociation, or
degradation of the polyplex.
Polyplex modification with PEG can solve several of those problems.
PEGylation has several significant advantages:
surface shielding
increase of solubility
reduction of interaction with blood cells and serum proteins
improvement of biocompatibility
increased blood circulation time
One way to avoid interactions with the extracellular matrix is the reduction of
polyplex charge ratio close to neutrality.
14. Inflammatory and Immunological Responses
The mammalian immune system is divided into two major branches:
the innate immune system and the acquired immune system.
The introduction of gene therapeutics into human body can trigger a
broad activation of the immune system.
Many problems occur due to this immune response including:
•decreased efficiency of vectors after readministration
•transient expression of therapeutic gene
•severe side effects in clinical trials
The activation of the nonspecific immune response represents a major
hurdle for efficient gene delivery.
15.
16. Cellular Targeting
The proper choice of a ligand for efficient gene transfer is importan.
In the selection of a ligand, several aspects must be considered:
•Being tissue specific
•Efficiency of internalization
•Carrying charges
Targeting ligands (conjugated to polycations):
•Transferrin (Tf) for Transferrin R
•EGF for EGF R
•folic acid for Folate R
•synthetic peptides with the arginine–glycine–aspartate (RGD) sequence for integrins
•synthetic peptides with the asparagine–glycine–arginine (NGR) sequence for aminopeptidase
N (APN; also known as CD13)
•Abs for antigens overexpressed by tumor cells or tumor cell-specific antigens (e.g. anti-CD3
antibody)
17. Cellular Targeting
An alternative way to incorporate ligands into polyplexes uses nucleic-acid-binding
domains derived from transcription factors to bind a protein component to polyplexes
(e.g. GAL4).
Cell binding, cell activation, and NP internalization are strongly influenced by the
selection of the ligand.
The combination of two or more ligands is an effective way for intracellular
delivery (e.g. RGD peptide for cell-surface binding and peptide B6 for intracellular
uptake).
Intracellular Delivery
Understanding cellular uptake and intracellular processing of nonviral gene
delivery systems is a key aspect in developing more efficient vectors.
Endocytosis is the major entry pathway into cells that consists of clathrin-
dependent (small particles (<200 nm)) and clathrin-independent pathways (large
18. In many cell types, positively charged polyplexes like PEI/pDNA polyplexes
internalize via adhesion to negatively charged transmembrane heparan sulfate
proteoglycans (HSPGs).
The productive endocytotic pathway varied in different cell types for examples:
• In COS-7 cells, the clathrin-dependent pathway
• In HeLa cells, the lipid-raft-dependent pathway
19. identification of protein transduction domains (PTDs) or cell-penetrating peptides
(CPPs) resulted in subsequent development of intracellular drug delivery (e.g. TATp
and VP22 (a major structural component of HSV-1))
Studies have shown that CPPs facilitate the intracellular delivery of various cargos.
Certain carrier molecules
NLS
The use of cell targeting ligands
Endosomal-releasing agents
20. Depending on the type of nucleic acid molecule (pDNA/ siRNA) and its site
of action, different hurdles have to be taken into account.
Intracellular Trafficking
Endosomal Release
Cytoplasmic and Nuclear Trafficking
Persistence of Gene Expression
The efficiency of gene transfer is greatly compromised by the entrapment and
degradation of the polyplex within intracellular vesicles.
Thus, after cellular internalization, the transferred gene must overcome the
degradation process due to pH changes within the vesicles and enzymatic
degradation.
21. Several strategies have been developed to ensure the protection and release of
polyplexes from intracellular vesicles:
• Lysosomotropic Agents and Fusogenic Peptides
• Photochemical Membrane Disruption
• Cationic Proton Sponge Polymers
Lysosomotropic agents are weak-base amines that can specifically inhibit lysosomal
function (e.g. ammonium chloride and alkylamines, Chloroquine)
pH rise and ion exchange are the most common mechanisms of lysosomotropic agents
triggering the release of polyplexes.
Fusogenic peptides, normally consisting of amphipathic sequences, are incorporated
into polyplexes (e.g. N-terminus of influenza virus hemagglutinin subunit HA-2,
melittin, GALA )
Under acidic pH within the endosome, the amphipathic sequences can interact with
lipid membranes inducing rupture of membranes resulting in release of polyplexes
into the cytosol
22. Photochemical Membrane
Disruption
Photochemical internalization (PCI)
is an approach to promote
endosomal release of
macromolecules into the cytosol
based on the use of photosensitive
compounds (e.g. phthalocyanine).
Cationic Proton Sponge Polymers
The two proton sponge polymers
PEI and PAMAM are
representatives of dynamic cationic
polymers that possess intrinsic
endosomolytic activity.
Not every cationic proton sponge is
an effective gene transfer vector.
23. Intracellular Trafficking
Endosomal Release
Cytoplasmic and Nuclear Trafficking
Persistence of Gene Expression
Cytoplasmic Trafficking
A limiting factor for cytoplasmic migration is the size of polyplexes.
Three transport phases were identified that were characterized by:
very slow actin-cytoskeleton-mediated movement (I)
increased velocities with normal diffusion (II )
very fast active transport of polyplexes within vesicles along the microtubules (III)
Nuclear Entry
The Possibility of polyplexes transfer to nucleus:
The entrance of polyplexes to nucleus via NPC
partial dissociation of polyplexes and transfer
passive nuclear uptake
•Cytoplasmic Trafficking
•Nuclear Entry
•Vector Unpacking
NLSs
• Cross-linking
• Electrostatic interaction
decrease transcription activity
the usage of a peptide nucleic acid
(PNA) as a bifunctional linker
DNA nuclear-targeting sequence
(DTS)
24. Intracellular Trafficking
Endosomal Release
Cytoplasmic and Nuclear Trafficking
Persistence of Gene Expression
Several factors threaten the persistence of transferred pDNA within the
nucleus:
1) degradation by intranuclear nucleases
2) pDNA loss (solutions: episomal vectors (EBV Ori P, EBNA-1) or insert pDNA into the
host genome (LTR sequences, integrase protein ))
3) loss of transfected cell due to apoptotic, inflammatory, or immune response
4) gene silencing by transcriptional shutoff
5) inefficient intranuclear trafficking
The inhibition of gene expression by RNA interference a high potential for
application in therapy of human diseases but these effects are only transient.
25. Characteristics of carriers that are required to overcome both extracellular
and intracellular barriers:
a) adaptable to any type of nucleic acid molecule
b) able to self-assemble with nucleic acids
c) viable carrier should avoid undesired interactions and degradation processes
(Bioresponsiveness and shielding)
d) easily escape from endosomes
e) when required, such in the case of pDNA, traffic through the cytoplasm and deliver
pDNA into the nucleus
f) stability and solubility
g) Non toxicity
h) The incorporation of ligands into carrier molecules (for cell targeting and
intracellular uptake)
26. Different strategies of designing functional polymers for polyplex formation:
(1) polyplex surface shielding (PEG or other hydrophilic polymers)
(2) interaction with lipid bilayers
(3) polyplex stability
Surface shielding of polyplexes results in:
(1) a prolonged blood life by providing protection from clearance
(2) decreased interaction with blood proteins
(3) protection from enzymatic degradation
(4)decreased transfection efficiency due to reduced polyplex interaction with target cell surface
and diminished endosomal release and irreversible stable surface shielding
Bioresponsive deshielding strategies are mediated by environmental changes such
as pH (pH-labile linkages), enzymatic activity (e.g. MMPs) ,or disulfide reducing
potential.
Crucial for intracellular delivery and endosomal escape is the interaction of
polyplexes with lipid bilayers.
27. Anionic lipids can be used to mask the undesirable positive surface charge of
polyplexes.
Lipidation of polyplexes generally reduces their toxicity and increases transfection
efficiency.
The variation in surrounding conditions outside the cell, within endosomal vesicles,
the cytosol, and the nucleus can be advantageous with regard to managing a
controlled dissociation of polyplexes to release nucleic acids.
To find a compromise between nucleic acid protection and release, variations
within molecular weight or length of the polymer chains were considered.
Results identified that transfection efficiency does not necessarily increase with a
higher nucleic acid/polymer affinity. Therefore, optimal conditions must be found.
28. Numerous pDNA and siRNA have shown encouraging anticancer effects in
vivo.
The therapeutic effects were aiming at:
• interfering with neoangiogenesis
• reducing tumor cell proliferation
• induction of apoptosis
• activation of the immune system
However, further improvements of nonviral vectors concerning stability,
biodegradability, toxicity profile, tissue specificity, and transfection
efficiency must be fulfilled for successful implementation into systemic
application of gene therapeutics.