diagnostic and therapeutic potential of small nucleic acid molecules mark e. sobel, m.d., ph.d....

Diagnostic and Therapeutic Potential of Small Diagnostic and Therapeutic Potential of Small

Nucleic Acid MoleculesNucleic Acid Molecules

Mark E. Sobel, M.D., Ph.D.Executive Officer

American Society for Investigative [email protected]

Penn State Milton S. Hershey Medical CenterAugust 11, 2009

This presentation will be available at http://www.asip.org/about/exec.htm

mailto:[email protected]

http://www.asip.org/about/exec.htm

Part I: The New GeneticsPart I: The New Genetics

Part II: Small RNAs

Part III: miRNA and Hodgkin Lymphoma

Part IV: Small Molecule Gene-Silencing Strategies

Dark Matter is 96% of the mass of the universe

The New Genetics: The Human GenomeThe New Genetics: The Human Genome

3.1 x 109 bp 500,000 stretches of DNA that are conserved

through evolution 22,000 genes based on current algorithms =

5% of genome 30% have instructions to make proteins 70% have instructions to regulate the

protein-coding genes

© The American Society for Investigative Pathology

RNA transcription

pre-mRNA

splicing

mRNA

ribosome

mRNA A(n)

exon intron exon intron exonGene

translation

protein

© The American Society for Investigative Pathology

Complexity of the Human GenomeComplexity of the Human Genome

The other 70% (regulating)• Ribosomal (rRNA)• Transfer RNA (tRNA)• Small nucleolar RNA (snoRNA)• Micro RNA (miRNA)

95% Junk DNA: Pseudogenes Other functions?

© American Society for Investigative Pathology

The Human Genome is NOT “The Human Genome is NOT “GreenGreen””

Inefficiencies can be an advantage Adapt quickly to rare, life-threatening

circumstances Fill up your gas tank and have it ready

at all times for a long trip Bleeding Starvation Extreme heat or cold


Small RNAsSmall RNAs

20-30 nt Associated with Argonaute (Ago) Family Proteins Three classes of small RNAs

miRNA: microRNAs control mRNA stability and translation

siRNA: small interfering RNAs suppress repetitive genes by cleaving their transcripts

piRNA: Piwi-interacting RNAs mediate RNA cleavage or heterochromatin formation of transposons


RNA Interference (RNA Interference (RNAiRNAi))

Nobel Prize in 2006: Andrew Fire and Craig Mellow In 1998, demonstrated double stranded RNA-

mediated degradation of target mRNA in Caenorhabditis elegans

Short interfering RNA (siRNA) of 20-23 nucleotides siRNAs are incorporated into an RNA-inducing

silencing complex (RISC)


siRNA

RISC

siRNA is bound by RISC and unwound by RNA helicase

RISC

Sense RNA strand degraded


RISC

AAAAAA….A

Target mRNA

mRNA cleavage and degradation

RISC-bound antisense strand directed to target mRNA

AAAAAA….A

Target mRNARISC


MicroRNAs (miRNA, miR)MicroRNAs (miRNA, miR)Molecular Diagnostics of the Future?Molecular Diagnostics of the Future?

A recently discovered class of naturally occurring siRNA molecules that function post-transcriptionally to regulate mRNA expression

Small non-coding RNAs (20-23 nucleotides) Excised from precursor molecules

Pre-miRNAs are 60-110 nucleotide foldback (double-stranded) RNA precursor structures

Recognized by RISC: RNA-Induced Silencing Complex


MicroRNA (miRNA, miR)MicroRNA (miRNA, miR)

Pleiotropic: each miR can regulate more than one target mRNA

“Silent” mutations in mRNAs such as conservative nucleotide changes may have effects

Two main mechanisms of action: Degradation of mRNA Efficiency of mRNA translation


cytoplasmic processing

Dicer (RNase III)

pre-miRNA

miRNA

Interact with target mRNA

RISCribosome

mRNA A(n)

RISC RISC

RISC RNA-Induced Silencing Complex


MicroRNA (miRNA, miR)MicroRNA (miRNA, miR)

Conserved through evolution Why did it take so long to discover?

Too small to isolate with usual techniques

Lethal mutations for development Coded throughout the genome, often at

chromosomal breakpoints


A

BC

DE

L

JF

GK

I

C

DE

BA

J

IH

FK

G

B

A

DC

AD

B

EC

G

C

BDF

AE

BAC

DGF

E

HI

GFEJDA

CB

B

E DC

F

A

ACFDBE

CG

DABFE

FBG

AHEDCI

BC

ADFE

DCB

EAA

B

EDC

ABCD

BC AA

B

AB

B

BA

miRs located at Fragile sitesmiRs located in other genomic regions

Adapted from Carlo Croce, Ohio State University

MicroRNA in Human DiseaseMicroRNA in Human Disease

Development Mental Retardation Cardiology Oncology

Altered expression levels in many tumor types

Solid Tumors Hematopoietic cancers

Germline sequence abnormalities Tumor suppressors Oncogenes


miRNA clusters in Chr. 8q24.1-24.3

Courtesy of Vladimir Valera-Romero and Maria Merino

MicroRNA as an Oncogene MicroRNA as an Oncogene

Higher expression of the miR will decrease the stability and translatability of the target tumor suppressor mRNA

Leads to decreased expression of the tumor suppressor

Increased tumor formation

Lower expression of the miR will increase the stability and translatability of the target oncogene mRNA

Leads to increased expression of the oncogene Increased tumor formation


An Approach to Identifying Novel Molecular Lesions and Potential Therapeutic Targets

MicroRNA-Mediated Down-Regulaton of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg Cells:

A Potential Pathogenetic Lesion in Hodgkin Lymphomas

Kui Nie, Mario Gomez, Pablo Pandgraf, Jose-Francisco Garcia, Yifang Liu, Leonard H.D. Tan, Amy Chadburn, Thomas Tuschl,

Daniel Knowles, and Wayne Tam

Am J Pathol 2008 173:242-2252; DOI: 10.2353/ajpath.2008.080009

PRDM1/Blimp-1

• PRDM gene family of transcription repressors• Contain Kruppel-type zinc fingers • Expressed as two isoforms, α and as a result of alternative promoter usage

– The form is functionally impaired (lacks amino-terminal acidic domain and part of the PR domain)

• PRDM1 plays a critical role in:– Terminal differentiation of lymphocytes and epidermal cells– Fate specification of primordial germ cells (and other cell types)– In B cells, key differentiation factor in post-germinal center (GC) cells-

“master regulator” of plasma cell differentiation• Tumor suppressor gene in large B-cell lymphoma diffuse (DLBCL)–

inhibition of terminal B-cell differentiation may play a role in pathogenesis• HRS cells and non-GCB DLBCL appear to be in similar differentiation stages• Sequence analysis has not identified inactivating mutations of PRDM1• Decrease in accumulation of PRDM1 may occur through quantitative

changes in synthesis or stability

Profiles of miRNA Expression in Hodgkin Lymphoma Cell Lines

Nie, K. et al, Am J Pathol 2008, 173:242-252Figure 1

miR-9 and let-7a

PRDM1 Harbors Putative Target Sites for miR-9 and let-7a

Nie, K. et al, Am J Pathol 2008, 173:242-252Figure 2A

Nie, K. et al, Am J Pathol 2008, 173:242-252Figure 2B

Complementarity Between miR-9 and

Conserved Putative Binding Sites in the Target mRNA (PRDM1 3’ UTR)

Nie, K. et al, Am J Pathol 2008, 173:242-252Figure 2C

Complementarity Between let-7a and Conserved Putative Binding Sites in the Target mRNA (PRDM1 3’ UTR)

Real-time PCR of PRDM1α mRNA


PEL- primary effusion lymphomaMM – multiple myeloma

Western Blot: PRDM1 protein levels are low in HL


Hodgkin/Reed-Sternberg Cells Harbor Low Levels of PRDM1:Better Correlation with miR-9 and let-7a Expression than with PRDM1 mRNA

Positive controls: PEL and MMNie, K. et al, Am J Pathol 2008, 173:242-252Figure 5C

Reactive GC in ahyperplastic tonsil

PRDM1-negative HRS cells Weakly positive HRS cells

IRF4/MUM1 positive

PRDM1 Immunohistochemistry (HL Cases)

Nie, K. et al, Am J Pathol 2008, 173:242-252, Figure 5D

Transfection of mir-9 or let-7a Alters Endogenous PRDM1 Expression

Nie, K. et al, Am J Pathol 2008, 173:242-252Figure 6A


Transfection with anti-micro RNA Increases PRDM1 Protein Levels

Hypothetical Model of microRNA-mediated PRDM1 Inactivation inPathogenesis of Hodgkin/Reed-Sternberg Cells:

PRDM1 protein levels do not accumulate despite induction of PRDM1mRNA transcripts because of translation repression by miRNAs

Nie, K. et al, Am J Pathol 2008, 173:242-252Figure 7

Oncogene miR Expression: Neoplastic Progression

transcription target mRNA Phenotypemutations

amplification

translocation to strong promoter

translocation to weak promoter

homozygous deletion

promoter hypermethylation

deletion + mutation

miR gene

down regulation of tumor suppressor

over expression of oncogene

Proliferation

Invasion

Angiogenesis


MicroRNA as a Tumor SuppressorMicroRNA as a Tumor Suppressor

Lower expression of the miR will increase the stability and translatability of the target tumor suppressor mRNA

Leads to increased expression of the tumor suppressor

Suppression of tumor formation

Higher expression of the miR will decrease the stability and translatability of the target oncogene mRNA

Leads to decreased expression of the oncogene Suppression of tumor formation


Gene-Silencing TherapeuticsGene-Silencing Therapeutics

siRNA DNA enzymes (DNAzymes) Antisense oligonucleotides Decoys Ribozymes Aptamers


siRNA for Gene-Silencing Therapy siRNA for Gene-Silencing Therapy

Major concerns: Tissue specificity Ability to withstand degradation

Clinical Trials: Prevent ocular neovascularization

Age-related macular degeneration Cand5 (VEGF siRNA) intravitreal injection

(Phase II) SiRNA-027 (Phase I)

Diabetic retinopathy


DNAzymesDNAzymes

Gene-specific molecular scissors Not observed in nature Advantages for therapeutics:

Lower production costs Serum stability

“10-23” subtype is best characterized Cation-dependent catalytic core of 15 dNTPs

Binds to target mRNA Cleaves target RNA between unpaired purine and

paired pyrimidine Flanking complementary binding arms

6 to 12 dNTPs Target mRNA specificity


DNAzyme binds target mRNA

mRNA cleavage and degradation

DNAzyme

AAAAAA….ATarget mRNA

protein

X


DNAzymesDNAzymes

Enhance stability Prevent exonuclease degradation: Incorporate 3’-3’

inverted nucleotide at 3’end Extend half-life with locked nucleic acids

LNA bases have a 2’-O 4-C methylene bridge Constraint on the ribose ring Increases affinity for complementary sequences Increased solubility

Egr-1: master regulator zinc finger transcription factor Egr-1 (animal models)

Restenosis via inhibition of smooth muscle cell hyperplasia

Intracoronary administration reduces neointima formation after stent implantation

Reduce size of myocardial infarct VEGF: Inhibit tumor growth via inhibition of angiogenesis


Antisense OligonucleotidesAntisense Oligonucleotides

Over 50 Clinical Studies Infection:

Fomivirsen (Vitravene): targets immediate - early RNA of human CMV DNA -- approved by US FDA to treat CMV retinitis

Inflammation: Alicaforsen: targets ICAM-1 to treat inflammatory

bowel disease Cancer:

Aprinocarsen: targets protein kinase C- to treat non-small cell lung carcinoma

Genasense (oblimersen): targets bcl-2 to treat CLL, AML, myeloma


DecoysDecoys

Short double-stranded oligonucleotides Binding elements for protein targets

Competitively inhibit promoter binding Inhibit gene expression

Reduction of neointima formation and atherosclerosis in animal models: E2F decoy

Edifoligide (PREVENT IV Trial) to prevent vein graft failure in coronary artery bypass grafting


RibozymesRibozymes

Catalytically active RNA molecules that cleave target mRNA in a site-specific manner

Can occur naturally HEPTAZYME: targets 5’-untranslated region of

hepatitis C virus– toxic Cancer therapy:

ANGIOZYME: targets VEGF receptor VEGF R1 (Flt-1) in solid tumors

HERzyme: targets human epidermal growth factor-2 in breast and ovarian cell carcinomas


Aptamers (Aptamers (aptusaptus= to fit)= to fit)

Synthetic oligonucleotides Bind target protein with high affinity and

specificity Chemical modifications for stability Pegaptanib: RNA aptamer targets VEGF165 to

treat neovascular age-related macular degeneration (US FDA approved)

Inhibit HIV-1


References References

1)1) Calin, GA and Croce, CM, Calin, GA and Croce, CM, MicroRNA signatures in human cancers.MicroRNA signatures in human cancers. 2006; 2006; Nature Reviews: CancerNature Reviews: Cancer 6:857-866. 6:857-866.

2)2) Cummins, JM and Velculescu, VE, Cummins, JM and Velculescu, VE, Implications of micro-RNA Implications of micro-RNA profiling for cancer diagnosis.profiling for cancer diagnosis. 2006; 2006; OncogeneOncogene 25:6220-6227. 25:6220-6227.

3)3) Bhindi, R, Fahmy, RG, et al., Bhindi, R, Fahmy, RG, et al., Brothers in Arms: DNA Enzymes, Brothers in Arms: DNA Enzymes, Short Interfering RNA, and the Emerging Wave of Small-Molecule Short Interfering RNA, and the Emerging Wave of Small-Molecule Nucleic Acid-Based Gene-Silencing Strategies.Nucleic Acid-Based Gene-Silencing Strategies. 2007; 2007; Am J PatholAm J Pathol 171:1079-1088.171:1079-1088.

4)4) Nie, K, Gomez, M, Landgraf, P, Garcia, J-F, Tan, L.C, Chadburn, A, Nie, K, Gomez, M, Landgraf, P, Garcia, J-F, Tan, L.C, Chadburn, A, Tuschi, T, Knowles, DM;, and Tam, W, Tuschi, T, Knowles, DM;, and Tam, W, MicroRNA-Mediated Down-MicroRNA-Mediated Down-Regulation of Regulation of PRDM1/Blimp-1PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg Cells: A in Hodgkin/Reed-Sternberg Cells: A Potential Pathogenetic Lesion in Hodgkin LymphomasPotential Pathogenetic Lesion in Hodgkin Lymphomas. 2008; . 2008; Am J Am J PatholPathol 173:242-252. 173:242-252.


The Future: MicroRNA (MiRNA, miR)The Future: MicroRNA (MiRNA, miR)

Expression profiles and mutation analysis of miRs will be standard procedure for determining the “molecular signature” of a diseased tissue.


This presentation will be available at http://www.asip.org/about/exec.htm



diagnostic and therapeutic potential of small nucleic acid molecules mark e. sobel, m.d., ph.d....

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