si-rna delivery

Gene Delivery Slide 1 of 100 December 2013

Tehran University of Medical SciencesSchool of Pharmacy

siRNA DeliveryAn Approach to Cancer Therapy



Definitions

Gene therapy refers to the transmission of nucleic acid (DNA and RNA) encoding a therapeutic gene of interest into the targeted cells or organs with consequent expression of the transgene

Gene Delivery is the process of introducing foreign genetic material into host cellsIn other words Gene Delivery is a key step to gene therapy

A Gene Delivery System (GDS) is a special purpose conjugate that consists of carry-over material and nucleic acids (payload)



Gene Therapy ApproachesSeveral Approaches may be used for correcting faulty

genesndash A normal gene may be inserted into the genome to compensate

for nonfunctional gene (the most common approach)ndash An abnormal gene is replaced for a normal genendash The abnormal gene could be repaired through selective reverse

mutationndash The regulation of a gene expression could be altered



Barriers to Gene Delivery In order to deliver genes into cell nucleus several barriers need to

be overcome

Extracellular barriersndash Nucleasesndash Plasma Proteins (aggregation)ndash Immune systemndash Glomerular filtration

Cellular barriersndash Cell Membranendash Endolysosomal entrapmentndash Cytosolic Sequestrationndash Nuclear Exclusion



Challenges to Gene Delivery Off-target Effect

siRNAs are capable of reducing expression of nontarget genes due to interaction of the siRNA guide strand with a partially complementary site on an ldquooff-targetrdquo mRNA

Careful selection of siRNA sequences to avoid off-target effects is an important issue



Challenges to Gene Delivery Immune stimulation

Innate immune activation is a significant undesirable side effect due to the toxicities associated with excessive cytokine release

The inflammatory response is mediated by toll-like receptors (TLRs) which are located in endosome compartments and recognize unmethylated CpG DNA as well as various moieties in double-stranded RNA or their degraded products

TLR-mediated recognition and concomitant immune stimulation can be inhibited by chemical modifications such as introduction of 2prime-O-methyl (2primeOMe)-modified nucleotides



Challenges to Gene DeliverySerum inactivation and enzyme degradation

Naked genetic material are rapidly degraded by nuclease in the serum

2prime-hydroxyl group of the ribose ring is not necessary for gene silencing by siRNAs common modifications are 2prime-fluoro 2prime-O-methyl and 2prime-amine conjugations

Naked genetic material lack specific tumor targeting and would be quickly excreted by the kidney upon systemic administration



Challenges to Gene DeliveryRES Recognition

In order to condense negatively charged nucleic acids into delivery vehicles most vehicles are cationic

The positive charge aids cellular uptake but also promotes nonspecific interactions with non target cells and extracellular components such as serum proteins and extracellular matrix

Binding of proteins is the primary mechanism for RES recognition

The most common way to decrease nonspecific interactions is to shield the nanoparticle surface with hydrophilic uncharged polymers such as polyethylene glycol (PEG)



Challenges to Gene DeliveryEntrance Into Cells

Delivery systems that are not degraded phagocytosed or cleared by the kidney must leave the bloodstream by traversing the endothelium to reach other tissues This occurs most readily in tissues whose endothelia are discontinuous (fenestrated) such as the liver and many solid tumours

Most siRNA delivery systems undergo cellular internalization through endocytosis

Various delivery systems aim to improve the rate of cellular uptake by incorporating targeting ligands that bind specifically to receptors on target cells to induce receptor-mediated endocytosis

Other systems use cell-penetrating peptides that can induce cell uptake through endocytosis or non-endocytic mechanisms




Commonly used targeting ligands include ndash Aptamersndash Cell penetrating peptidesndash Antibodiesndash Peptides or proteinsndash Small molecule ligands



Challenges to Gene DeliveryEndosome Escape

The delivery of nonviral gene vehicles almost invariably involves endocytosis and escaping from endosomes before they traffic into lysosomes is an essential step to avoid enzymatic degradation

Cationic lipid complexes can bind to anionic lipids on the endosome membrane and form neutral ion pairs These ion-pairs destabilize the endosome membrane and promote de-assembly of the lipoplex

Polymers and peptides with high buffer capacity between pH 72 and 50 such as PEI or peptides containing the cationic amino group lysine arginine and histidine could buffer the endosome This would cause more protons to enter into the endosomes followed by chloride ions leading to increased osmotic pressure and endosome rupture (ldquoproton sponge effectrdquo)

Stimuli other than pH have been used to destabilize endosome membranes Lipid or polymer derivatives which are sensitive to sulfhydryl reduction and enzymatic cleavage have been used to construct nonviral gene vectors






Challenges to Gene DeliveryNuclear Entry

The final target destination of antisense oligonucleotides siRNAmiRNA and mRNA is the cytoplasm whereas pDNA must be transported into the nucleus for gene expression

Nuclear transport generally occurs through nuclear pore complexes(NPCs) however nucleic acid condensates are impermeable through nuclear pore complexes due to their large size

In dividing cells the nuclear envelope disassembles during mitosis pDNA transfection can only occur at this stage

For nondividing cells the mechanisms of DNA nuclear transport are of critical importance



Nuclear Pore Complex



The NPC is freely permeable to small molecules metabolites and ions but acts as a highly efficient molecular sieve for macromolecules

Transport of almost all macromolecules into and out of the nucleus is achieved requires the assistance of soluble nuclear transport factors (NTFs) and transport signals

The NTFs specifically bind transport signals found on their cognate substrates and translocate them through the NPC channel

The best-studied transport signals are found on nuclear protein cargoes Such signals consist of short amino acid sequences called nuclear localization sequences (NlSs for import) or nuclear export sequences (NESs for export)





To facilitate nuclear targeting many nuclear localization signal (NLS) peptides have been studied to allow DNA nuclear entry through nuclear pore complexes by active transport

NLSs are short clusters of amino acids that can bind to cytoplasmic receptors known as importins NLS peptides can bind to DNA either through noncovalent electrostatic interaction or by covalent attachment

The most well-known and popularly used NLS is from the large tumor antigen of simian virus 40 (SV40)

Some DNA sequences themselves have nuclear import activity based on their ability to bind to cell-specific transcription factors



Gene Delivery Systems The most important and most difficult challenge in gene therapy is

the issue of delivery

Not only must the therapy evade the RES as it circulates after systemic administration but it must also cross several barriers before it arrives in the cytoplasm or nucleus of its target cells

So there is a need to develop a safe and efficient gene transfection therapy system

Carry-over material of a GDS can pass through the cell membrane and in some instances enter the nucleus

Carry-over material are called vectors



Vectors

A vector can be described as a system fulfilling several functions including

ndash Enabling delivery of genes into the target cells and their nucleus ndash Providing protection from gene degradationndash Ensuring gene transcription in the cell

Gene therapy vectors should not only be targeted and safe but also protected from degradation sequestration or immune attack

However it has to be inexpensive and easy to produce and purify in large amounts and at high concentrations



Categorization

Gene delivery systems can be classified into two categories based on the origin of the gene carrier

ndash Viral gene delivery systems use recombinant viruses as carriersndash Non-viral gene delivery system

bull Physical (carrier-free gene delivery)bull Chemical (synthetic vector-based gene delivery

Each vector has its own advantages and disadvantages

None of these types of vectors has been found to be ideal for both safe and efficient gene transfer and stable and sufficient gene expression



Viral Vector Delivery Systems

Viral techniques use various classes of viruses for gene delivery

Viruses introduce their genome into the cells with high efficiency

However there are limitations including a strong immune response triggered by the expression of viral genes oncogenic insertions into the genome and unstable maintenance of viruses in the host cell

Gene therapy vectors are being developed by genetic modification of retroviruses adenoviruses poxviruses parvoviruses (adeno-associated viruses) herpesviruses and others



Viral Vector Replication These vectors are engineered by deleting the essential genes

which allow replication assembling or infection

But on the other hand vectors need to be produced in large amounts of virus particles

Specialized cell lines called ldquopackaging cell linesrdquo (PCLs) engineered to replace a function of a deleted viral gene Removed virus genes are inserted into the genome of the packaging cells and can be expressed there

The process by which the function of the deleted viral gene is supplied by a protein encoded in the PCL genome is called ldquocomplementationrdquo



Viral Vectors



Non-Viral Delivery Systems Non-viral techniques of gene transfer represent a simple and more

importantly safer alternative to viral vectors

Because of their relatively simple quantitative production and low immunogenicity these vectors are attractive tools in gene therapy

Ongoing studies and the development of new vectors that show transfection efficiency just like that of viral vectors point towards their great potential

The main advantage is a virtually unlimited clone capacity low toxicity and immunogenicity of non-viral vectors and the ability of repeated application



Challenges to Non-viral Gene Delivery

Viruses are naturally occurring being which are specialized for transfection

So they inherently can pass the barriers to gene delivery and transfer their genetic material into host

For non-viral delivery systems these abilities should be designed in the vector



Non-viral Gene Delivery Systems Non-viral gene delivery system can be categorized to

ndash Physical (carrier-free gene delivery)bull Naked DNAbull Direct Injectionbull Electroporationbull Gene-Gun

ndash Chemical (synthetic vector-based gene delivery)bull Lipoplex Vectorsbull Polyplexesbull Dendrimeric Vectorsbull Polypeptide Vectorsbull Inorganic Nanoparticles



Non-Viral Delivery Systems Lipoplexes and Polyplexes

Synthetic vectors improving the admission of DNA into the cell and protecting it from undesirable degradation were created

The most used are derived from lipids or synthetic polymers

Plasmid DNA can be covered by lipids into organized structures such as liposomes or micelles This complex of DNA with lipids is called a lipoplex

Vectors based on a complex of polymers with DNA are called polyplexes



LipoplexesLipoplexes can be divided into two types

ndash Anionic and neutral liposomes are characterized by safety compatibility with body fluids and the possibility of tissue-specific gene transfer but the level of transduced cell expression is relatively low

ndash Cationic and Ionizable liposomes naturally create complexes with negatively charged DNA Their positive charge allows interactions with the negatively charged cell membrane

Targeted transfection can be gained to some extent by the addition of tissue-specific target ligand



Polyplexes

Most of Polyplexes consist of cationic polymers and their production is regulated by ionic interactions

In contrast to Lipoplexes some Polyplexes (poly lysin) are not able to release intracellular DNA into the cytoplasm

Polymers such as polyethylenimine have a mechanism of endosome disruption and there is thus no need for transfection with endosome-lytic agents



Polyplexes Poly(L-Lysine) or PLL Polylysine has an exceptional capacity to condense DNA

At physiological pH all primary-amino groups of PLL are protonated yielding a structure with no buffering capacity to aid in endosomal escape

Polylysine structures with molecular weightsgt3000 Da can effectively condense DNA indicating the significance of primary amine number for complex formation

But these heavier PLLs exhibit relatively high cytotoxicity

This toxicity has been reduced with the incorporation of imidazole group into the PLL chain or through the use of dendritic PLLs



Polyplexes Poly(L-Lysine) or PLL High MWt PLLDNA complexes also have a tendency to aggregate

and precipitate depending on the ionic strength of the solution

One method used to overcome the formation of insoluble precipitates is to form block copolymers of PLL with PEG

To overcome nonspecific cell targeting various research groups have derivatized PLL with targeting moieties



Polyplexes Polyethylenimine (PEI) Polyethylenimine is often considered the gold standard of gene

transfection

Polyethylenimine exists as both a branched and linear structure

The transfection efficiency of PEI is due at least in part to the ldquoproton spongerdquo nature of it

At a physiological pH approximately 80 of the amines remain unprotonated compared to less than 50 unprotonated nitrogens at a pH of 5

This buffering capacity allows PEI polyplexes to avoid lysosomal trafficking and degredation once inside the cell



Polyplexes Polymethacrylate Due to its inherent cationic charge poly[2-(dimethylamino)ethyl

methacrylate] (PDMAEMA) offers significance as a gene transfer agent

The successful in vitro transfection efficiency of PDMAEMA polyplexes is attributed to the ability of the polymer to destabilize endosomes as well as to dissociate easily from the plasmid once delivered into the cytosol

The mechanism of gene transfer for methacrylate polyplexes has been shown to proceed by both clathrin- and caveolae-dependent pathways

Caveolae-dependent uptake appears to be vital for effective gene transfer of PDMAEMA polyplex



Carbohydrate Based Polymers β-cyclodextrins

To overcome the cytotoxicity incorporating β-cyclodextrin into cationic polymers has been studied

The length of the alkyl chain(n) between cyclodextrin monomer units affects polyplex cytotoxicity

ndash Cytotoxicity generally decreases with longer chain lengths (decreased charge density)

ndash With alkyl chain lengths (n) ranging from 4 to 10 cytotoxicity was lowestndash Transfection efficiency was highest with 6 7 or 8 methylene units ndash The high toxicitylow transfection efficiency of the polymer with 10 methylene

units was attributed to lower solubility

Like most other cationic vector systems in vivo use of β-cyclodextrin is hindered by aggregation at high ionic strengths While PEGylation can generally reduce the formation of aggregates



Carbohydrate Based Polymers Chitosan

The biodegradability biocompatibility and cationic potential of chitosan has helped it become one of the most prominent naturally derived nonviral vectors for gene transfer

Chitosan is produced by deacetylation of chitin to form a polymer composed of D-glucosamine and N-acetyl D-glucosamine subunits linked by β(14) glycosidic bonds

Molecular weight of chitosan polymers can strongly influence gene transfer efficiency

Regardless of the increased polyplex size high molecular weight chitosan forms more stable complexes with DNA due to a chain entanglement effect




In addition to molecular weight several other factors have been shown to affect the transfection efficiency of chitosan polyplexes including the NP ratio pH and the degree of deacetylation

Optimal transfection efficiency of chitosan polyplexes can be achieved between pH 68 and 70

Above pH 75 DNA was shown to dissociate from the complex

Below pH 65 cellular uptake was significant but transfection efficiency was low possibly due to hindered endosomal release



Dendrimer Based Vectors PAMAM Polyamidoamine Dendrimers (PAMAM)

ndash Due to ease of synthesis and commercial availability they have become the most utilized dendrimer-based vectors for gene transfer

ndash High generation PAMAM dendrimers (G5-G7) induce lipid mixing and leakage from phospholipid vesicles Which was attributed to the ability of the spherical PAMAM structure to bend the anionic membrane through electrostatic forces and induce packing stresses leading to lipid mixing

ndash Following cellular uptake endosomal chloride accumulation increased significantly and the pH also increased indicating the occurrence of endosomal swellinglysis (proton sponge effect)

ndash Various alterations to the basic PAMAM dendrimer structure have been investigated in an effort to improve transfection efficiency or PAMAMDNA complex formation



Polypeptide Vectors Peptide-oligonucleotide conjugates offer a strategy of delivering

genetic material into cells with high efficiencies and cell-specificity

These amino acid sequences called cell-penetrating peptides(CPPs)

are generally divided into two classesndash lysine-rich peptides such as the amphipathic MPG peptide and transportanndash arginine-rich peptides such as the homeodomain of antennapedia (Antp) and

trans-activating transcriptional activators (TAT)

Generally the peptide is covalently linked to the oligonucleotide construct rather than complexed via electrostatic interactions (Complexation of peptides and siRNA can be either electrostatic or covalent)



Polypeptide Vectors TAT-Based Peptides

TAT protein is an 86-102 amino acid sequence organized into three domains

ndash cationic regions involved in controlling the rate of gene expressionndash cysteine-rich regions involved in DNA binding andndash basic amino acid regions involved in promoting the crossing of the membrane

The cellular uptake of free Tat-peptides has been shown to proceed by an energy-independent pathway but the transfection of Tat-DNA complexes may proceeds by endocytosis

As an example By covalently attaching the Tat-peptide with anti-MDR antisense oligonucleotide it was shown that in vitro P-glycoprotein expression could be significantly inhibited



Polypeptide Vectors Antennapedia Homeodomain Peptide

Antennapedia homeodomain is a 60-amino acid polypeptide corresponding to the Drosophilia melanogaster antennapedia homeobox sequence

While the third alpha-helix of the pAntp is involved in promoting translocation the 60-amino acid structure could be reduced to a 16-mer peptide

Cellular uptake of pAntp proceeds by a nonendocytic pathway(Even at 4ordmC)

The replacement of several amino acids with proline disrupted the alpha-helical structure of pAntp but did not hinder cellular uptake suggesting that the alpha-helical conformation is not required



Polypeptide Vectors MPG peptide

The MPG peptide is a synthetic compound containing a hydrophobic N-terminal region derived from the fusion sequence of HIV and a hydrophilic region derived from the NLS of the SV40 large T antigen

Complexation of MPG with oligonucleotides proceeds via electrostatic interaction between the basic residues of the NLS region and the phosphonate backbone of the oligonucleotide

When MPG peptide is complexed with oligonucleotides the hydrophobic region partially folds into a β-sheet structure which promotes cellular uptake by inserting into the plasma membrane and forming a transmembrane pore-like structure



Mechanisms of membrane translocation

Theories of CPP translocation can be classified into three main entry mechanisms

Direct penetration in the membranendash Most likely involved a direct electrostatic interaction with negatively

charged phospholipids ndash interactions between cell-penetrating peptides and the phosphate groups on both

sides of the lipid bilayer the insertion of charged side-chains that nucleate the formation of a transient pore followed by the translocation of cell-penetrating peptides by diffusing on the pore surface

Endocytosis-mediated entry

Formation of a transitory structurendash Formation of the inverted micellesndash Transmembrane structures



Inorganic Nanoparticles

The application of inorganic nanoparticles in gene delivery is an emerging field too because they can be prepared and surface-functionalized in many different ways Examples of such systems are

ndash Metallic nanoparticles (Gold nanoparticles are inert and are easily functionalized)

ndash Iron oxide (superparamagnetic cytotoxic used in polymer-coated form)

ndash calcium phosphate magnesium phosphate manganese phosphate

ndash carbon nanotubes (are surface functionalized and used)

ndash quantum dots (employed for tracking of delivery systems)



Nanoparticles Iron Oxide Nanoparticles Iron oxide nanoparticles (IONPs) superparamagnetic material

sized between approximately 1 and 100 nm have a long history of investigation

In order to produce IONPs that are highly efficient for gene delivery the IONPs should have an enhanced cationic surface with cationic polymers such as PEI PLL and chitosan

PEI-IONPs are limited for in vivo applications due to cellular toxicity

In order to overcome cellular toxicity various polymers have been utilized to coat or conjugate to the IONPs (PEG for example)



Nanoparticles Carbon nanotubes Carbon nanotubes can be used as a GDS when they are

chemically modified

They can be either covalently functionalized by oxidation and subsequent 1 3-dipolar cyclo-addition reaction or non-covalently functionalized with hydrophobic or π-π stacking between the CNT and another non-polar ring such as the backbone of DNA

In vitro and in vivo studies have suggested that PEG-modified CNTs have favorable pharmacokinetic and toxicology profiles



Nanoparticles Silica nanoparticles Silica nanoparticles (SiNPs) have been used as drug and gene

delivery agents because they can be easily modified

SiNPs need to be modified with an anchoring group and charge transfer functional group to allow for DNA binding by electrostatic interactions for efficient cellular delivery

The regular arrangement of pores or hollow cavities in the silica nanoparticles easily accommodates siRNA molecules

Silica materials are usually toxic but their toxicity may be reduced through surface modifications For example surface-coating with PEI makes positively charged SiNPs which can pass through the cell membrane more easily and have significantly reduced toxicity



Nanoparticles Gold nanoparticles Gold nanoparticles (AuNPs) consist of colloidal gold suspended in

liquids sized from 1 to 150 nm

Gold nanoparticles have emerged as an attractive and widely used nanomaterial for GDSs because they are inert and essentially nontoxic to cells

Furthermore AuNPs can be easily functionalized by anchoring thiol linkers Conjugate materials used for facilitating cellular uptake include peptides proteins antibodies oligosaccharides and nucleic acids



Nanoparticles Gold nanoparticles Antisense oligodeoxynucleotide (ASODN)-modified gold

nanoparticles have higher affinity for complementary nucleic acids than their unmodified oligonucleotide counterparts

Furthermore AuNP-ASODNs are less susceptible to degradation by nuclease activity exhibit greater than 99 cellular uptake and are less toxic to the cells under the studied conditions



Challenges in Non-viral GDS Non-viral GDSs lack mechanisms for integration into the host

chromosome

So the expression of delivered genes will be transient because it gradually becomes inactivated

One the inactivation mechanisms is so-called ldquogene dilutionrdquo This process takes place mainly in dividing cells with a plasmid vector that is not integrated and the number of copies of extrachromosomal DNA reduces with each cell cycle



Challenges in Non-viral GDS For this reason combination of DNA transposon-based vectors is

required for gene delivery

A transposon is a DNA sequence that can change its position within the genome through a direct cut-and-paste mechanism

A simple transposon is organized by terminal inverted repeats embracing a gene encoding transposase an enzyme required for its transposition

Engineered DNA transposons have the desired features of naked DNA and plasmidsas well as the ability to insert transgenes into host chromosomes for long-term transgene expression



Transposition



RNA Interference RNA interference (RNAi) gained international attention in 1998

when Fire Mello and colleagues discovered the ability of double-stranded RNA to silence gene expression in the nematode worm Caenorhabditis elegans

Three years later Tuschl et al published their experiment demonstrating that synthetic siRNA could achieve sequence-specific gene knockdown in a mammalian cell line

The first successful use of siRNA for gene silencing in mice was achieved for a hepatitis C target shortly thereafter



RNA Interference There are numerous classes of small non-coding RNA in eu Cells

involved in RNA splicing editing and modification (catalytic RNAs)

Very small RNAs or micro RNAs (miRNA) are gen expression regulators found in most eu cells

Human genome hast an estimated 1000 genes that code for miRNAs that participate in RNA interference(RNAi) (half from coding genes and half from ncRNAs or even pseudogenes)

This is a general mechanism to repress gene expression usually but not always at the translation level



RNA Interference These miRNAs go by a number of names and are sometimes

called short temporal RNAs (stRNA) because of their role in development

Piwi-associated RNAs(piRNA) are found in germ cells

Small interfering RNAs(siRNA) are typically produced during a viral infection

Both can be used to control the expression of transposable elements



RNA Interference RNAi is triggered by the presence of long pieces of double-stranded

RNA which are cleaved into the fragments known as siRNA (21ndash23 nucleotides long) by the enzyme Dicer

In practice siRNA can be synthetically produced and then directly introduced into the cell thus circumventing Dicer mechanics








Once siRNA is present in the cytoplasm of the cell it is incorporated into a protein complex called the RNA induced silencing complex(RISC) composed of argonauts(ago) protein family

RISC has endonuclease activity that cleaves the passenger strand the one which will not be used in the duplex siRNA or miRNA



RNA Interference The degree of base pairing and the sequence of the ends

The RISC complex is now in a position to use the mature siRNA to guide it to its target mRNA

siRNA searches mRNAs for small regions of homology usually found in AU-reach region in 3rsquo-UTR

Two primary mechanisms used to control mRNA expression ndash Degredation of the mRNA (common in plant cells)ndash Inhibition of translation (common in animals)

The choice is primarily determined by the degree of base-pairing between the siRNA and mRNA The higher base paring the more likely that the target mRNA will be degraded



RNA Interference The activated RISC complex can destroy additional mRNA

targets which further propagates gene silencing This extra potency ensures a therapeutic effect for 3ndash7 days in rapidly dividing cells and for several weeks in non-dividing cells

Theoretically when using appropriately designed siRNA the RNAi machinery can be exploited to silence nearly any gene in the body giving it a broader therapeutic potential than typical small-molecule drugs

In order for these advances to be implemented in a clinical setting safe and effective delivery systems must be developed



Delivery Systems Naked chemically modified siRNA has shown efficacy in certain

physiological settings such as the brain and the lung

But most tissues in the body require an additional delivery system to facilitate transfection(enzymes large size(~13 kDa) too negatively charged)

The issue of effective and non-toxic delivery is a key challenge and serves as the most significant barrier between siRNA technology and its therapeutic application



Delivery Systems localized siRNA delivery

ndash Application of siRNA therapy directly to the target tissuendash Several tissues are amenable to topical or localized therapy

including the eye skin mucus membranes and local tumorsndash well-suited for the treatment of lung diseases and infections

The direct instillation of siRNA into the lung through intranasal or intratracheal routes



siRNA Modification Chemical modifications can be introduced

into the siRNA molecule to evade immune defense and nucleases in vivo

Incorporation of 2prime-O-methyl or 2prime-Fluoro or 2-methoxyethyl modifications into the sugar structure of selected nucleotides within both the sense and antisense strands

Replacement of the phosphodiester(PO4)group with phosphothioate(PS) at the 3-end

The locked nucleic acid substitution containing a methylene linkage between the 2- and 4-positions



Polymer Vectors siRNA differs from pDNA in Mwt charge ratio stability and action

However both are nucleic acids

Polymer-mediated DNA delivery systems would provide a lot of knowledge for the development of polymer-based siRNAs

Cationic polymers usually form a complex with negatively charged siRNA upon simple mixing

Categoriesndash Synthetic PEI PLL cyclodextrin-based polycationsndash Natural Chitosan atelocollagen and polypeptides



Cyclodextrin Polymer Vectors Cyclodextrin polymers are polycationic oligomers (n asymp 5)

synthesized by a step-growth polymerization between diamine-bearing cyclodextrin monomers and dimethyl suberimidate yielding oligomers with amidine functional groups

The strong basicity of these amidine groups mediates efficient condensation of nucleic acids

End-capping of the polymer termini with imidazole functional group can aid endosomal escape

Both adamantanendashPEG (ADndashPEG) and adamantanendashPEGndashtransferrin (ADndashPEGndashTf) were incorporated to improve particle properties



Cyclodextrin Polymer Vectors






Lipid Vectors Liposomes studied as delivery vectors for DNA-based drugs

because of their ability both to protect entrapped oligonucleotides from nucleases and renal clearance and to promote cellular uptake and endosomal escape

Cationic and ionizable lipidsndash Improve the entrapment of the negatively charged siRNA increase cellular

uptake and aid endosomal escapendash Several studies have determined that cationic lipids are less efficacious and

more toxic than ionizable lipids whose charge is dependent on the pHndash Thus recent work has focused on the development of new ionizable lipids ndash The composition of these lipids is generally divided into three parts

an amine head group a linker group and hydrophobic tails



Lipid Vectors Ionizable Lipids To minimize toxicity without sacrificing efficacy the pKa of an

ionizable lipid should be low enough for it to remain unprotonated during circulation but high enough for it to become protonated in either the early or late endosome (54-76)

Lipid transition temperature refers to the temperature at which lipid membranes shift from the more stable lamellar phase to the less stable hexagonal phase

This transition promotes destabilization of the endosomal membrane and release of siRNA



Lipid Vectors Ionizable Lipids Lipids with lower transition

temperatures which more readily shift from lamellar to hexagonal to promote endosomal release have small polar head groups and large unsaturated hydrophobic tails Lipids with large polar head groups and fully saturated hydrophobic tails are more likely to adopt the stable lamellar phase Successful lipid formulations are engineered to remain in the lamellar phase during circulation and to transition to the hexagonal phase within endosomal compartments



Lipid Vectors Shielding lipids Lipid-anchored PEG is a common component in liposomes PEG groups serve many purposes they prevent aggregation

increase circulation time and reduce uptake by unintended targets such as RBCs and macrophages

Shielding lipids can reduce cellular uptake and efficacy After endocytosis PEG can sterically and electrostatically block the

interaction between the liposome and the endosomal membrane hindering membrane fusion and preventing endosomal release

One strategy for improving the efficacy of PEGylated nanoparticles involves incorporation of acid-sensitive bonds connecting PEG to the liposome



Lipid Vectors Shielding Lipids Another method to reduce the negative effects of shielding involves

the use of a pH-sensitive modified PEG that binds to liposomes through ionic interactions

The liposomal core consists of 12-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) as well as 2-hydroxyethyl methacrylate(HEMA)ndashlysine-modified cholesterol

PEG is covalently modified with HEMAndashhistidinendashmethacrylic acid

At neutral pH the PEG copolymer has a net negative charge whereas the liposomal core has a net positive charge In the endosome imidazole and methacrylic acid residues become protonated and the net charge of the PEG becomes positive



Lipid Vectors Cholesterol Many liposomal formulations include cholesterol which can

associate with lipid bilayers Increase in the cholesterol content lowers the transition temperature

of liposomal membranes containing conical-shaped lipids



Lipid Vectors Targeting ligands

To improve the biodistribution of liposomes many formulations use endogenous or exogenous targeting ligands

ndash Endogenous targeting ligand often a serum protein binds to the liposome during circulation and directs it to the ligandrsquos natural target

bull The lipoprotein ApoE has been used as an endogenous targeting ligand by DLinndashKC2ndashDMA-based ionizable liposomes

bull Retinol binding protein (RBP) is also used an endogenous targeting ligand This serum protein binds vitamin A and transports it to cells expressing the RBP receptor including hepatic stellate and pancreatic stellate cells

ndash Exogenous targeting ligands are added to liposomal formulations before injection to bind desired surface proteins on target cells

bull folate which has been used to target delivery to rapidly dividing cancer cells is an example of exogenous ligands



Lipid Vectors SNALPs For in vivo siRNA delivery stable nucleic acidndashlipid particles

(SNALPs) have been formulated

A SNALP consists of a lipid bilayer containing a mixture of cationic and fusogenic lipids that enables the cellular uptake and endosomal release of siRNA

The surfaces of SNALPs were coated with a polyethylene glycolndashlipid conjugate that provides a neutral hydrophilic exterior and stabilizes the particle during formulation



Conjugate delivery systemsDirectly conjugating delivery material to the siRNA cargo

The first conjugate delivery systems to show efficacy in vivo consisted of siRNA conjugated to cholesterol and other lipophilic molecules

Other conjugate delivery systems have been developed by attaching siRNA to polymers peptides antibodies aptamers and small molecules

ndash Dynamic PolyConjugatesndash GalNAcndashsiRNA



Conjugate Delivery Systems Dynamic Polyconjugates

siRNAndashpolymer conjugate delivery systems designed to respond to intracellular environments

The siRNA cargo is attached to a membrane-disrupting polymer by a hydrolysable disulphide linker cleaved in the reducing environment of the cytosol releasing the siRNA from the delivery polymer

The activity of the polymer is masked by PEG side chains while the system is in circulation The PEG is designed to be shed in the acidic environment inside the endosome

To induce uptake by target cells through receptor-mediated endocytosis ligands are incorporated GalNAc ligands which bind to the asialoglycoprotein receptor (ASGPR) on hepatocytes

the siRNA itself is chemically modified to improve nuclease stability and to reduce off-target effects






Conjugate Delivery Systems Triantennary GalNAcndashsiRNA

In this system the 3ʹ terminus of the siRNA sense strand is attached to three GalNAc molecules by means of a triantennary spacer

Multivalency of the sugar ligand greatly improves cell uptake and spacing of the sugar moieties also plays a role In a study of triantennary galactose ligands binding affinity increased with spacer length over a range of 4ndash20 Aring



Oligonucleotide Vectors The construction of three-dimensional nanoparticles of defined

composition from nucleic acids has generated interest ndash Molecularly identical nanoparticles with strictly defined characteristics

Oligonucleotide nanoparticles (ONPs) were composed of complementary DNA fragments designed to hybridize into predefined three-dimensional structures

constructing DNA tetrahedra was adapted by incorporating single-stranded overhangs on each edge

Short interfering RNAs were modified by extension of the 3ʹ sense strands with DNA overhangs



Oligonucleotide Vectors



Oligonucleotide Vectors Oligonucleotide nanoparticles modified with folate ligands were

used to study the minimum number of targeting ligands required

These questions are difficult or impossible to address using many other nanoparticle systems



Cancer Worldwide between 100 and 350 of each 100000 people die of cancer

each year

Genetic control systems regulate the balance between cell birth and death in response to growth signals growth-inhibiting signals and death signals

The losses of cellular regulation that give rise to most or all cases of cancer are due to genetic damage

Mutations in two broad classes of genes have been implicated in the onset of cancer proto-oncogenes and tumorsuppressor genes

Many of the genes in both classes encode proteins that help regulate cell birth (entry into and progression through the cell cycle) or cell death by apoptosis others encode proteins that participate in repairing damaged DNA



Cancer Seven types of proteins that

participate in controlling cell growth and proliferation

ndash Extracellular signaling molecules (I) ndash Signal receptors (II) ndash Signal-transduction proteins (III)ndash Transcription factors (IV) ndash Apoptotic proteins (V)ndash Cell cycle control proteins (VI)ndash DNA-repair proteins (VII)





Tumor Targeting Can be categorized to Passive Targeting and Active Targeting

Passive Targeting

Enhanced Permeability and Retention (EPR) effectndash For most tumors nanoparticles with a mean size around 100 nm are

attractive for tumor targetingndash Particles larger than 400 nm can not easily enter the capillary gaps in the

tumor vasculature whereas particles smaller than 70 nm are able to access the parenchymal cells in the liver



Tumor Targeting Active Targeting

Grafting markers or ligands at the surface of the delivery system for receptors only expressed or at least overexpressed in tumors

Tumor homing peptides are able to react with different receptorsndash Integrin receptors with RGD peptide (ArgeGlyeAsp)

Alpha V integrin proteins overexpressed on tumoral vesselsndash AsneGlyeArg (NGR) motif interact with aminopeptidase N receptor (CD13) The expression of this receptor was correlated with cancerous angiogenic

property and cell mobilityndash Unsaturated fatty acids folic or hyaluronic acids Antibodies etc



Targets of siRNA in Cancer TherapyCell Cycle Cell cycle progression is strictly controlled by numerous effectors

and checkpoints

Main effectors of cell cycle progression are cyclins and cyclin-dependant kinases (cdk)

Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer

When activated by a bound cyclin CDKs performs phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle



Cell Cycle Control



Targets of siRNA in Cancer Therapy cyclin B1cdk1 complex is the main effector implicated in G2M transition

Numerous studies have proven the overexpression of cyclin B1 in various cancers as oesophageal squamous cancer non-small lung cancer renal cancer prostate adenocarcinoma and breast cancer

Cyclin B1 siRNA inhibition strategy was employed in prostate and lung cancer in vivo with peptide-based delivery systems

Polo-like-kinase1(Plk1) plays key roles during mitosis notably in the activation of cyclin B1cdk1 complex

Plk1 is overexpressed in multiple cancer types including melanoma nonsmall cell lung cancer colorectal cancer and breast cancer

Plk1 siRNA delivery systems are being studied



Targets of siRNA in Cancer Therapy Proliferation pathways

Cancer cells are characterized by sustained proliferation resulting from increased proliferative signals decrease of negative feed-back signals and replicative immortality acquisition

Cancer cells may produce or force their environment to produce excessive growth factors like insulin growth factor (IGF) or epidermal growth factor (EGF)

Such external stimuli activate intracellular cascades like the MAPK pathways or the phosphoinositide-3-kinase (PI3K)serinethreonin kinase AKT one that promotes proliferation division and survival and were clearly described in cancerogenesis mechanism



Targets of siRNA in Cancer Therapy Cell death and survival Pathways involved in cell death and survival have important role in

cancer development

Cancer cells display acquired or innate cell death resistance mechanisms (to apoptosis or other cell death pathways)

Angiogenesis Without angiogenesis cancer could not grow up to more than 2 mm of

diameter

Vascular endothelium growth factors(VEGFs and VEGFA in particular) are the most described secreted molecules implicated in angiogenesis of human cancers

Apoptotic Pathways



Some of Main References












Thank Youfor Your Kind Attention

Slide 1

Definitions

Gene Therapy Approaches

Barriers to Gene Delivery



Challenges to Gene Delivery Serum inactivation and enzyme degr

Challenges to Gene Delivery RES Recognition

Challenges to Gene Delivery Entrance Into Cells

Challenges to Gene Delivery Entrance Into Cells (2)

Challenges to Gene Delivery Endosome Escape

Challenges to Gene Delivery Endosome Escape (2)

Challenges to Gene Delivery Nuclear Entry


Nuclear Pore Complex (2)

Challenges to Gene Delivery Nuclear Entry (2)

Gene Delivery Systems

Vectors

Categorization


Viral Vector Replication

Viral Vectors

Non-Viral Delivery Systems


Non-viral Gene Delivery Systems


Lipoplexes

Polyplexes

Polyplexes Poly(L-Lysine) or PLL

Polyplexes Poly(L-Lysine) or PLL (2)

Polyplexes Polyethylenimine (PEI)

Polyplexes Polymethacrylate



Carbohydrate Based Polymers Chitosan (2)

Dendrimer Based Vectors PAMAM

Polypeptide Vectors






Nanoparticles Iron Oxide Nanoparticles

Nanoparticles Carbon nanotubes

Nanoparticles Silica nanoparticles

Nanoparticles Gold nanoparticles

Nanoparticles Gold nanoparticles (2)

Challenges in Non-viral GDS

Challenges in Non-viral GDS (2)

Transposition

RNA Interference

RNA Interference (2)



Slide 55


Slide 57


Slide 59


Delivery Systems

Delivery Systems (2)

siRNA Modification

Polymer Vectors


Cyclodextrin Polymer Vectors (2)


Lipid Vectors

Lipid Vectors Ionizable Lipids

Lipid Vectors Ionizable Lipids (2)

Lipid Vectors (2)

Lipid Vectors Shielding Lipids

Lipid Vectors (3)

Lipid Vectors (4)

Lipid Vectors SNALPs

Conjugate delivery systems


Conjugate Delivery Systems Dynamic Polyconjugates (2)



Oligonucleotide Vectors (2)


Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)

Targets of siRNA in Cancer Therapy

Cell Cycle Control

Targets of siRNA in Cancer Therapy (2)



Slide 93


Some of Main References (2)

Slide 96

Slide 97

Slide 98

Slide 99

Slide 100



Definitions

Gene therapy refers to the transmission of nucleic acid (DNA and RNA) encoding a therapeutic gene of interest into the targeted cells or organs with consequent expression of the transgene

Gene Delivery is the process of introducing foreign genetic material into host cellsIn other words Gene Delivery is a key step to gene therapy

A Gene Delivery System (GDS) is a special purpose conjugate that consists of carry-over material and nucleic acids (payload)










be overcome


















































































Vectors







Categorization






















Viral Vectors


































Polyplexes




















transfection


































































































chemically modified
























chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100










be overcome


















































































Vectors







Categorization






















Viral Vectors


































Polyplexes




















transfection


































































































chemically modified
























chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100
















































































Vectors







Categorization






















Viral Vectors


































Polyplexes




















transfection


































































































chemically modified
























chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100



Categorization






















Viral Vectors


































Polyplexes




















transfection


































































































chemically modified
























chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100

















Viral Vectors


































Polyplexes




















transfection


































































































chemically modified
























chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100


































Polyplexes




















transfection


































































































chemically modified
























chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100

















transfection


































































































chemically modified
























chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100






























































































chemically modified
























chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100






















chromosome












Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100










Transposition



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100



















































































































































































each year















Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100











Passive Targeting














and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100











and checkpoints






Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100



Cell Cycle Control


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100


















cancer development



diameter


Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100

Apoptotic Pathways
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100
















Slide 1

Definitions
















Vectors

Categorization



Viral Vectors





Lipoplexes

Polyplexes









Polypeptide Vectors













Transposition

RNA Interference




Slide 55


Slide 57


Slide 59


Delivery Systems


siRNA Modification

Polymer Vectors




Lipid Vectors



Lipid Vectors (2)


Lipid Vectors (3)

Lipid Vectors (4)









Cancer

Cancer (2)

Slide 85

Tumor Targeting

Tumor Targeting (2)


Cell Cycle Control




Slide 93



Slide 96

Slide 97

Slide 98

Slide 99

Slide 100

si-rna delivery

Health & Medicine