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Nanoscale Constructs for Therapy

Nanoscale Constructs for Therapy. Milan Mrksich. The University of Chicago and HHMI. Examples from the Northwestern CCNE molecular diagnostics—PSA nano -flares for mRNA detection gold nanoparticles for gene regulation validating targets in acetylation biology.

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Nanoscale Constructs for Therapy

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  1. Nanoscale Constructs for Therapy Milan Mrksich The University of Chicago and HHMI Examples from the Northwestern CCNE molecular diagnostics—PSA nano-flares for mRNA detection gold nanoparticles for gene regulation validating targets in acetylation biology

  2. Prostate Cancer and PSA: CCNE Past Success Background: • Prostate cancer is the most common (200,000 dx/year) and 2nd most deadly cancer among American men (30,000 deaths/year) • 70,000 men / year experience PSA failure after undergoing surgery for prostate cancer (40%), remaining 60% don’t objectively know they are cured! • Problem: • Conventional PSA immunoassays • (LOD 0.1 ng/ mL) do not have the • requisite sensitivity to: • Define disease cure (modify/ • define surveillance protocols) • Diagnose recurrence early • for enrollment in clinical trials • Assess biological response • to adjuvant/ salvage therapy • Innovation: The Bio-Barcode Assay • LOD 0.3 pg/ mL = >300X Increase

  3. Retrospective Clinical Validation of Post-Operative PSA Kinetics Cure Translation: • Retrospective Trial (ongoing) • ~410 patients following surgery (T=0) • ~750 samples • Patients w and w/o recurrence • All conventional PSA ≤ 0.1 ng/mL • NanoPSA (red) • Up to 15 years follow-up • Multi-Institution (NU, Wash U) Early Recurrence Salvage Response Early Recurrence Radiation

  4. Nano-Flares for Intracellular mRNA Detection Target Induced Flare Release Flares are Quenched Target mRNA Non-Complementary Nano-Flare (targeting survivin) 20 µm Patient prognostication, treatment response measurement, stem cell isolation

  5. Gold Nanoparticle Agents for Gene Regulation DNA/ siRNA transfected by Gold Nanoparticle

  6. Antisense-DNA Knockdown of GFP Cy5.5 GFP Transmission Overlay Normalized Fraction eGFPexpression 6 nM Au NP 24 nM Au NP 6 nM Au NP 6 nM Au NP

  7. Translation of Gene Regulating DNA/ siRNA AuNPs Indications Glioblastoma (Brain Tumors) Local Delivery Following Tumor Removal—Clean Up Pancreatic Cancer Local Delivery Following Tumor Resection—Clean Up Innovation Indication-Specific Targeted siRNA AuNP New Properties/ New Opportunities Beyond Cells Organ Level Efficacy Organ Level βgal Knockdown (right) in ovaries using anti-βgal siRNA AuNPs Deep siRNA AuNP Penetration Into Target Organs (Red) In Vivo Efficacy Demonstrated: Breast cancer, skin disorders (15 current collaborations, 6 in animal models)

  8. Translational Efficacy of siRNAAuNPs in Breast Cancer In Targeted siRNA- AuNP Treated Tumors, and Increase in Apoptosis and Decrease in Proliferation is observed Survivin NP-treated tumor Untreated tumor Caspase 3 staining for apoptosis Ki67 staining For cell proliferation (+) (-) (-) (+)

  9. Mass Spectrometry and NanoMaterials for Label-Free Assays Detector Alkanethiolate LASER MS Alkyldisulfide m/z

  10. Ac Ac Label-Free Deacetylase Assay Ac-GAKRHRKVC-NH2 1.0 1992.9 Ac-GAKRHRKVC-NH2 Relative Intensity 0.5 0 KDAC8 Ac-GAKRHRKVC-NH2 1.0 1950.8 Relative Intensity m/z 42 0.5 1992.9 0 1930 1958 1986 2014 m/z

  11. Lack of Suitable KDAC Activity Assays Parent peptide 1.0 0.5 0 Deacetylated peptide SAMs on gold patterned glass Rel. Int. 1.0 0.5 0 m/z Peptides arrayed Trypsin KDAC Rel. Int. 42 m/z m/z 1.0 0.5 0 Solution of KDAC 1. Rinse / dry 2. SAMDI Rel. Int. E D C B A 42 m/z 1 2 3 4 5 6 7 8 9 10 Applied Biosystems 4800 MALDI TOF/TOF m/z

  12. Specificity Profiles for Deacetylases X Position Ac-GXKAcZGC-NH2 Z Position N N N N E E E E Q Q Q Q K K K K R R R R H H H H S S S S T T T T M M M M W W W W F F F F Y Y Y Y V V V V I I I I L L L L A A A A G G G G P P P P D D D D Z Position D D D D KDAC8, SIRT1 specific for RKF and RKR, respectively Overlapping activity on active substrates Non-selective substrates N N N N E E E E Q Q Q Q K K K K R R R R KDAC8 SIRT1 X Position KDAC3/SMRT H H H H S S S S T T T T M M M M KDAC1 and 3 were not active on any peptides in this array, despite showing activity on fluorescently-labeled substrates. W W W W F F F F Y Y Y Y V V V V I I I I L L L L A A A A G G G G P P P P KDAC2 1 0

  13. Many Possible Combinations, Few Observed P Me Me H4 S G R G K G G K G L G K G G A K R H R K V Ac Ac H4: 7 Modified Residues = 512 Possible Combinations! H3 Tail: 13 Modified Residues = 25,600,000 Possible Combinations! P Ac P Ac R K T R S G K P K L T A R S P H3 A T Q A K T G A R Q A K A K A Histone Code: Modifications act sequentially or in combination to create a ‘code’ read by other proteins to regulate cellular events. Me1 Me1 P Me Me Me2 Me2sym Me Me Me Me Me Me Me Me P Me2unsym Me3 Ac Ac Ac Ac Ac R K

  14. Acetylation of a H3-Derived Peptide 2067 Ac-TARK9STGGK14APC Ac-TARK9STGGK14AcAPC PCAF/Ac-CoA KDAC8 2109 Ac 42 Da m/z

  15. Addition of KDAC Gives MORE Acetylation 2067 --K9--K14-- -S PCAF --K9--K14Ac-- + 2109 PCAF KDAC8 ---K9Ac--K14--- -S --K9Ac--K14Ac-- + --K9--K14Ac-- 2151 + --K9Ac--K14--

  16. Kinetic Model for Increased Acetylation H3 9 KAc K KAc K 14 K KAc KAc K kcat/Km ~ 0 mM-1·min-1 94 mM-1·min-1 H3 H3 9 9 14 14 12 mM-1·min-1 1mM-1·min-1 H3 KDAC8 9 14 PCAF

  17. Relative Kinetics Dictates Deacetylation Pathway Starting Acetylation State 1Ac 3Ac # Ac groups after KDAC8 Rxn 0Ac 2Ac H4 Histone “Tail” Sequence: SGRGKGGKGLGKGGAKRHRC

  18. Relative Kinetics Dictates Deacetylation Pathway Starting Acetylation State 1Ac 3Ac # Ac groups after KDAC8 Rxn 0Ac 2Ac H4 Histone “Tail” Sequence: SGRGKGGKGLGKGGAKRHRC

  19. Role of Centers in Translational Research A strongly integrated partnership between the International Institute for Nanotechnology and the Robert H. Lurie Comprehensive Cancer Center Funded by the NCI Alliance for Nanotechnology in Cancer U54-CA119341 • Established in 2005 • Mission • To advance discovery and knowledge within the field of nanotechnology through focused and collaborative research leading to the design and testing of nanomaterials and nanodevices for their translational application into the clinic • Supports • 6 interdisciplinary translational research projects • 2 seed projects each year with university funds • 37 nano-scientists, cancer biologists, engineers, and clinicians • 26 postdoctoral associates • 10 undergraduate researchers

  20. Translation Through Industrial Partnerships • Nanosphere, Inc. • Retrospective study for development of ultra-sensitive PSA detection • Takara Biosciences • Scanometric detection and nanoparticle-based electrical and optical transduction mechanisms • NanoInk, Inc. • Commercialization of DPN technology portfolio • PreDx, Inc. • Transform conventional diagnostic imaging techniques, e.g. MRI, from their current role as anatomical imaging tools into metabolic and theranostic probes • Bracco Corp and Siemens Inc. • New probe development • Neopharm Pharmaceuticals • Polymer coated liposome (PCL) technology • Altor Biosciences • Targeting technology • Integrated DNA Technologies • Studies of DNA thermodynamics • Nanotope • Multiplatform therapies in medicine • Baxter • Assays for the quantification of cell “polarizability” independent of the cell asymmetry

  21. Example of Technology Heading to the Clinic ATO nanobin inhibits breast tumor growth • Summer 2009- NCI/NCL Anticipated Completion of Preclinical Work • August 1, 2009Submission to Rapid Access to Intervention Development (NCI-RAID) • (GMP scale up, toxicity and pharmacokinetic testing) • Winter 2010- Pre IND meeting with FDA • Mid Summer 2011- Investigational New Drug Application • (Enables Phase 1 Clinical Trials) PBS NB(NaCl) ATO NB(Ni,As) At completion of NCI-RAID IND will be filed Anticipated entry into clinical trials Summer 2011

  22. Nanoscale Constructs for Diagnostics and Therapy Activity depends on shape, size, composition knock-down with gold NPs Nanomaterials have novel, and tunable properties enable label-free assays Centers critical to translational work nanoassemblies in the clinic partnerships with other Centers, Industry

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