for or Can Cance cer r The heragn gnos osis is Div Divisi - - PowerPoint PPT Presentation

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for or Can Cance cer r The heragn gnos osis is Div Divisi - - PowerPoint PPT Presentation

Apr 04, 2024 257 likes •488 views

Ver ersa satili tility ty of of Nu Nucleic leic Acid Acid for or Can Cance cer r The heragn gnos osis is Div Divisi ision on of of Con Conver ergence gence Tec echnolog hnology Resear esearch h Instit Institute ute of

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Div Divisi ision

  • n of
  • f Con

Conver ergence gence Tec echnolog hnology Resear esearch h Instit Institute ute of

  • f Na

National C tional Cancer C ancer Center enter, , Rep

  • ep. of

. of K Kor

  • rea

ea Yun un-Hee Kim Hee Kim

Ver ersa satili tility ty of

  • f Nu

Nucleic leic Acid Acid for

  • r Can

Cance cer r The heragn gnos

  • sis

is

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Requirements for development of cancer gene therapeutics in vivo Imaging Nucleic acid-based therapy

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Therapeutic RNA

Replacement Replaced protein

RISC

mRNA Protein

Target

Protein sysnthesis

Results

Inhibition of protein function Protein sysnthesis mRNA

 Gene Silencing: antisense RNA , ribozyme, RNAi, miRNA  RNA replacement: trans-splicing ribozyme, spliceosome  Modulating protein function: aptamer

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4

General strategy of Nucleic acid-therapeutics

DNA

Transcription Translation Post translational modification

Membrane Protein Protein Protein

  • Antisense oligonucleotides
  • Ribozymes
  • Small interfering RNAs (siRNA)
  • Short hairpin RNAs (shRNA)
  • Micro RNA (miRNA)
  • r

replaced protein mRNA

1) Inhibition of protein biosynthesis

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5

DNA mRNA Protein

Transcription Translation Post translational modification

Protein Membrane Protein

Aptamer

2) Capturing agents to bind protein

Strategy of Nucleic acid-therapeutics

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RNA replacement : trans-splicing Ribozyme

transcription genome

translation Trans-cleaving Trans-splicing Transgene expression

G G

Rz

G G U

Target RNA

5’ Rz

U

5’ Therapeutic RNA Therapeutic RNA expression by trans-splicing

Trans-splicing ribozyme

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Multifun ltifunctional ctional Tumor

  • r Targeting

eting Devic ice e by y trans ans-sp splici licing ng ribozy

  • zyme

me

Ad Ad Ad Ad

Dual Targeting

Tissue specific Tumor promoting

Dual Therapy

Promoter Ribozyme Therapeutic gene

Target RNA knock-down Cytotoxic effect Anti-sense effect Direct anti-tumor effect

  • HSV-Tk
  • Cytosine deaminase
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hTERT : compo ponen nent t of t telomer

  • merase

ase gene e

  • potent

ntial ial anti-cance cancer r therapeu peutic tic target et -

  • 1. Ribonucleoprotein enzyme that maintains

the protective telomere structures (tandomly repeated (TTAGGG)n seq.) in eukaryotic chromosome

  • 2. Expression level of hTERT relates with

telomerase activity

  • 3. Selective expression in highly proliferative

cells (bone marrow stem cells, germ cells…) and ~90% of cancer cells

He Herpes pes simpl implex x th thymi ymidin dine kinas inase (HS HSVtk tk) )

  • Cell death

h by bystander ander effect

  • Well-known mechanism of action
  • Strong cell killing potential due to neighboring effect
  • No toxic effect without GCV
  • Expression can be monitored by PET in vivo

GCV : Ganciclovir

Target Therapeutic gene

Target et & therape peut utic ic gene e for r replac lacement ement in trans ns-spli plici cing ng riboz

  • zyme

yme

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9

Multifunctional Devices(trans-splicing ribozyme) for Hepa patocel

  • cellular

ular Carcinoma cinoma Ther erapy

* I Image-aide ided d in vivo Eval alua uatio tion

Y-H Kim et al. Theranostics (2016)

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Multifunctional Devices(trans-splicing ribozyme) for Pancr crea eatic tic Cancer cer Therapy

Target RNA:

PAUF (pancreatic adenocarcinoma up-regulated factor) * I Image-aide ided d in vivo Eval alua uatio tion

Y-H Kim et al. Cancer Letters (2014)

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1. Target (tumor specificity, natural conformation,…) 2. Specific-Targeting efficiency 3. Penetration into tumor tissue 4. Easy manipulation for optimization 5. Speed & Cost for development 6. Multiple targeting (simultaneously)

  • Complexity of tumor microenvironment

Aptamer

Trends in Pharmacological Sciences. 2008. 29(2);57 - 61

<Advantages of aptamers>

  • Specifically bind a target of interest

(KD = pM ~ nM)

  • Produced by chemically process (in vitro)
  • Conjugation chemistries are easy
  • Smaller size allows tumor tissue penetration
  • Able to select for cell-surface targets
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Modulating protein function : Aptamer

  • -Nucleic acid-based antibody

 Latin: “aptus” – to fit  Oligonucleic acid (DNA or RNA) or peptide molecules that bind a specific target molecule  Aptamer can be thought of as the nucleic acid antibodies  Sequences are selected from very large pools (1015 or greater)  Discovered through in vitro selection (SELEX) to recognize and specifically bind a target of interest

SELEX : systematic evolution of ligands by exponential enrichment

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Cloning and Sequencing : protein, sugar,

cell ,…

Known target Known target Unknown target

Validation: target-specificity Optimization

SELEX * Discovery of surface markers the specific cell types * Therapeutic aptamer development known cell surface proteins

Protein as it appears Cell itself as a target

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PAUF-targeting Aptamer

Protein-based SELEX  Aptamer

Fc LBL313-Fc 5 cycles Fc LBL313-Fc 10 cycles Fc LBL313-Fc 18 cycles

L1, L2, L4, L6, L7, L8, L9, L10, L11, L14, L17, L18, L19 CCTGTAAATACACGCATCGTATCTCGATTCGTATCCTTGACC L3 CCTGTAAATACACGCATCGTATCTCGATTCATATCCTTGACC L12 CCTGTAAATACACGCATCGTATCTCGATTCGCATCCCTGACT L15, L20 CCTGTAAATACATTCTTCCGCGTGTGATACCGTCCGTGACTA L5 CCTGTAAATACACTGATCTTCCGATATGTTCCTGCCATCATAC

PAUF targeting aptamer – in vivo function

PBS Neutralizing antibody 12-FR2 Aptamer Reverse sequence (CTL) Tumor: CFPAC-1-Luc (pancreatic tumor cell) Aptamer: 10 day (every 2day injection)

for known secretory target

Y-H Kim et al. Cancer Letters (2012)

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Pancreatic cancer -targeting Aptamer

for unknown cell surface marker

  • 1. Target identification (in case of unknown target)
  • binding to target & pulldown & purification
  • 2. Specific recognition for target protein
  • 3. Easy optimization for therapeutic utilization
  • 4. Small size benefits-penetration, imaging probe,…

1. Cell surface target without destruction of 3- dimentional structure (SELEX in live cells) 2. Recognition ability to small sized epitope 3. Recognition to tumor status-dependent modification

  • f target

&

Why Cell-SELEX ?

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Aptamer candidates

Negative control aptamer

Control cell (HPNE) Target cell (Lu-M1)

<Internalization> SQ7 SQ8

SQ8 SQ7 SQ1 library

unpublished

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Antibo ntibody dy Apta ptamer mer (O (Oli ligo gomer) mer)

Ol Oligob igobod

  • dy

High penetration High stability in blood High penetration High stability in blood

Win-Win Win : Antibody ibody-ba based sed delivery ery of Aptame tamer

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Increase pharmacokinetics Increase tumor tissue penetration Inhibit tumor growth

Heo et al. J Contl Release (2016) Anti-cotinine (Antibody) Anti-VEGF (Aptamer)

Proof-of-Concept of Oligobody

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Ab

Cot Aptamer1 Cot Aptamer2 Cot Aptamer3 Cot Aptamer4 Cot Aptamer5

antibody as Novel delivery tool of aptamer

Target 1 Target 2 Target 3

Ab

Hapten

Linking tool

. . . . .

Ab Ab

Apt1 Apt2 Apt3

Aptamers

Targeting tool

Anti-Cotinine humanized antibody High affinity against Cotinine (KD = 4.9 X 10-12 M) Increase pharmacokinetics of aptamer-conjugate Need just one antibody!! Link between aptamer and antibody Cotinine is not toxic Easy to synthesize with aptamer Bind to a specific target molecule Fast generation, Low cost Low molecular weight (tissue penetration) Chemistry for Conjugation

Oligobody: a novel aptamer-antibody hybrid complex

Simultaneously multi-targeting

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Systemic administration Ref:Theranostics 04: 0931, No3

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<Aptamer>

  • Dr. In-Hoo Kim
  • Dr. Kyun Heo
  • Dr. Sun Il Choi (Ph.D)
  • Dr. Jun-Ho Jung (SNU)
  • Dr. Jin Sook Jeong (Dong-A Univ)
  • Dr. Sang-Jin Lee
  • Dr. Eun Sook Lee
  • Dr. Ho Jin Sung
  • Dr. Kyoung Ho Choi (SNU)
  • Dr. Seok-ki Kim

* All lab. members * Research Core Center, NCC <Pancreatic Cancer>

  • Dr. Sang Myoung Woo
  • Dr. Sang Jae Park
  • Dr. Woo Jin Lee,
  • Dr. Sung Sik Han
  • Dr. Eun Kyoung Hong
  • Dr. Sun-Young Kong