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Víctor M. González - Introducción a la tecnología de aptámeros y posibles aplicaciones terapéuticas
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Víctor M. González - Introducción a la tecnología de aptámeros y posibles aplicaciones terapéuticas

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Introducción a la tecnología de aptámeros y posibles aplicaciones terapéuticas

Introducción a la tecnología de aptámeros y posibles aplicaciones terapéuticas

By Víctor M. González

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Víctor M. González - Introducción a la tecnología de aptámeros y posibles aplicaciones terapéuticas Víctor M. González - Introducción a la tecnología de aptámeros y posibles aplicaciones terapéuticas Presentation Transcript

  • Introduction to aptamer technology and possible therapeutic applications APTÁMEROS: NUEVAS POSIBILIDADES DE DESARROLLO Y APLICACIONES EN BIOTECNOLOGÍA 17 de Noviembre de 2011 Introducción a la tecnología de aptámeros y posibles aplicaciones terapéuticas Víctor M. González Servicio de Bioquímica-Investigación Hospital Ramón y Cajal (IRYCIS)
  • Introduction to aptamer technology and possible therapeutic applications APTAMERS: NEW DEVELOPMENT POSSIBILITIES AND APPLICATIONS IN BIOTECHNOLOGY November 17, 2011 Introduction to aptamer technology and possible therapeutic applications Víctor M. González Servicio de Bioquímica-Investigación Hospital Ramón y Cajal (IRYCIS)
  • Introduction to aptamer technology and possible therapeutic applications Number of publications related to aptamers (PubMed) 600 500 Publications 400 300 200 100 0 Year View slide
  • Introduction to aptamer technology and possible therapeutic applications What is an aptamer?ssDNA TGATCC ATTCGGATCAAGCTAGC RNA UGAUCC AUUCGGAUCAAGCUAGC ATTCGGATCAAGCTAGC CCTAGT View slide
  • Introduction to aptamer technology and possible therapeutic applications Secondary structures Chastain, M. and Tinoco Jr., I., (1991) Prog. Nucleic Acid Res. Mol. Biol. 41, 131-177.
  • Introduction to aptamer technology and possible therapeutic applications Tertiary structure The folded aptamers adopt stable conformations that allow molecular recognition of the target Overview of the Vitamin B12 aptamer structure Nature Structural Biology, 7(1):53-57
  • Introduction to aptamer technology and possible therapeutic applications Aptamers are obtained… • …from libraries of oligonucleotides with random sequences • …by an in vitro method called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) consisting of successive rounds of selection- amplification
  • Introduction to aptamer technology and possible therapeutic applications Oligonucleotide library Random positions Oligo 1 20-90 N Oligo 2 PCR amplification Theoretical : N = 40 440 = ~ 1024 different molecules Real: ~ 1015 different molecules
  • Introduction to aptamer technology and possible therapeutic applications Libraries used for SELEX • Classical library • Libraries with structural constraints – The variable region is located between conserved sequences that produce defined structures (hairpin, G-Quartett, pseudoknot) • Libraries based on known sequences – It has a known sequence including small amounts (1-30%) of all other nucleotides allow certain variations along the sequence • Libraries free of fixed sequences – Method: tailored SELEX – Very short conserved sequences to which an adapter is added that is removed after each PCR • Libraries based on genomic sequences – The genomic DNA is fragmented (50-500 nt) and the conserved sequences are added
  • Introduction to aptamer technology and possible therapeutic applications General scheme for a SELEX procedure Target DNA: Strand separation RNA: transcription Formation of DNA/RNA-target complexesPCR or RT-PCR amplification Separation of bound aptamers Target Target
  • Introduction to aptamer technology and possible therapeutic applications Methods of separation • Target binding affinity to a support (SELEX) • Plates or resins conjugated to streptavidin, antibodies, etc. • Colloidal gold, etc. • Filtering using nitrocellulose membranes. • Microfluidic systems (M-SELEX) • Chromatography (MonoLEX) • Centrifugation (Cell SELEX) • Large targets (cells, viruses, etc). • Gel electrophoresis under native conditions (PhotoSELEX) • Capillary electrophoresis (CE-SELEX)
  • Introduction to aptamer technology and possible therapeutic applications SELEX Target DNA: Strands separation RNA: transcription Formation of the ssDNA/RNA-target complexesPCR or RT-PCR amplification Separation of bound aptamers Target Target
  • Introduction to aptamer technology and possible therapeutic applications Preparation of aptamers PCR or RT-PCR ssDNA pool dsDNA RNA pool• Labeling with 5‘ phosphate and L- exonuclease treatment• Biotin labeling and separation with streptavidin• Thermal denaturation• PAGE separation• Asymmetric PCR
  • Introduction to aptamer technology and possible therapeutic applications Polyclonal and monoclonal aptamers3-15 rounds of selection-amplificationSet of aptamers withdifferent degrees ofaffinity for the target Comparable tomolecule polyclonal antibodies Cloning and Comparable to characterization of monoclonal antibodies aptamer
  • Introduction to aptamer technology and possible therapeutic applications Main advantages of aptamers over antibodies Aptamers Antibodies Aptamers are produced by chemical Antibodies often suffer from batch to batch synthesis resulting in little or no batch to variation batch variation Aptamers are identified through an in vitro Requires the use of animals process not requiring animals Aptamers may be obtained against non- Antibodies may be obtained only against immunogenic proteins and toxins immunogenic proteins but not against target representing constituents of the body and toxic substances Denatured aptamers can be regenerated Antibodies have limited shelf life and are within minutes, aptamers are stable to sensitive to temperature and may undergo long term storage and can be transported denaturation at ambient temperature Selection conditions can be manipulated Identification of antibodies that recognize to obtain aptamers stable in a wide range targets under conditions other than of environmental conditions including pH physiological is not feasible and temperature Reporter molecules can be attached to Labelling of antibodies can cause loss in aptamers at precise locations not involved affinity in binding They are not immunogenic They are usually immunogenic Aptamers can be modified increasing their The stability of the antibodies cannot be stability easily altered Their small size allows for more efficient Their larger size limits their access to entry into the cell and its compartments cellular compartments
  • Introduction to aptamer technology and possible therapeutic applications
  • Introduction to aptamer technology and possible therapeutic applications Potential targets of the aptamers • Aptamers are capable of binding: – small organic or inorganic molecules – nucleotides – nucleic acids – peptides and proteins – membranes, cells and whole organisms (virus)
  • Introduction to aptamer technology and possible therapeutic applications Applications of aptamers • biotechnological tools • diagnostic systems • therapeutic agents
  • Introduction to aptamer technology and possible therapeutic applications Applications of aptamers as therapeutic• Aptamers targeting coagulation factors e.g against factor IXa• Aptamers targeting growth factors or hormones e.g against VEGF• Aptamers targeting antibodies involved in autoimmune diseases e.g. auto-antibodies against nicotinic AChRs (for m gravis)• Aptamers targeting inflammation markers e.g against elastase• Aptamers targeting neuropathological targets e.g against synthetic β-amyloid peptide (Alzheimer)• Aptamers against infectious diseases e.g against gp120 or HA• Aptamers targeting membrane biomarkers e.g against CTL-4• Aptamers targeting whole organisms e.g against CMV
  • Introduction to aptamer technology and possible therapeutic applications Aptamers in use or in clinical trials
  • Introduction to aptamer technology and possible therapeutic applications Mechanism of action of MACUGEN• Macugen is a chemically synthesized aptamer that binds to and inhibits the function of VEGF.• VEGF is a protein that plays an important role in the abnormal growth of blood vessels associated with AMD (age-related macular degeneration) or DME (diabetic macular edema).
  • Introduction to aptamer technology and possible therapeutic applications Mechanism of action of MACUGEN Macugen VEGF Endotelial cell VEGF receptors
  • Introduction to aptamer technology and possible therapeutic applications Projects of the laboratory• Biotechnological or diagnostic tool – Aptamers against proteins of Leishmania (LiKmp-11, LiH2A, LiH3, LiPABP) (Dr. Manuel Soto, Centro de Biología Molecular Severo Ochoa-UAM, Madrid; Aptus Biotech) – Aptamers against NL1Tc endonuclease of T. cruzi (Dr. Manuel Carlos López, Instituto de Parasitología y Biomedicina “López Neyra”, Granada) – Aptamers against CD3, CD4 and CD8 (Dr. Ernesto Roldán, Hospital Ramón y Cajal, Madrid) – Aptamers against β-amiloid peptide and “tau” protein (Drs. Ginés Lifante and Juan Jiménez, Universidad Autónoma de Madrid) – Aptamers against bacteria (Bioapter SL) – Aptamers against Apo A IV (Dr. Mª Dolores López Tejero. Facultad de Biología. Universidad de Barcelona y CEREMET-UB, Barcelona; Aptus Biotech)• Therapeutic agents – Aptamers against proteins involved in translation (4E-BP1, eIF4E and Mnk1 kinase) (Dr. M. Elena Martín, Hospital Ramón y Cajal, Madrid) – Aptamers against TLR-4 (Dr. Ignacio Lizasoaín, U. Complutense, Madrid; Aptus Biotech) – Aptamers against purinergic receptors T2X (Dr. Juan M. Gómez, Hospital Ramón y Cajal, Madrid) – Aptamers against abscisic acid (ABA) (Dr. Elena Zocchi, Universidad de Génova, Italia)
  • Introduction to aptamer technology and possible therapeutic applications Selection of aptamers against LiH2A RND40 (ssDNA) HIS-LiH2A DNA: Strands separation by Ni2+ resin thermal denaturation Formation of ssDNA/RNA-targetPCR amplification 3 rounds of complexes selection Selection by centrifugation
  • Introduction to aptamer technology and possible therapeutic applications Binding affinity of SELH2A for LiH2A proteinA BC D SELH2A 2.5 μg/mL 10 μg/mL 5 μg/mL 500 ng BSA 500 ng 100 ng H2A 50 ng 25 ng -
  • Introduction to aptamer technology and possible therapeutic applications Mapping of the SELH2A-protein interactionA PEPTIDE #1 SEQUENCE MATPRSAKKAVRKSGSKSAK C pept5 #2 SKSAKCGLIFPVGRVGGMMR #3 GGMMRRGQYARRIGASGAVY #4 SGAVYLAAVLEYLTAELLEL #5 ELLELSVKAAAQSGKKRCRL pept8 #6 KRCRLNPRTVMLAARHDDDI #7 HDDDIGTLLKNVTLSHSGVV #8 HSGVVPNISKAMAKKKGGKK #9 KGGKKGKATPSA DB 7500 b b pept8 b Abs 405nm 5000 c 2500 pept5 0 - 2A pe 1 pe 2 pe 3 pe 4 pe 5 pe 6 pe 7 pe 8 9 pt pt pt pt pt pt pt pt pt H pe
  • Introduction to aptamer technology and possible therapeutic applications Binding capabilityA 15000 B 12500 Abs 405 nm 10000 17500 15000 12500 7500 Abs 405nm Rd3 10000 LiAPT1 LiAPT2 7500 5000 5000 2500 0 0 25 50 100 200 400 800 800 2500 H2A BSA Protein (ng/well) 0 0 100 200 300 400 500 600 700 800 900 Protein (ng/well) C 10000 Rd3 AptLiH2A#1 8000 AptLiH2A#2 Abs 405nm 6000 4000 2000 0 0 10 20 30 40 50 60 70 time (min)
  • Introduction to aptamer technology and possible therapeutic applications Specificity of the SELH2A aptamers AptLiH2A#1 AptLiH2A#2 T C N R T C N R H2A
  • Introduction to aptamer technology and possible therapeutic applications Sequences of aptamers from SELH2AAptLiH2A#1 5´-GCG GAT GAA GAC TGG TGT TGT GCA ATG ATT TTT CCG GTT GAC CAG GTA GGA ATT GTA GGC CCT AAA TAC GAG CAA C-3´AptLiH2A#2 5´-GCG GAT GAA GAC TGG TGT GGA GTC TAC CCT GTT TTC TAG TCT GCC ATC CCT ATC CCA TGC CCT AAA TAC GAG CAA C-3´
  • Introduction to aptamer technology and possible therapeutic applications Mapping of the H2A-aptamer interactionA PEPTIDE #1 SEQUENCE MATPRSAKKAVRKSGSKSAK #2 #3 SKSAKCGLIFPVGRVGGMMR GGMMRRGQYARRIGASGAVY C pept5 #4 SGAVYLAAVLEYLTAELLEL #5 ELLELSVKAAAQSGKKRCRL #6 KRCRLNPRTVMLAARHDDDI pept8 #7 HDDDIGTLLKNVTLSHSGVV #8 HSGVVPNISKAMAKKKGGKK #9 KGGKKGKATPSA B 10000 b b 7500 b DO 405nm 5000 b b 2500 0 l 2A 1 2 3 4 5 6 7 8 9 ro pt pt pt pt pt pt pt pt pt nt H pe pe pe pe pe pe pe pe pe co Rd3 AptLiH2A#1 AptLiH2A#2
  • Introduction to aptamer technology and possible therapeutic applications Aptamers are able to specifically bind LiH2A A Bound B Bound AptLiH2A#1 AptLiH2A#2 Unbound AptLiH2A#1 AptLiH2A#2 Lysate Unbound Lysate kDa M M 94 67 43 30 20 LiH2A 14 rLiH2A 29% 79% 97%
  • Introduction to aptamer technology and possible therapeutic applicationsLaboratory of Aptamers (Hospital Ramón yCajal)- Dr. Víctor M. González- Dr. M. Elena Martín- Eva M. García-Recio- M. Isabel Pérez-Morgado- Marta García- Dr. Natalia Guerra (2005-2010)- Edurne Ramos (2004-2009)Aptus Biotech- Dr. Gerónimo F. Gómez-Chacón- Marta SánchezCentro de Biología Molecular (CSIC-UAM)- Dr. Manuel Soto