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Oligonucleotide Oligonucleotide Therapeutics: Basic Therapeutics: Basic
Principles and Delivery Principles and Delivery StrategiesStrategies
Ashish SarodeAshish SarodeFebruary 17, 2006February 17, 2006
OutlineOutline
IntroductionIntroductionBasic PrinciplesBasic PrinciplesDelivery StrategiesDelivery StrategiesSummarySummaryConclusionConclusion
DefinitionsDefinitions
Oligonucleotide (ON) is a molecule Oligonucleotide (ON) is a molecule composed of 25 or fewer nucleotidescomposed of 25 or fewer nucleotides
ON strategies designed to treat ON strategies designed to treat disease by altering gene expression disease by altering gene expression of an affected individualof an affected individual
Past and PresentPast and Present1,21,2
First synthetic oligonucleotide – Zamecnik First synthetic oligonucleotide – Zamecnik and Stephenson (1978) – RS virusand Stephenson (1978) – RS virus
First oligonucleotide drug – Fomivirsen First oligonucleotide drug – Fomivirsen (Vitravene) – FDA approval (1998/99) – (Vitravene) – FDA approval (1998/99) – Cytomegalovirus (CMV) retinitis – AIDSCytomegalovirus (CMV) retinitis – AIDS
Approximately 70 to 80 oligonucleotides Approximately 70 to 80 oligonucleotides are currently in clinical trialsare currently in clinical trials
Gene Therapy vs. ON Gene Therapy vs. ON TherapyTherapy55
Gene TherapyGene TherapyMissing or defective Missing or defective
genes are added or genes are added or
replaced with replaced with functional functional
versionsversions
ON TherapyON TherapyExisting but Existing but
abnormally abnormally
expressed genes are expressed genes are
modulatedmodulated
Modulation of gene Modulation of gene expressionexpression2,62,6
Antisense Antisense technologytechnologyONs are synthesized ONs are synthesized
that that
are complementary are complementary to to
the RNA of interestthe RNA of interest
(Control of (Control of translation)translation)
Antigene Antigene technologytechnologyONs are synthesized ONs are synthesized
for for
direct binding to DNAdirect binding to DNA
(Control of (Control of transcription)transcription)
Antisense TechnologyAntisense Technology66
Specificity is mediated through Specificity is mediated through Watson-Crick base pairingWatson-Crick base pairing
J. Dagle, D Weeks 2001
Antigene TechnologyAntigene Technology88
Intramolecular triplex formationIntramolecular triplex formation
Intermolecular triplex formationIntermolecular triplex formation
R. Guntaka, B. Varma, K. Webber2003
Antisense vs. AntigeneAntisense vs. Antigene22
PotencyPotency Gene expressionGene expression Selective modificationSelective modification Chemical modificationChemical modification ppHH sensitivity of C-GC sensitivity of C-GC KK++ sensitivity of GA or sensitivity of GA or
GTGT Low target accessibility Low target accessibility
and affinityand affinity No antigene ON in No antigene ON in
clinical trialsclinical trialsS. Buchini, C. Leumann 2003
Steps in successful ON Steps in successful ON therapytherapy99
1.1. Design and chemistryDesign and chemistry
2.2. StabilityStability
3.3. Cell association and entryCell association and entry
4.4. Net accumulation (influx > efflux)Net accumulation (influx > efflux)
5.5. Avoid compartmentalizationAvoid compartmentalization
6.6. Localization at active sitesLocalization at active sites
7.7. Exert activityExert activity
Designing ONsDesigning ONs99
Gene-walkingGene-walking RNaseH mappingRNaseH mapping Scanning Scanning
combinatorial ON combinatorial ON arraysarrays
S. Akhtar 2000
Delivery StrategiesDelivery Strategies99
1.1. LiposomesLiposomes
2.2. DendrimersDendrimers
3.3. Carrier peptide-mediatedCarrier peptide-mediated
4.4. Receptor-mediatedReceptor-mediated
5.5. Polymers (microsphere Polymers (microsphere formulations)formulations)
LiposomesLiposomes1010
1.1. Anionic liposomesAnionic liposomes
2.2. pH sensitive liposomespH sensitive liposomes
3.3. ImmunoliposomesImmunoliposomes
4.4. Fusogenic liposomesFusogenic liposomes
5.5. Cationic liposomesCationic liposomes
Anionic LiposomesAnionic Liposomes
Low encapsulation Low encapsulation efficiencyefficiency
Phosphatidylserine Phosphatidylserine and calciumand calcium
Cardiolipin (MVE) Cardiolipin (MVE) techniquetechnique
Dipalmitoyl-Dipalmitoyl-phosphatidylglycerphosphatidylglycerol (DPPG)ol (DPPG) O. Zelphati, F.
Szoka1996
pH-sensitive LiposomespH-sensitive Liposomes Principle of action – Principle of action –
enveloped virusesenveloped viruses DioleylphosphatidylethanolDioleylphosphatidylethanol
-amine (DOPE)-amine (DOPE) Non-specific electrostatic Non-specific electrostatic
adsorptionadsorption 90% of the contents is 90% of the contents is
degraded in lysosomesdegraded in lysosomes Plasma and serum Plasma and serum
instability – incorporation instability – incorporation of cholesterol / ganglioside of cholesterol / ganglioside / cholesterol hemisuccinate / cholesterol hemisuccinate (CHEMS) – loss of (CHEMS) – loss of encapsulation capacityencapsulation capacity O. Zelphati, F.
Szoka1996
ImmunoliposomesImmunoliposomes Double specificity – Double specificity –
antibodyantibody Amount of binding Amount of binding
depends on density of depends on density of targeted cell targeted cell membrane moleculesmembrane molecules
Endocytic pathwayEndocytic pathway Poor encapsulation Poor encapsulation
capacity – ONs capacity – ONs coupled to cholesterol coupled to cholesterol via disulfide linkagevia disulfide linkage
Lysosomal destructionLysosomal destruction ImmunogenicImmunogenic O. Zelphati, F.
Szoka1996
Fusogenic LiposomesFusogenic Liposomes
Liposomes merge with Liposomes merge with cell membranescell membranes
Fusogenic agents – Fusogenic agents – PEG, glycerol, Poly-PEG, glycerol, Poly-vinyl alcohol, vinyl alcohol, reconstituted viral reconstituted viral membranesmembranes
ImmunogenicityImmunogenicity Poor cellular specificityPoor cellular specificity Instability in plasma Instability in plasma
and serumand serum O. Zelphati, F. Szoka1996
Cationic LiposomesCationic Liposomes
No encapsulation step No encapsulation step – electrostatic – electrostatic interaction between interaction between ONs and cationic lipidsONs and cationic lipids
Charge ratio is critical Charge ratio is critical for efficiencyfor efficiency
Fusion of cationic Fusion of cationic lipids with anionic cell lipids with anionic cell membranesmembranes
Enocytosis but Enocytosis but uncoated vesicles – uncoated vesicles – acidification is not acidification is not requiredrequired O. Zelphati, F.
Szoka1996
DendrimersDendrimers99
Supermolecular delivery systems – Supermolecular delivery systems – Polymerization – monodisperse, Polymerization – monodisperse, reproducible productreproducible product
Several functional groups – versatileSeveral functional groups – versatile Polyamidoamine (PAMAM) starburst Polyamidoamine (PAMAM) starburst
dendrimers – hydrocarbon core – charged dendrimers – hydrocarbon core – charged surface amino groupssurface amino groups
Stable complex – plasma and serumStable complex – plasma and serum Reduce degradation of ONs in serum and Reduce degradation of ONs in serum and
lysosomeslysosomes
Carrier peptide-mediatedCarrier peptide-mediated5,95,9
Poly-L-lysine (PLL) – polycationic drug Poly-L-lysine (PLL) – polycationic drug carriercarrier
Non-specific adsorptive endocytosisNon-specific adsorptive endocytosisPLL interacts nonspecifically with PLL interacts nonspecifically with
negatively charged molecules on the negatively charged molecules on the cell membranecell membrane
Cytotoxicity and nonspecificityCytotoxicity and nonspecificitySpecific peptides can be conjugated to Specific peptides can be conjugated to
PLLPLL
Receptor-mediatedReceptor-mediated55
Affinity of the receptor target may Affinity of the receptor target may increase ON-cellular associationincrease ON-cellular association
Combination approach – endosome Combination approach – endosome disrupting agents and labile linkages disrupting agents and labile linkages between ON and targeting moietybetween ON and targeting moiety
Y. Rojanasakul 1996
PolymersPolymers99
ONs are encapsulated in biodegradable ONs are encapsulated in biodegradable polymers – copolymers of lactic acid and polymers – copolymers of lactic acid and glycolic acid (PLA and PLGA)glycolic acid (PLA and PLGA)
Entrapment provides nuclease protectionEntrapment provides nuclease protection Controlled release – size of microspheres, Controlled release – size of microspheres,
length of ONs, and Mlength of ONs, and Mww of the polymer of the polymer Triphasic profiles – initial ‘burst effect’ (48 Triphasic profiles – initial ‘burst effect’ (48
hrs) – sustained release – increased hrs) – sustained release – increased release (due to bulk degradation of release (due to bulk degradation of microspheres)microspheres)
SummarySummary
ON therapeutics has a potential to ON therapeutics has a potential to specifically alter gene expressionspecifically alter gene expression
However ON activity is restricted by However ON activity is restricted by lack of target cell recognition, low lack of target cell recognition, low cellular uptake, and nuclease cellular uptake, and nuclease degradationdegradation
Chemical modification of ONs and Chemical modification of ONs and delivery strategies explored to delivery strategies explored to overcome these drawbacks show overcome these drawbacks show promising results at some levelpromising results at some level
ConclusionConclusion
ON therapeutics can be used to treat ON therapeutics can be used to treat diseases such as cancer, viral diseases such as cancer, viral infections, inflammatory and genetic infections, inflammatory and genetic disordersdisorders
There is a wide scope to improve the There is a wide scope to improve the available delivery strategies as well available delivery strategies as well as to invent new strategies for as to invent new strategies for successful application of ON successful application of ON therapeuticstherapeutics
ReferencesReferences1.1. J. Rossi. A society of our own. Oligonucleotides. 2005; 15:71-71J. Rossi. A society of our own. Oligonucleotides. 2005; 15:71-712.2. S. Buchini, C. Leumann. Recent improvements in antigene technology. S. Buchini, C. Leumann. Recent improvements in antigene technology.
Current opinion in chemical biology. 2003; 7:717-726Current opinion in chemical biology. 2003; 7:717-7263.3. Isis Pharmaceuticals 2003 annual reportIsis Pharmaceuticals 2003 annual report4.4. Isis Pharmaceuticals 2004 annual reportIsis Pharmaceuticals 2004 annual report5.5. Y. Rojanasakul. Antisense oligonucleotide therapeutics: drug delivery Y. Rojanasakul. Antisense oligonucleotide therapeutics: drug delivery
and targeting. ADDR. 1996; 18:115-131and targeting. ADDR. 1996; 18:115-1316.6. J. Dagle, D Weeks. Oligonucleotide-based strategies to reduce gene J. Dagle, D Weeks. Oligonucleotide-based strategies to reduce gene
expression. Differentiation. 2001; 69:75-82expression. Differentiation. 2001; 69:75-827.7. E. Devor. ID Tutorial: Antisense Technologies. Integrated DNA E. Devor. ID Tutorial: Antisense Technologies. Integrated DNA
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modulators of gene expression. IJBCB. 2003; 35:22-31modulators of gene expression. IJBCB. 2003; 35:22-319.9. S. Akhtar, M. Hughes, A. Khan, M. Bibby, M. Hussain, Q. Nawaz, J Double, S. Akhtar, M. Hughes, A. Khan, M. Bibby, M. Hussain, Q. Nawaz, J Double,
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antisense oligonucleotides: a real or magic bullet? Journal of controlled antisense oligonucleotides: a real or magic bullet? Journal of controlled release. 1996; 41:99-119release. 1996; 41:99-119