ADVANCED MOLECULAR BIOLOGY

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MOLECULAR MEDICINE David
Blicq dblicq_at_rrc.mb.ca Chemical Bioscience
Technology                 
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A Fantastic Voyage?
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Or the future of Medicine?
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Topics

• Diagnostics for Infectious Disease
• Diagnostics for Genetic Disease
• Gene Therapy
• Stem Cells
• Nanomedicine

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1. Diagnosis of Infectious Disease

• Recent developments have significantly altered
  the monitoring and diagnosis of infectious
  diseases
• There are two general methods for examining
  infectious disease
• microbial phenotype characterization
• nucleic acid techniques

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Shift to DNA-based testing
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Old School Diagnosis

• Microbial Phenotying (examine physical
  characteristics)
• Biotyping (grow organisms on media)
• Protein content (of pathogen)
• Bacteriophage profiles (virus analysis)
• Chromatography (membrane lipids)
• Antibiotic (susceptibility testing)

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Old School Diagnosis

• Biotyping (grow them)
• examines the physical / morphological
  characteristics
• includes growth media, biochemical uptake /
  usage, staining, etc.
• produces a "biogram" (a combination of analytical
  information)
• not always definitive, not always stable

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Old School Diagnosis

• Antibiotics / Resistograms
• test an organism's resistance to specific
  antibiotics
• develop a "resistogram" or "antibiogram" (a
  detailed profile of antibiotic resistance to a
  range of compounds)
• Problem! - common resistance to an antibiotic
  does not always indicate organisms are related!

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Old School Diagnosis

• Challenges
• Time have to culture and grow pathogen
• Lab Safety need to keep many pathogenic
  cultures alive in lab
• Accuracy not always definitive
• Limited Info no information on antibiotic
  resistance or virulence factors

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Molecular Diagnosis

• Can detect disease-causing agents without having
  to grow them (directly from sample)
• Can detect slow / hard to culture microbes
• Can amplify DNA to get more accurate results
• ID sub-species (excellent discrimination)
• ID genes that impart drug resistance (i.e. target
  the treatments)
• Fast results - automated systems available

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Molecular Diagnosis

• Overall  - PCR and nucleic acid amplification
  technology has one enormous benefit bacterial
  growth is no longer necessary to detect and
  characterize microorganisms!
• "Amplicons" (amplified products) are
  characterized by
• nucleic acid probe hybridization (labeled probes)
  
• analysis of fragments after restriction digestion
  
• direct sequence analysis

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Molecular Diagnosis

• Nucleic Acid Techniques
• Plasmid profiling (when characteristic plasmids
  are available)
• RFLP analysis (restriction fragment length
  polymorphisms - use RE to produce characteristic
  gel fragments)
• PCR (amplify key sequences which distinguish
  target microorganisms)

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Molecular Diagnosis

• Plasmid Analysis
• plasmids are small, self-replicating circular DNA
  molecules found in many bacteria
• plasmids often code for resistance to antibiotics
  and certain virulence factors
• widely-used for tracking resistance in disease
  outbreaks / pandemics
• can track transfer of resistance between
  hospitals, organisms, and countries
• weakness - can transfer between microbes

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Molecular Diagnosis

• Restriction Enzyme Pattern
• Cut DNA at specific location using natural
  enzymes restriction endonucleases
• get characteristic fragments RFLP's /
  restriction fragment length polymorphisms
• see fragments via electrophoresis
• very accurate can ID between microbial strains

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Molecular Diagnosis

• Uses of Restriction Enzyme Patterns
• identification of bacterial populations
• epidemiology (spread of disease through
  population), pandemic science
• study of Tuberculosis in HIV-positive patients
• combined with DNA fingerprinting (southern blot,
  etc.) it is a very powerful tool

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Molecular Diagnosis

• PCR Polymerase Chain Reaction
• Making copies of a DNA sequence
• PCR is conducted in vitro (beaker / test tube)
• 20 cycles of PCR can allow for a 1,000,000 X
  amplification of DNA samples
• Typical PCR reactions use small (ng-mg)
  quantities of DNA and go through 30-40
  amplification cycles
• PCR has revolutionized RD in biology / medicine
  and helped refine criminology and law

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PCR
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Molecular Diagnosis

• Nucleic Acid Probes - MicroArray
• can identify organisms at, or below species level
  
• can detect fastidious organisms directly
  (bacteria, viruses, mycobacteria, fungi and
  parasites)
• Commercial kits available Gen-probe, Microprobe,
  Digene etc. (all FDA approved)
• procedures are well-standardized
• use short, synthetic DNA probes for well
  understood characteristic sequences

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Molecular Diagnosis Probe/Microarray
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Summary - Molecular Diagnosis

• many different nucleic-acid based methods
• don't have to culture / grow organism (excellent
  for dangerous / fastidious organisms)
• can detect disease-causing gene mutations (in
  humans, etc.)
• can track drug resistance
• fast, sensitive, and improving all the time
• limit - contamination and amplification of
  contaminants

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2. Diagnostics for Genetic Disease

• Genetic diseases are transferred through families
• Often seemingly random
• Early awareness can lead to early therapy

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Genetic Disease - Trends

• reduced prevalence of infectious diseases due to
  vaccination, antibiotics and improved sanitation
• increased prevalence of genetic diseases due to
  increased life expectancies reduced prevalence of
  infectious diseases

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Why study Genetic Disease?

• may provide the biochemical basis to the disease
  (help designing therapies)
• can devise a screening program
• Identify mutant genes in individuals who are
  carriers
• To find the approximate position of the gene in
  the human genome (ex. breast cancer)
• Most have no family history of the disease in
  some a predisposition to acquiring the disease

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Methods of studying Genetic Disease

• Linkage analysis  - Comparing the inheritance
  pattern for the target gene with the inheritance
  patterns for healthy individuals
• Pedigree analysis need to obtain DNA samples
  from at least three generations of each family
• Example breast cancer research at HSC

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Pedigree / Linkage Analysis
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3. Gene Therapy

• Techniques that aim to cure an inherited disease
  by providing the patient with a correct copy of
  the defective gene
• Can include gene addition or gene subtraction

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Gene Therapy for Inherited Diseases

• Germline therapy uses a fertilized egg, so the
  gene is present in all cells of the resulting
  individual
• Somatic cell therapy healthy cells are removed
  from an organism then placed back in the body
  (with a retrovirus-based vector)

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Gene Therapy for Inherited Diseases

• Somatic cell therapy - good for inherited blood
  diseases (e.g., haemophilia, thalassemia stem
  cells from the bone marrow) and lung diseases
  (e.g., cystic fibrosis periodic inhaling of DNA
  in rats)
• no good method available yet for replacing a
  defective gene (necessary for a dominant one)
• Current therapies add a gene but cant replace
  the defective information yet

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Gene Therapy and Cancer

• gene therapy can be applied not only to inherited
  / infectious diseases but also cancer
• specific killing of cancer cells using
  cancer-specific promoters and toxin genes
• cause tumor cells to synthesize strong antigens
  that are efficiently recognized by the immune
  system
• suitable delivery methods to the cancerous cells
  are not yet available

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Gene Therapy and Cancer
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4. Stem Cells

• Stem cells are poised to revolutionize medical
  science
• Re-grow damaged tissues
• Fix otherwise lethal abnormalities
• Potential to repair birth defects before symptoms
  ever appear
• Cure incurable diseases (MS, ALS, etc.)

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Stem Cells

• There are two core characteristic of stem cells
  that set them apart from other cell and tissue
  types
• Differentiation - they can differentiate into
  many different cell types
• Replication - they continue to grow and replicate
  to replace tissues, etc.

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Stem Cells
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Somatic Stem Cells

• Somatic stem cells occur in different types
• Haematopoietic stem cells blood-forming stem
  cells are found in the bone marrow as well as the
  umbilical cord of newborn babies.
• Stromal stem cells (bone marrow cells) can
  differentiate into cartilage, fat/adipocytes and
  bone.
• Neural stem cells can differentiate into various
  neural cells including neurons and the
  myelin-sheath producing oligodendrocytes.

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Embryonic Stem Cells

• Derived from cells of the inner cell mass of the
  blastocyst (lt5 day old embryonic cell mass)
  -typically has less than 160 cells in total.
• Like Somatics, Embryonic stem cells have two core
  characteristics
• an  unlimited capacity to self-replicate
•  the capability (potency) of differentiating into
  any one of the more than two hundred identified
  tissue types found in the human body. 

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Save Your Cells!

• Currently, more than 45 disorders can be treated
  with with therapies that involve the use of stem
  cells from umbilical cord blood
• A number of independent companies now offer to
  "bank" a baby's umbilical cord blood as a
  potential source of stem cells which could one
  day combat currently untreatable disorders 

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Stem Cell Therapy Requirements

• Proliferation - stem cells must replicate in
  quantities that make them therapeutically
• Differentiation - stem cells must possess the
  appropriate level of differentiation
• Biocompatibility - stem cells must not illicit an
  antigenic response from the
• Longevity - therapeutic stem cells must survive
  as long as the cells they are intended to
  replace.

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Therapy Research
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5. Nanomedicine

• Nanotechnology is the study and interaction with
  materials and systems at the nanoscale level - or
  approximately 0.1-100 x 10-9 meters. 
• At this microscopic scale, scientists and
  engineers are interacting with materials at the
  molecular and even atomic level, where the
  chemical, physical, electrical and biological
  properties  are being viewed and understood as
  never before

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Nano technologies

• nanomedicine
• biological sensors and diagnostics
• micro-mechanical devices and nano-machinery
• detection and treatment of disease
• molecular-level assembly and manufacturing
• nano-factories and production
• self-replicating mico-machines
• manipulation of events "atom by atom"

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Nano benefits

• Enhanced Diagnostics - diagnosis and repair
  before the disease occurs
• Sensitivity and Resolution - small, even
  molecular-level conditions can be observed and
  assessed at the cellular level
• Automation - self-directing nano-machines will
  find diseased cells and initiating repairs
• Artificial Immunology - micromachines could be
  set to track down and eliminate specific diseases

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Example
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Free Online Resources - click to visit site
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Free Online Resources - click to visit site
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Free Online Resources - click to visit site
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Free Online Resources - click to visit site
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Free Online Resources - click to visit site
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Free Online Resources - click to visit site
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MOLECULAR MEDICINE David
Blicq dblicq_at_rrc.mb.ca Chemical Bioscience
Technology