molecular medicine presentation

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


MOLECULAR MEDICINEMOLECULAR MEDICINE

David Blicq [email protected] Chemical Bioscience TechnologyDavid Blicq [email protected] Chemical Bioscience Technology


A Fantastic Voyage?A Fantastic Voyage?


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


TopicsTopics

1.1. Diagnostics Diagnostics for Infectious for Infectious Disease Disease

2.2. Diagnostics Diagnostics for Genetic for Genetic DiseaseDisease

3.3. Gene TherapyGene Therapy4.4. Stem CellsStem Cells5.5. NanomedicineNanomedicine


1. Diagnosis of Infectious 1. Diagnosis of Infectious DiseaseDisease Recent developments have Recent developments have

significantly altered the significantly altered the monitoring and diagnosis of monitoring and diagnosis of infectious diseasesinfectious diseases

There are two general methods for There are two general methods for examining infectious disease:examining infectious disease:

microbial phenotype microbial phenotype characterization characterization

nucleic acid nucleic acid techniques techniques


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


““Old School” DiagnosisOld School” Diagnosis

Microbial Phenotying (examine Microbial Phenotying (examine physical characteristics)physical characteristics)

BiotypingBiotyping (grow organisms on media) (grow organisms on media) Protein content Protein content (of pathogen)(of pathogen) BacteriophageBacteriophage profiles (virus profiles (virus

analysis)analysis) ChromatographyChromatography (membrane lipids) (membrane lipids) AntibioticAntibiotic (susceptibility testing) (susceptibility testing)



Biotyping (“grow them”)Biotyping (“grow them”) examines the physical / morphological examines the physical / morphological

characteristics characteristics includes growth media, biochemical includes growth media, biochemical

uptake / usage, staining, etc. uptake / usage, staining, etc. produces a produces a "biogram""biogram" (a combination (a combination

of analytical information) of analytical information) notnot always definitive, not always always definitive, not always

stablestable



Antibiotics / ResistogramsAntibiotics / Resistograms test an organism's resistance to test an organism's resistance to

specific antibiotics specific antibiotics develop a "resistogram" or develop a "resistogram" or

"antibiogram" (a detailed profile of "antibiogram" (a detailed profile of antibiotic resistance to a range of antibiotic resistance to a range of compounds) compounds)

Problem!Problem! - common resistance to an - common resistance to an antibiotic does antibiotic does not not always indicate always indicate organisms are related! organisms are related!



Challenges:Challenges:

TimeTime – have to culture and grow – have to culture and grow pathogenpathogen

Lab Safety Lab 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 Info Limited Info – no information on – no information on

antibiotic resistance or virulence antibiotic resistance or virulence factorsfactors


Molecular DiagnosisMolecular Diagnosis

Can detect disease-causing agents Can detect disease-causing agents without without having to grow themhaving to grow them (directly from sample) (directly from sample)

Can detect Can detect slowslow / hard to culture/ hard to culture microbes microbes Can Can amplifyamplify DNA to get more accurate results DNA to get more accurate results ID sub-species ID sub-species (excellent discrimination) (excellent discrimination) ID genes that ID genes that impart drug resistance impart drug resistance (i.e. (i.e.

target the treatments) target 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: bacterial bacterial growth is no longer necessary to detect and growth is no longer necessary to detect and characterize microorganisms!characterize microorganisms!

"Amplicons" "Amplicons" (amplified products)(amplified products) are are 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 digestion analysis of fragments after restriction digestion

3.3. direct sequence analysis direct sequence analysis



Nucleic Acid Techniques:Nucleic Acid Techniques:

Plasmid profiling Plasmid profiling (when (when characteristic plasmids are available) characteristic plasmids are available)

RFLP analysis RFLP analysis (restriction fragment (restriction fragment length polymorphisms - use RE to length 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)


Molecular DiagnosisMolecular DiagnosisPlasmid AnalysisPlasmid Analysis plasmids are small, self-replicating circular plasmids are small, self-replicating circular

DNA molecules found in many bacteria DNA molecules found in many bacteria plasmids often code for plasmids often code for resistance to resistance to

antibiotics antibiotics and certain and certain virulencevirulence factors factors widely-used for tracking resistance in widely-used for tracking resistance in

disease outbreaks / pandemicsdisease outbreaks / pandemics can track can track transfer of resistancetransfer of resistance: between : between

hospitals, organisms, and countries hospitals, organisms, and countries weaknessweakness - can transfer between microbes - can transfer between microbes



Restriction Enzyme PatternRestriction Enzyme Pattern Cut DNA at specific location using natural Cut DNA at specific location using natural

enzymes: enzymes: restriction endonucleasesrestriction endonucleases get characteristic fragments: get characteristic fragments: “RFLP's / “RFLP's /

restriction fragment length restriction fragment length polymorphismspolymorphisms” ”

see fragments via electrophoresissee fragments via electrophoresis veryvery accurate – can ID between microbial accurate – can ID between microbial

strains strains



Uses of Restriction Enzyme Patterns:Uses of Restriction Enzyme Patterns: identification of bacterial populations identification of bacterial populations epidemiology (spread of disease through epidemiology (spread of disease through

population), pandemic science population), pandemic science study of Tuberculosis in HIV-positive study of Tuberculosis in HIV-positive

patients patients combined with DNA fingerprinting combined with DNA fingerprinting

(southern blot, etc.) it is a very powerful (southern blot, etc.) it is a very powerful tool 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 PCR is conducted in vitro (beaker / test

tube)tube) 20 cycles of PCR can allow for a 1,000,000 20 cycles of PCR can allow for a 1,000,000

X amplification of DNA samplesX amplification of DNA samples Typical PCR reactions use small (ng-mg) Typical PCR reactions use small (ng-mg)

quantities of DNA and go through 30-40 quantities 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 medicine and helped refine criminology and lawlaw



PCRPCR


Nucleic Acid Probes - MicroArrayNucleic Acid Probes - MicroArray can identify organisms at, or below can identify organisms at, or below speciesspecies

level level can detect fastidious organisms directly can detect fastidious organisms directly

(bacteria, viruses, mycobacteria, fungi and (bacteria, viruses, mycobacteria, fungi and parasites) 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-standardized procedures are well-standardized use short, synthetic DNA probes for well use short, synthetic DNA probes for well

understood characteristic sequences understood characteristic sequences



Molecular Diagnosis – Molecular Diagnosis – Probe/MicroarrayProbe/Microarray


many different nucleic-acid based methods many different nucleic-acid based methods don't have to culture / grow organism don't have to culture / grow organism

(excellent for dangerous / fastidious (excellent for dangerous / fastidious organisms) organisms)

can detect disease-causing gene mutations can detect disease-causing gene mutations (in humans, etc.) (in humans, etc.)

can track drug resistance can track drug resistance fast, sensitive, and improving all the time fast, sensitive, and improving all the time limit - contamination and amplification of limit - contamination and amplification of

contaminantscontaminants

Summary - Molecular Summary - Molecular DiagnosisDiagnosis


Genetic diseases are transferred Genetic diseases are transferred through familiesthrough families

Often “seemingly random”Often “seemingly random” Early awareness can lead to early Early awareness can lead to early

therapytherapy

2. 2. Diagnostics for Genetic Diagnostics for Genetic DiseaseDisease


reducedreduced prevalence of prevalence of infectious diseasesinfectious diseases due to due to vaccination, antibiotics and vaccination, antibiotics and improved sanitation improved sanitation

increasedincreased prevalence of prevalence of genetic genetic diseasesdiseases due to increased life due to increased life expectancies reduced prevalence expectancies reduced prevalence of of infectious diseasesinfectious diseases

Genetic Disease - TrendsGenetic Disease - Trends


may provide the biochemical basis to the may provide the biochemical basis to the disease (help designing therapies) disease (help designing therapies)

can devise a screening program can devise a screening program Identify mutant genes in individuals who Identify mutant genes in individuals who

are carriersare carriers To find the approximate position of the gene To find the approximate position of the gene

in the human genome (ex. breast cancer) in the human genome (ex. breast cancer) Most have no family history of the disease; Most have no family history of the disease;

in some a predisposition to acquiring the in some a predisposition to acquiring the diseasedisease

Why study Why study Genetic Disease?Genetic Disease?


Linkage analysis Linkage analysis - Comparing the - Comparing the inheritance pattern for the target gene inheritance pattern for the target gene with the inheritance patterns for healthy with the inheritance patterns for healthy individuals individuals

““Pedigree analysis”Pedigree analysis” need to obtain DNA need to obtain DNA samples from at least three generations samples from at least three generations of each family of each family

Example – breast cancer research at HSCExample – breast cancer research at HSC

Methods of studying Methods of studying Genetic Genetic DiseaseDisease


Pedigree / Linkage AnalysisPedigree / Linkage Analysis


Techniques that aim to cure an Techniques that aim to cure an inherited disease by providing the inherited disease by providing the patient with a correct copy of the patient with a correct copy of the defective genedefective gene

Can include “gene addition” or Can include “gene addition” or “gene subtraction” “gene subtraction”

3. Gene Therapy3. Gene Therapy


Germline therapy Germline therapy – uses a – uses a fertilized egg, so the gene is present fertilized egg, so the gene is present in all cells of the resulting individual in all cells of the resulting individual

Somatic cell therapy Somatic cell therapy – healthy – healthy cells are removed from an organism cells are removed from an organism then placed back in the body (with a then placed back in the body (with a retrovirus-based vector)retrovirus-based vector)

Gene Therapy for Inherited Gene Therapy for Inherited DiseasesDiseases


Somatic cell therapy Somatic cell therapy - good for inherited - good for inherited blood diseases (e.g., haemophilia, blood diseases (e.g., haemophilia, thalassemia; stem cells from the bone thalassemia; stem cells from the bone marrow) and lung diseases (e.g., cystic marrow) and lung diseases (e.g., cystic fibrosis; periodic inhaling of DNA in rats) fibrosis; periodic inhaling of DNA in rats)

no good method available yet for no good method available yet for replacingreplacing a defective gene (necessary for a defective gene (necessary for a dominant one) a dominant one)

Current therapies Current therapies add a gene add a gene but can’t but can’t replace the defective information yetreplace the defective information yet

Gene Therapy for Inherited Gene Therapy for Inherited DiseasesDiseases


gene therapy can be applied not only to gene therapy can be applied not only to inherited / infectious diseases but also inherited / infectious diseases but also cancer cancer

specific killing of cancer cells using cancer-specific killing of cancer cells using cancer-specific specific promoters and toxin genes promoters and toxin genes

cause tumor cells to synthesize cause tumor cells to synthesize strong strong antigens antigens that are efficiently recognized by that are efficiently recognized by the immune system the immune system

suitable delivery methods to the cancerous suitable delivery methods to the cancerous cells are not yet available cells are not yet available

Gene Therapy and CancerGene Therapy and Cancer


Gene Therapy and CancerGene Therapy and Cancer


Stem cells are poised to revolutionize Stem cells are poised to revolutionize medical science:medical science:

Re-grow damaged tissuesRe-grow damaged tissues Fix otherwise lethal abnormalitiesFix otherwise lethal abnormalities Potential to repair birth defects Potential to repair birth defects

before symptoms ever appearbefore symptoms ever appear Cure “incurable diseases” (MS, ALS, Cure “incurable diseases” (MS, ALS,

etc.)etc.)

4. Stem Cells4. Stem Cells


There are two core characteristic of There are two core characteristic of stem cells that set them apart from stem cells that set them apart from other cell and tissue types:other cell and tissue types:

DifferentiationDifferentiation - they can - they can differentiate into many different cell differentiate into many different cell typestypes

Replication Replication - they continue to grow - they continue to grow and replicate to replace tissues, etc. and 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 –blood-forming

stem cells are found in the bone marrow as stem cells are found in the bone marrow as well as the umbilical cord of newborn babies. well as the umbilical cord of newborn babies.

Stromal stem cellsStromal stem cells – (bone marrow cells) can – (bone marrow cells) can differentiate into cartilage, fat/adipocytes and differentiate into cartilage, fat/adipocytes and bone. bone.

Neural stem cellsNeural stem cells –can differentiate into –can differentiate into various neural cells including neurons and the various neural cells including neurons and the myelin-sheath producing myelin-sheath producing oligodendrocytesoligodendrocytes. .

Somatic Stem CellsSomatic Stem Cells


Derived from cells of the inner cell mass of the Derived 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 Like Somatics, Embryonic stem cells have two core characteristics:core characteristics:

an an unlimited unlimited capacity to self-replicate capacity to self-replicate the capability (potency) of the capability (potency) of differentiatingdifferentiating

into any one of the more than two hundred into any one of the more than two hundred identified tissue types found in the human identified tissue types found in the human body. body.

Embryonic Stem CellsEmbryonic Stem Cells


Currently, more than 45 disorders can Currently, more than 45 disorders can be treated with with therapies that be treated with with therapies that involve the use of stem cells from involve the use of stem cells from umbilical cord bloodumbilical cord blood

A number of independent companies A number of independent companies now offer to now offer to "bank" "bank" a baby's umbilical a baby's umbilical cord blood as a potential source of cord blood as a potential source of stem cells which could one day combat stem cells which could one day combat currently untreatable disorders currently untreatable disorders

Save Your Cells!Save Your Cells!


Proliferation Proliferation - stem cells must replicate in - stem cells must replicate in quantities that make them therapeuticallyquantities that make them therapeutically

DifferentiationDifferentiation - stem cells must possess - stem cells must possess the appropriate level of differentiationthe appropriate level of differentiation

Biocompatibility Biocompatibility - stem cells must not - stem cells must not illicit an antigenic response from theillicit an antigenic response from the

LongevityLongevity - therapeutic stem cells must - therapeutic stem cells must survive as long as the cells they are survive as long as the cells they are intended to replace. intended to replace.

Stem Cell Therapy Stem Cell Therapy RequirementsRequirements


Therapy ResearchTherapy Research


Nanotechnology Nanotechnology is the study and is the study and interaction with materials and systems at interaction with materials and systems at the nanoscale level - or approximately 0.1-the nanoscale level - or approximately 0.1-100 x 10100 x 10-9-9 meters. meters.

At this microscopic scale, scientists and At this microscopic scale, scientists and engineers are interacting with materials at engineers are interacting with materials at the molecular and even atomic level, where the molecular and even atomic level, where the chemical, physical, electrical and the chemical, physical, electrical and biological properties are being viewed and biological properties are being viewed and understood as never beforeunderstood as never before

5. Nanomedicine5. Nanomedicine


nanomedicinenanomedicine biological sensors and diagnosticsbiological sensors and diagnostics micro-mechanical devices and nano-micro-mechanical devices and nano-

machinerymachinery 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 - diagnosis and repair before the disease occursrepair before the disease occurs

Sensitivity and ResolutionSensitivity and Resolution - small, even - small, even molecular-level conditions can be observed molecular-level conditions can be observed and assessed at the cellular leveland assessed at the cellular level

AutomationAutomation - self-directing nano-machines - self-directing nano-machines will find diseased cells and initiating repairswill find diseased cells and initiating repairs

Artificial Immunology Artificial Immunology - micromachines - micromachines could be set to track down and eliminate could be set to track down and eliminate specific diseasesspecific diseases

““Nano” benefits:Nano” benefits:


Example:Example:


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MOLECULAR MEDICINEMOLECULAR MEDICINE

David Blicq [email protected] Chemical Bioscience TechnologyDavid Blicq [email protected] Chemical Bioscience Technology

molecular medicine presentation

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