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

IntroductionIntroduction

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

Current ResearchCurrent Research33

Isis Pharmaceuticals 2003 annual report

Clinical TrialsClinical Trials44

Isis Pharmaceuticals 2004 annual report

Basic PrinciplesBasic Principles

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

Antisense TechnologyAntisense Technology77

E. Devor 2005

Antigene TechnologyAntigene Technology66

J. Dagle, D Weeks 2001

Antigene TechnologyAntigene Technology88

Intramolecular triplex formationIntramolecular triplex formation

Intermolecular triplex formationIntermolecular triplex formation

R. Guntaka, B. Varma, K. Webber2003

Antigene TechnologyAntigene Technology22

S. Buchini, C. Leumann 2003

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

StabilityStability22

Nuclease digestionNuclease digestion

S. Buchini, C. Leumann 2003

Delivery StrategiesDelivery Strategies

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

Cationic LiposomesCationic Liposomes

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

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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

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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,

P. Sayyed. The delivery of antisense therapeutics. ADDR. 2000; 44:3-21P. Sayyed. The delivery of antisense therapeutics. ADDR. 2000; 44:3-2110.10. O. Zelphati, F. Szoka. Liposomes as a carrier for intracellular delivery of O. Zelphati, F. Szoka. Liposomes as a carrier for intracellular delivery of

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AcknowledgementAcknowledgement

Dr. S. KislaliogluDr. S. KislaliogluDr. H. ZiaDr. H. Zia