Artificial Viruses: A Nanotechnological Approach to Gene Delivery Enrico Mastrobattista, Marieke A.E - PowerPoint PPT Presentation

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Artificial Viruses: A Nanotechnological Approach to Gene Delivery Enrico Mastrobattista, Marieke A.E

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Title: Artificial Viruses: A Nanotechnological Approach to Gene Delivery Enrico Mastrobattista, Marieke A.E


1
Artificial Viruses A Nanotechnological Approach
to Gene DeliveryEnrico Mastrobattista, Marieke
A.E.M. van der Aa, Wim E. Hennink and Daan J.A.
Crommelin
  • Trisha Blood
  • April 26, 2007

2
Introduction
  • Two approaches to clinical medicine
  • Gene therapy treatment of physiological disorder
    at the genetic level
  • Genetic vaccination induction of specific
    immunity via delivery of genetic material
  • Nucleic acids (DNA, antisense oligonucleotides,
    small interfering RNAs) utilized to combat
    gene-based diseases
  • Correct missing genes, replace defective genes,
    downregulate abberant gene expression
  • A carrier, or smart vehicle, necessary for
    implementation of such strategies

3
Genetic Delivery Systems
  • Synthetic, or man-made
  • Self-assembling DNA complex including positively
    charged molecules (polymers, peptides, lipids)
  • Nanometer scale (40-150nm)
  • Net positive surface charge
  • Viral vectors
  • Introduction of genetic
  • material into viral genome
  • while eliminating replication ability

4
Viral Vectors
  • A model system viruses superior to synthetic
    genetic carriers in transfection abilities
  • Restrictions outweigh benefits
  • Limited carrying capacity of therapeutic
  • nucleic acids
  • Lack of target cell specificity
  • Immunogenicity
  • Insertional mutagenesis
  • Large scale production problems

5
Smart Vehicles A marvel of Nanotechnology
  • Living organisms composed of cells approximately
    10mm in diameter
  • Organelles in sub-micron region
  • Proteins even smaller with typical
  • measurements of 5nm across
  • Nanoparticles of a comparable size, thus
    providing a means of delivering therapeutic genes
    to cell

6
Nanobiotechnology
  • Nano-bio science at the interface of
    nanotechnology and molecular biology
  • Combines aspects of chemistry, physics, molecular
    biology, electrical engineering, materials
    science to manipulate
  • cellular machinery
  • Construction on nanometer
  • scale leads to devices for
  • use in biology and medicine

7
Nanotechnology
  • Quantum mechanics replaces classical mechanics as
    governing factor in nanoparticle formation
  • Two opposing methods
  • top-down manufacture of nanoscale arrays via
    machining, templating and lithographic techniques
  • bottom-up atom-by-atom or molecule-by-molecule
    self-assembly of organic and inorganic materials
    into precise structures

8
Artificial Virus An Example of Nanotechnology
9
Challenges
  • Systemic, or biological, barriers
  • Hamper transport to affected organ sites
    following administration
  • Intravenous administration exposure to blood
    components resulting in premature destabilization
    of nucleic acid
  • Mononuclear phagocytic system (MPS) recognizes
    nanoparticles as foreign and removes from blood
    circulation
  • Local administration transport of gene carriers
    mediated by diffusion and convection slowed by
    nonspecific interactions or elevated interstitial
    fluid pressure

10
Intracellular Barriers
  • Endosomal compartment
  • Cytosolic environments
  • Nuclear envelope
  • Gene transcription regulatory elements
  • Immune response elicited by introduction of
    foreign DNA into mammalian cells

11
Requirements
  • Bio-compatible, bio-degradable, bio-invisible
  • Cell-binding and internalization
  • Endosomal escape
  • Cytosolic trafficking
  • Nuclear import
  • Controllable and sustained transgene expression

12
Nanoparticle Composition
  • Bio-compatable
  • Cationic molecules employed to condense DNA
  • Bio-degradable
  • Peptides derived from L-amino acids
  • Bio-invisible
  • Hydrophilic coat around carrier to prevent
    unwanted immune reactions

13
Cell Binding and Internalization
  • Conjugation of ligands to hydrophilic
  • coat for specific binding to cell-surface
  • receptors
  • Peptides, antibodies, and vitamins
  • Receptor binding leads to receptor-mediated
    endocytosis as preferred route of uptake into
    cell

14
Endosomal Escape
  • Dissociation of carrier from receptors triggering
    internalization process
  • Destabilization of endosomal membrane to allow
    translocation of carriers into cytosol
  • Examples
  • Peptides imitating amino-terminal fusion portion
    of influenza virus hemagglutinin
  • Protein listeriolysin O from Listeria
    monocytogenes

15
Cytosolic Trafficking
  • Molecular motors utilized for intracellular
    transport
  • Dyneins transport cargo along microtubules in
    retrograde fashion
  • Gene carriers expose peptides/proteins for
    interaction with dynein molecular motor complex

16
Nuclear Import
  • Limiting step in therapeutic gene delivery
  • Passive diffusion through nuclear pore complexes
    (NPC) prohibited
  • Nuclear import machinery utilized in manner
    similar to that of karyophylic proteins
  • Synthetic nuclear localization sequences (NLS)
    attached to imported DNA
  • NLS recognized by cytoplasmic karyopherins of
    import machinery to facilitate docking to NPC

17
Proposed Nanoparticle Structure
  • Core-shell artificial virus system with three
    components
  • Plasmid vector
  • Artificial virus core (pDNA, condensing agents,
    functional peptides)
  • Hydrophilic shell with attached targeting ligands

18
Nanoparticle Assembly
  • Plasmid DNA condensed with cationic compounds
  • Self-assembly process via formation of interchain
    electrostatic bonds
  • Important factors to consider pH, ionic
    strength, temperature, medium composition, DNA
    concentration
  • Plasmid DNA coated with functional groups and
    further fused to DNA-binding proteins
  • Core coated with hydrophilic polymer layer by
    acid-labile bonds
  • Targeting ligands attached to distal ends of
    polymer shell

19
Artificial Virus Life Cycle
  • Specific binding to target cell surface receptors
  • Endocytosis
  • Release of shell polymer due to low pH in
    endosome
  • Disintegration of core complex due to reduced
    redox potential of cytoplasm
  • Docking to molecular motors and transport to
    nucleus
  • Expression of transgene in nucleus

20
Antisense Therapy
  • Regulate transcription of disease-related genes
    in vivo
  • Antisense oligonucleotide 15-20 bp fragment of
    deoxynucleotides complementary to portion of
    target mRNA
  • Mechanism of action interfere with gene
    expression by preventing mRNA translation into
    required proteins
  • Antisense binding to target mRNA and subsequent
    cleavage of antisense-mRNA hybrid by RNaseH
    enzyme activity

21
Mechanism of Action Antisense Therapy
22
Nanoparticles and siRNA
  • What is siRNA?
  • Double-stranded RNA approximately 21 nucleotides
    in length with 2-nucleotide 3 overhangs per end
  • Each strand bears a 3 hydroxyl and 5 phosphate
    group
  • Gene silencing by targeting identical sequence of
    target mRNA for catalytic degradation degraded
    message no longer functional

23
siRNA Cancer Treatment
  • Triangular nanoparticle composed of packaging RNA
    (pRNA, discovered by Guo) from bacteria-infecting
    virus phi29
  • pRNA as delivery vehicle and siRNA as therapeutic
    agent
  • Folic acid as targeting agent

24
siRNA Delivery to Mammalian Cells
25
Nanotechnology Present and Future
  • Nanotechnology relevant in medical diagnosis,
    therapeutic delivery systems and cell/tissue
    engineering
  • Nanoparticles encapsulating DNA provide novel
    technique for introducing therapeutic nucleic
    acids into the body.
  • Nanomedicine holds great promise for instant
    pathogen diagnosis and destruction plus
    chromosome replacement and cell surgery

26
References
  • Chowdhury, E.H. Akaike, T. Curr. Gene Ther.
    2005, 5, 669.
  • Mastrobattista, E. van der Aa, M.A.E.M.
    Hennink, W.E. Crommelin, D.J.A. Nature Reviews
    Drug Discovery. 2006, 5, 115.
  • Salata, O.V. J. Nanobiotechnol. 2004, 23.
  • Kubik, T. Bogunia-Kubik, K. Sugisaka, M. Curr.
    Pharm. Biotechnol. 2005, 6, 17.
  • NCI Alliance for Nanotechnology in Cancer.
    http//nano.cancer.gov/news_center/monthly_feature
    _2006_august.asp
  • Wettig, S. University of Saskatchewan.
    http//homepage.usask.ca/sdw132/wettig_research_g
    enetherapy.htm
  • www.bergen.org/ACADEMY/Bio/cellbio/cellbio
  • www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Ill
    ustration/adenovirus.cfm?keyadenovirus
  • www.enchantedlearning.com/subjects/animals/cell
  • http//pbm.tnw.utwente.nl/research/dd.doc
  • www.answers.com/topic/dynein
  • www.boc.uu.se/boc14www/res_proj/pictures/antisense
    _big.JPG

27
Questions
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