Molecular medicine presentation

D.Blicq Red River College 2010 Prepared for STAM / SAG

MOLECULAR MEDICINEMOLECULAR MEDICINE
David Blicq dblicq@rrc.mb.ca Chemical Bioscience TechnologyDavid Blicq dblicq@rrc.mb.ca Chemical Bioscience Technology

A Fantastic Voyage?A Fantastic Voyage?

Or the future of Medicine?Or the future of Medicine?

TopicsTopics
1.1. Diagnostics forDiagnostics for
Infectious DiseaseInfectious Disease
2.2. Diagnostics forDiagnostics for
Genetic DiseaseGenetic Disease
3.3. Gene TherapyGene Therapy
4.4. Stem CellsStem Cells
5.5. NanomedicineNanomedicine

1. Diagnosis of Infectious Disease1. Diagnosis of Infectious Disease
 Recent developments have significantlyRecent developments have significantly
altered the monitoring and diagnosis ofaltered the monitoring and diagnosis of
infectious diseasesinfectious diseases
There are two general methods forThere are two general methods for
examining infectious disease:examining infectious disease:
 microbial phenotypemicrobial phenotype characterizationcharacterization
 nucleic acidnucleic acid techniquestechniques

Shift to DNA-based testing:Shift to DNA-based testing:

““Old School” DiagnosisOld School” Diagnosis
Microbial Phenotying (examineMicrobial Phenotying (examine
physical characteristics)physical characteristics)
 BiotypingBiotyping (grow organisms on media)(grow organisms on media)
 Protein contentProtein content (of pathogen)(of pathogen)
 BacteriophageBacteriophage profiles (virus analysis)profiles (virus analysis)
 ChromatographyChromatography (membrane lipids)(membrane lipids)
 AntibioticAntibiotic (susceptibility testing)(susceptibility testing)

Biotyping (“grow them”)Biotyping (“grow them”)
 examines the physical / morphologicalexamines the physical / morphological
characteristicscharacteristics
 includes growth media, biochemical uptakeincludes growth media, biochemical uptake
/ usage, staining, etc./ usage, staining, etc.
 produces aproduces a "biogram""biogram" (a combination of(a combination of
analytical information)analytical information)
 notnot always definitive, not always stablealways definitive, not always stable

Antibiotics / ResistogramsAntibiotics / Resistograms
 test an organism's resistance to specifictest an organism's resistance to specific
antibioticsantibiotics
 develop a "resistogram" or "antibiogram"develop a "resistogram" or "antibiogram"
(a detailed profile of antibiotic resistance to(a detailed profile of antibiotic resistance to
a range of compounds)a range of compounds)
 Problem!Problem! - common resistance to an- common resistance to an
antibiotic doesantibiotic does notnot always indicatealways indicate
organisms are related!organisms are related!

Challenges:Challenges:
 TimeTime – have to culture and grow pathogen– have to culture and grow pathogen
 Lab SafetyLab Safety – need to keep many– need to keep many
pathogenic cultures alive in labpathogenic cultures alive in lab
 AccuracyAccuracy – not always definitive– not always definitive
 Limited InfoLimited Info – no information on– no information on
antibiotic resistance or virulence factorsantibiotic resistance or virulence factors

Molecular DiagnosisMolecular Diagnosis
 Can detect disease-causing agentsCan detect disease-causing agents withoutwithout
having to grow themhaving to grow them (directly from sample)(directly from sample)
 Can detectCan detect slowslow / hard to culture/ hard to culture microbesmicrobes
 CanCan amplifyamplify DNA to get more accurate resultsDNA to get more accurate results
 ID sub-speciesID sub-species (excellent discrimination)(excellent discrimination)
 ID genes thatID genes that impart drug resistanceimpart drug resistance (i.e. target(i.e. target
the treatments)the treatments)
 Fast resultsFast results - automated systems available- automated systems available

 OverallOverall - PCR and nucleic acid amplification - PCR and nucleic acid amplification
technology has one enormous benefit:technology has one enormous benefit: bacterialbacterial
growth is no longer necessary to detect andgrowth is no longer necessary to detect and
characterize microorganisms!characterize microorganisms!
"Amplicons""Amplicons" (amplified products)(amplified products) areare
characterized by:characterized by:
1.1. nucleic acid probe hybridization (labeled probes)nucleic acid probe hybridization (labeled probes)
2.2. analysis of fragments after restriction digestionanalysis of fragments after restriction digestion
3.3. direct sequence analysisdirect sequence analysis

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

Plasmid AnalysisPlasmid Analysis
 plasmids are small, self-replicating circular DNAplasmids are small, self-replicating circular DNA
molecules found in many bacteriamolecules found in many bacteria
 plasmids often code forplasmids often code for resistance to antibioticsresistance to antibiotics
and certainand certain virulencevirulence factorsfactors
 widely-used for tracking resistance inwidely-used for tracking resistance in diseasedisease
outbreaks / pandemicsoutbreaks / pandemics
 can trackcan track transfer of resistancetransfer of resistance: between: between
hospitals, organisms, and countrieshospitals, organisms, and countries
 weaknessweakness - can transfer between microbes- can transfer between microbes

Restriction Enzyme PatternRestriction Enzyme Pattern
 Cut DNA at specific location using naturalCut DNA at specific location using natural
enzymes:enzymes: restriction endonucleasesrestriction endonucleases
 get characteristic fragments:get characteristic fragments: “RFLP's /“RFLP's /
restriction fragment length polymorphismsrestriction fragment length polymorphisms””
 see fragments via electrophoresissee fragments via electrophoresis
 veryvery accurate – can ID between microbial strainsaccurate – can ID between microbial strains

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

PCR: Polymerase Chain ReactionPCR: Polymerase Chain Reaction
 Making copies of a DNA sequenceMaking copies of a DNA sequence
 PCR is conducted in vitro (beaker / test tube)PCR is conducted in vitro (beaker / test tube)
 20 cycles of PCR can allow for a 1,000,000 X20 cycles of PCR can allow for a 1,000,000 X
amplification of DNA samplesamplification of DNA samples
 Typical PCR reactions use small (ng-mg)Typical PCR reactions use small (ng-mg)
quantities of DNA and go through 30-40quantities of DNA and go through 30-40
amplification cyclesamplification cycles
 PCR has revolutionized R&D in biology /PCR has revolutionized R&D in biology /
medicine and helped refine criminology and lawmedicine and helped refine criminology and law

PCRPCR

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

Molecular Diagnosis – Probe/MicroarrayMolecular Diagnosis – Probe/Microarray

 many different nucleic-acid based methodsmany different nucleic-acid based methods
 don't have to culture / grow organism (excellentdon't have to culture / grow organism (excellent
for dangerous / fastidious organisms)for dangerous / fastidious organisms)
 can detect disease-causing gene mutations (incan detect disease-causing gene mutations (in
humans, etc.)humans, etc.)
 can track drug resistancecan track drug resistance
 fast, sensitive, and improving all the timefast, sensitive, and improving all the time
 limit - contamination and amplification oflimit - contamination and amplification of
contaminantscontaminants
Summary - Molecular DiagnosisSummary - Molecular Diagnosis

 Genetic diseases are transferred through familiesGenetic diseases are transferred through families
 Often “seemingly random”Often “seemingly random”
 Early awareness can lead to early therapyEarly awareness can lead to early therapy
2.2. Diagnostics for Genetic DiseaseDiagnostics for Genetic Disease

 reducedreduced prevalence ofprevalence of infectious diseasesinfectious diseases
due to vaccination, antibiotics anddue to vaccination, antibiotics and
improved sanitationimproved sanitation
 increasedincreased prevalence ofprevalence of genetic diseasesgenetic diseases
due to increased life expectancies reduceddue to increased life expectancies reduced
prevalence ofprevalence of infectious diseasesinfectious diseases
Genetic Disease - TrendsGenetic Disease - Trends

 may provide the biochemical basis to the diseasemay provide the biochemical basis to the disease
(help designing therapies)(help designing therapies)
 can devise a screening programcan devise a screening program
 Identify mutant genes in individuals who areIdentify mutant genes in individuals who are
carrierscarriers
 To find the approximate position of the gene inTo find the approximate position of the gene in
the human genome (ex. breast cancer)the human genome (ex. breast cancer)
 Most have no family history of the disease; inMost have no family history of the disease; in
some a predisposition to acquiring the diseasesome a predisposition to acquiring the disease
Why studyWhy study Genetic Disease?Genetic Disease?

 Linkage analysis Linkage analysis - Comparing the inheritance- Comparing the inheritance
pattern for the target gene with the inheritancepattern for the target gene with the inheritance
patterns for healthy individualspatterns for healthy individuals
 ““Pedigree analysis”Pedigree analysis” need to obtain DNAneed to obtain DNA
samples from at least three generations of eachsamples from at least three generations of each
familyfamily
 Example – breast cancer research at HSCExample – breast cancer research at HSC
Methods of studyingMethods of studying Genetic DiseaseGenetic Disease

Pedigree / Linkage AnalysisPedigree / Linkage Analysis

 Techniques that aim to cure an inherited diseaseTechniques that aim to cure an inherited disease
by providing the patient with a correct copy ofby providing the patient with a correct copy of
the defective genethe defective gene
 Can include “gene addition” or “geneCan include “gene addition” or “gene
subtraction”subtraction”
3. Gene Therapy3. Gene Therapy

 Germline therapyGermline therapy – uses a fertilized egg, so the– uses a fertilized egg, so the
gene is present in all cells of the resultinggene is present in all cells of the resulting
individualindividual
 Somatic cell therapySomatic cell therapy – healthy cells are– healthy cells are
removed from an organism then placed back inremoved from an organism then placed back in
the body (with a retrovirus-based vector)the body (with a retrovirus-based vector)
Gene Therapy for Inherited DiseasesGene Therapy for Inherited Diseases

 Somatic cell therapySomatic cell therapy - good for inherited blood- good for inherited blood
diseases (e.g., haemophilia, thalassemia; stem cellsdiseases (e.g., haemophilia, thalassemia; stem cells
from the bone marrow) and lung diseases (e.g.,from the bone marrow) and lung diseases (e.g.,
cystic fibrosis; periodic inhaling of DNA in rats)cystic fibrosis; periodic inhaling of DNA in rats)
 no good method available yet forno good method available yet for replacingreplacing aa
defective gene (necessary for a dominant one)defective gene (necessary for a dominant one)
 Current therapiesCurrent therapies add a geneadd a gene but can’t replacebut can’t replace
the defective information yetthe defective information yet
Gene Therapy for Inherited DiseasesGene Therapy for Inherited Diseases

 gene therapy can be applied not only togene therapy can be applied not only to
inherited / infectious diseases but also cancerinherited / infectious diseases but also cancer
 specific killing of cancer cells using cancer-specificspecific killing of cancer cells using cancer-specific
promoters and toxin genespromoters and toxin genes
 cause tumor cells to synthesizecause tumor cells to synthesize strong antigensstrong antigens
that are efficiently recognized by the immunethat are efficiently recognized by the immune
systemsystem
 suitable delivery methods to the cancerous cellssuitable delivery methods to the cancerous cells
are not yet availableare not yet available
Gene Therapy and CancerGene Therapy and Cancer

Gene Therapy and CancerGene Therapy and Cancer

Stem cells are poised to revolutionize medicalStem cells are poised to revolutionize medical
science:science:
 Re-grow damaged tissuesRe-grow damaged tissues
 Fix otherwise lethal abnormalitiesFix otherwise lethal abnormalities
 Potential to repair birth defects beforePotential to repair birth defects before
symptoms ever appearsymptoms ever appear
 Cure “incurable diseases” (MS, ALS, etc.)Cure “incurable diseases” (MS, ALS, etc.)
4. Stem Cells4. Stem Cells

There are two core characteristic of stem cells thatThere are two core characteristic of stem cells that
set them apart from other cell and tissue types:set them apart from other cell and tissue types:
 DifferentiationDifferentiation - they can differentiate into- they can differentiate into
many different cell typesmany different cell types
 ReplicationReplication - they continue to grow and- they continue to grow and
replicate to replace tissues, etc.replicate to replace tissues, etc.
Stem CellsStem Cells

Stem CellsStem Cells

 Somatic stem cells occur in different types:Somatic stem cells occur in different types:
 HaematopoieticHaematopoietic stem cellsstem cells –blood-forming stem–blood-forming stem
cells are found in the bone marrow as well as thecells are found in the bone marrow as well as the
umbilical cord of newborn babies.umbilical cord of newborn babies.
 Stromal stem cellsStromal stem cells – (bone marrow cells) can– (bone marrow cells) can
differentiate into cartilage, fat/adipocytes and bone.differentiate into cartilage, fat/adipocytes and bone.
 Neural stem cellsNeural stem cells –can differentiate into various–can differentiate into various
neural cells including neurons and the myelin-sheathneural cells including neurons and the myelin-sheath
producingproducing oligodendrocytesoligodendrocytes..
Somatic Stem CellsSomatic Stem Cells

 Derived from cells of the inner cell mass of theDerived from cells of the inner cell mass of the
blastocyst (<5 day old embryonic cell mass)blastocyst (<5 day old embryonic cell mass)
-typically has less than 160 cells in total.-typically has less than 160 cells in total.
 Like Somatics, Embryonic stem cells have two coreLike Somatics, Embryonic stem cells have two core
characteristics:characteristics:
 an an unlimitedunlimited capacity to self-replicatecapacity to self-replicate
 the capability (potency) ofthe capability (potency) of differentiatingdifferentiating intointo
any one of the more than two hundred identifiedany one of the more than two hundred identified
tissue types found in the human body. tissue types found in the human body.
Embryonic Stem CellsEmbryonic Stem Cells

 Currently, more than 45 disorders can be treatedCurrently, more than 45 disorders can be treated
with with therapies that involve the use of stemwith with therapies that involve the use of stem
cells fromcells from umbilical cord bloodumbilical cord blood
 A number of independent companies now offer toA number of independent companies now offer to
"bank""bank" a baby's umbilical cord blood as aa baby's umbilical cord blood as a
potential source of stem cells which could one daypotential source of stem cells which could one day
combat currently untreatable disorders combat currently untreatable disorders
Save Your Cells!Save Your Cells!

 ProliferationProliferation - stem cells must replicate in- stem cells must replicate in
quantities that make them therapeuticallyquantities that make them therapeutically
 DifferentiationDifferentiation - stem cells must possess the- stem cells must possess the
appropriate level of differentiationappropriate level of differentiation
 BiocompatibilityBiocompatibility - stem cells must not illicit an- stem cells must not illicit an
antigenic response from theantigenic response from the
 LongevityLongevity - therapeutic stem cells must survive as- therapeutic stem cells must survive as
long as the cells they are intended to replace.long as the cells they are intended to replace.
Stem Cell Therapy RequirementsStem Cell Therapy Requirements

Therapy ResearchTherapy Research

 NanotechnologyNanotechnology is the study and interaction withis the study and interaction with
materials and systems at the nanoscale level - ormaterials and systems at the nanoscale level - or
approximately 0.1-100 x 10approximately 0.1-100 x 10-9-9
meters. meters.
 At this microscopic scale, scientists and engineersAt this microscopic scale, scientists and engineers
are interacting with materials at the molecular andare interacting with materials at the molecular and
even atomic level, where the chemical, physical,even atomic level, where the chemical, physical,
electrical and biological properties are beingelectrical and biological properties are being
viewed and understood as never beforeviewed and understood as never before
5. Nanomedicine5. Nanomedicine

 nanomedicinenanomedicine
 biological sensors and diagnosticsbiological sensors and diagnostics
 micro-mechanical devices and nano-machinerymicro-mechanical devices and nano-machinery
 detection and treatment of diseasedetection and treatment of disease
 molecular-level assembly and manufacturingmolecular-level assembly and manufacturing
 nano-factories and productionnano-factories and production
 self-replicating mico-machinesself-replicating mico-machines
 manipulation of events "atom by atom"manipulation of events "atom by atom"
““Nano” technologies:Nano” technologies:

 Enhanced DiagnosticsEnhanced Diagnostics - diagnosis and repair- diagnosis and repair
before the disease occursbefore the disease occurs
 Sensitivity and ResolutionSensitivity and Resolution - small, even- small, even
molecular-level conditions can be observed andmolecular-level conditions can be observed and
assessed at the cellular levelassessed at the cellular level
 AutomationAutomation - self-directing nano-machines will- self-directing nano-machines will
find diseased cells and initiating repairsfind diseased cells and initiating repairs
 Artificial ImmunologyArtificial Immunology - micromachines could be- micromachines could be
set to track down and eliminate specific diseasesset to track down and eliminate specific diseases
““Nano” benefits:Nano” benefits:

Example:Example:

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