Receptor-Targeted Receptor-Targeted Polyplexes for Polyplexes for

pDNA and siRNA pDNA and siRNA Delivery Delivery

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.

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.

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.

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

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

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

histones and protamineProtein-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.

siRNA versus pDNA: Similarities and Differences in Deliverystructural 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.

Physical Restrictions of PolyplexesNanoparticle (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) ratiolarge particle sizes and a high aggregation tendency were observed at a low N/P ratiohigh 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.

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 shieldingincrease of solubilityreduction of interaction with blood cells and serum proteinsimprovement of biocompatibilityincreased blood circulation timeOne way to avoid interactions with the extracellular matrix is the reduction of polyplex charge ratio close to neutrality.

Inflammatory and Immunological ResponsesThe 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.

Cellular TargetingThe 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)

Cellular TargetingAn 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 DeliveryUnderstanding 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 particles (>500 nm)).

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

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

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.

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

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.

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)

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 nucleases2) 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 response4) gene silencing by transcriptional shutoff5) 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.

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 acidsc) 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 solubilityg) Non toxicityh) The incorporation of ligands into carrier molecules (for cell targeting and

intracellular uptake)

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.

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.

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.

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