Personalized Medicine - scientific and commercial aspects

0. Executive Summary ................. 16

1. Basic Aspects...... 18

Definition of personalized medicine  18

Historical evolution of medical concepts .............. 19

Molecular biological basis of personalized medicine ................ 21

The human genome....21

Chromosomes............22

Genes.22

The genetic code...22

Gene expression ...22

DNA sequences and structure .....23

Single nucleotide polymorphisms 23

Genotype and haplotypes...........24

Non-SNP genetic variations .............24

Insertions and deletions in the human genome ..24

Large scale variation in human genome ............25

Variation in copy number in the human genome.25

Structural variations in human genome.............26

Basics technologies for developing personalized medicine ...... 26

Definitions of technologies relevant to personalized medicine........26

Problems with the ICH definitions of pharmacogenomcis and pharmacogenetics...27

Relationship of various technologies to personalized medicine.......27

Conventional medicine versus personalized medicine.............. 28

Role of genetics in future approaches to healthcare ................ 29

Genetic medicine........29

Human disease and genes...............29

Mass analysis of DNA from whole populations .........30

Role of genetics in development of personalized medicines...........30

Genetic databases.30

Genetic epidemiology.................31

Limitations of medical genetics and future prospects ..............31

Genetics vs. epigenetics.............32

Role of systems biology in personalized medicine 32

A personalized approach to environmental factors in disease.. 32

Reclassification of diseases............. 33

2. Molecular Diagnostics in Personalized Medicine ............... 36

Introduction............... 36

Molecular diagnostic technologies .. 36

Direct dideoxy DNA sequencing (DDS)...................37

Single-stranded conformation polymorphism (SSCP) analysis .......37

Heteroduplex analysis (HA) .............37

The WAVE System......38

DirectLinear™ Analysis38

Denaturing high-performance liquid chromatography (DHPLC)......38

Denaturing Gradient Gel electrophoresis (DGGE).....38

Chemical cleavage mismatch (CCM) .39

Multiplex Allele-Specific Diagnostic Assay ...............39

Representational oligonucleotide microarray analysis...................39

Cleavase Fragment Length Polymorphism (CFLP) ....39

Restriction fragment length polymorphism (RFLP) ...39

Arrayed primer extension (APEX) .....40

Enzymatic Mutation Detection (EMD) 40

T cells and molecular diagnosis ........40

The chorioallantoic membrane assay 41

Sequencing ...............41

Multiplex DNA sequencing ..........41

Sequencing in microfabricated high-density picoliter reactors ..42

Nanopore-based sequencing .......42

Electrophoresis in nanochannels..42

Whole genome sequencing ..............42

Biochips and microarrays................ 43

Application of biochip technology in developing personalized medicine ................43

Standardizing the microarrays .........45

Biochip technologies ...45

GeneChip .............45

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AmpliChip CYP450 .45

Microfluidics ..............47

Lab-on-a-chip .......48

Micronics' microfluidic technology48

LabCD..................48

Microfluidic automated DNA analysis using PCR..48

Integrated microfluidic bioassay chip ................49

Electronic detection of nucleic acids on microarrays .49

Strand displacement amplification on a biochip .......49

Rolling circle amplification on DNA microarrays .......50

Universal DNA microarray combining PCR and ligase detection reaction ..............50

Protein biochips .........50

ProteinChip...........51

LabChip for protein analysis........51

TRINECTIN proteome chip..........52

Protein expression microarrays ...52

Microfluidic devices for proteomics-based diagnostics .............53

New developments in protein chips...................53

Protein chips for personalized medicine.............54

SNP genotyping .......... 54

Haplotyping..............55

Haplotype Specific Extraction......55

Computation of haplotypes.........56

HapMap project.....56

Predictingdrug response with HapMap...............57

Companies developing haplotyping technology...57

Technologies for SNP analysis..........57

Biochip and microarray-based detection of SNPs .....58

SNP genotyping by MassARRAY...58

Biochip combining BeadArray and ZipCode technologies..........59

SNP-IT primer-extension technology.................59

OmniScan SNP genotyping .........60

Affymetrix Variation Detection Arrays ...............60

Use of NanoChip for detection of SNPs ..............60

DNA sequencing.........60

Electrochemical DNA probes ............61

Single base extension-tag array .......61

Laboratory Multiple Analyte Profile ...61

PCR-CTPP (confronting two-pair primers) ...............62

SNP genotyping on a genome-wide amplified DOP-PCR template...62

TaqMan real-time PCR 62

Non-Enzymatic Amplification Technology................63

SNP genotyping with gold nanoparticle probes ........63

Locked nucleic acid.....63

Molecular inversion probe based assays .................64

Pyrosequencing..........64

Reversed enzyme activity DNA interrogation test ....65

Smart amplification process version 265

Zinc finger proteins ....65

UCAN method (Takara Biomedical)...66

Mitochondrial SNPs.....66

Limitations of SNP in genetic testing.66

Concluding remarks on SNP genotyping .................67

Companies involved in developing technologies/products for SNP analysis...........67

Impact of SNPs on personalized medicine...............68

Optical Mapping .......... 69

Role of nanobiotechnology in molecular diagnostics................ 69

Cantilevers for personalized medical diagnostics .....70

Nanopore-based technology for single molecule identification .......70

Role of biomarkers in personalized medicine ....... 71

Biomarkers for diagnostics ..............71

Biomarkers for drug development ....72

Application of proteomics in molecular diagnosis  72

Proteomic strategies for biomarker identification .....72

Proteomic technologies for detection of biomarkers in body fluids..................73

Protein patterns .........73

Layered Gene Scanning ..................73

Comparison of proteomic and genomic approaches in personalized medicine .......74

Gene expression profiling ............... 74

DNA microarrays........75

Analysis of single-cell gene expression...................76

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Gene expression profiling based on alternative RNA splicing .........76

Whole genome expression array ......77

Tangerine™ expression profiling.......77

Gene expression analysis on biopsy samples ..........78

Serial analysis of gene expression (SAGE)..............78

Multiplexed Molecular Profiling .........79

Gene expression analysis using competitive PCR and MALDI TOF MS ..................79

Monitoring in vivo gene expression by magnetic resonance imaging ...................79

Companies involved in gene expression analysis .....80

Monitoring in vivo gene expression by molecular imaging ....... 81

Molecular imaging and personalized medicine ..... 81

Glycomics-based diagnostics .......... 81

Combination of diagnostics and therapeutics....... 82

Companies combining diagnostics and therapeutics.82

Point-of-care diagnosis................... 84

Companies developing point-of-care diagnostic technologies ........85

Point-of-care diagnosis of infections .87

Advantages versus disadvantages of point-of-care diagnosis ........87

Future prospects of point-of-care diagnosis ............88

Role of diagnostics in integrated healthcare ........ 88

Concept of integrated healthcare......88

Components of integrated healthcare 89

Screening.............89

Disease prediction .89

Early diagnosis......90

Prevention............90

Therapy based on molecular diagnosis ..............90

Monitoring of therapy ................90

Advantages and limitations of integrated healthcare90

Commercially available systems for integrated healthcare ............91

Future of molecular diagnostics in personalized medicine ....... 91

3. Pharmacogenetics ................... 92

Basics of pharmacogenetics ............ 92

Role of molecular diagnostics in pharmacogenetics................. 93

Role of pharmacogenetics in pharmaceutical industry ............. 94

Study of the drug metabolism and pharmacological effects...........94

Causes of variations in drug metabolism ................94

Enzymes relevant to drug metabolism ...................95

Pharmacogenetics of phase I metabolism...............95

CYP450................95

P450 CYP 2D6 inhibition by selective serotonin reuptake inhibitors ................97

Lansoprazole and cytochrome P450 ..................98

Glucose-6-phosphate dehydrogenase................98

Pharmacogenetics of phase II metabolism..............99

N-Acetyltransferase ...................99

Uridine diphosphate-glucuronosyltransferase .....99

Measurement of CYP isoforms........ 100

Polymorphism of drug transporters. 100

Genetic variation in drug targets .... 101

Polymorphisms of kinase genes. 101

Effect of genetic polymorphisms on disease response to drugs.... 102

Ethnic differences in drug metabolism.................. 102

Gender differences in pharmacogenetics .............. 103

Role of pharmacogenetics in drug safety .............. 103

Adverse drug reactions ............ 103

Role of pharmacogenetics in warfarin therapy.. 104

Role of pharmacogenetics in carbamazepine therapy ............ 105

Malignant hyperthermia ........... 105

ADRs related to toxicity of chemotherapy ........ 106

FDA consortium linking genetic biomarkers to serious adverse events.......... 106

Therapeutic drug monitoring, phenotyping, and genotyping........ 107

Therapeutic drug monitoring..... 107

Phenotyping ....... 107

Genotyping......... 109

Genotyping vs phenotyping ...... 109

Phenomics............... 110

Limitations of genotype-phenotype association studies ......... 110

Molecular toxicology in relation to personalized medicines.......... 110

Toxicogenomics .. 110

Companies involved in molecular toxicology .... 111

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Gene expression studies .......... 112

Transcriptome profiling studies . 112

Genomics and the prediction of xenobiotic toxicity................ 113

Pharmacogenetics in clinical trials .. 113

Postmarketing pharmacogenetics ... 114

Clinical implications of pharmacogenetics.......... 114

Examples of use of pharmacogenetics in clinical pharmacology ... 114

Linking pharmacogenetics with pharmacovigilance  116

Recommendations for the clinical use of pharmacogenetics ........ 116

Limitations of pharmacogenetics .. 116

Academic research in pharmacogenetics ........... 117

Future role of pharmacogenetics in personalized medicine.... 118

4. Pharmacogenomics................ 120

Introduction............. 120

Basics of pharmacogenomics ........ 120

Pharmacogenomics and drug discovery ............. 120

Preclinical prediction of drug efficacy .............. 122

Pharmacogenomics and clinical trials ................ 122

Impact of genetic profiling on clinical studies ........ 124

Limitations of the pharmacogenomic-based clinical trials............ 125

Pharmacogenomic aspects of major therapeutic areas .......... 125

Oncogenomics ......... 125

Oncogenes ......... 126

Tumor suppressor genes .......... 126

Cardiogenomics ....... 127

Neuropharmacogenomics .............. 129

Pharmacogenomics of Alzheimer's disease....... 130

Pharmacogenomics of depression................... 130

Pharmacogenomics of schizophrenia............... 131

Companies involved in neurogenomics-based drug discovery. 131

5. Role of Pharmacoproteomics . 134

Basics of proteomics . 134

Proteomic approaches to the study of pathophysiology of diseases............ 134

Single cell proteomics for personalized medicine ... 135

Diseases due to misfolding of proteins ................. 135

Therapies for protein misfolding  136

Significance of mitochondrial proteome in human disease .......... 137

Proteomic technologies for drug discovery and development  137

Role of reverse-phase protein microarray in drug discovery ........ 137

Role of proteomics in clinical drug safety.............. 137

Toxicoproteomics ..... 138

Application of pharmacoproteomics in personalized medicine  139

6. Role of Metabolomics in Personalized Medicine .............. 140

Metabolomics and metabonomics . 140

Metabolomics bridges the gap between genotype and phenotype .............. 140

Metabolomics, biomarkers and personalized medicine........... 141

Metabolomic technologies............. 141

Urinary profiling by capillary electrophoresis......... 142

Lipid profiling........... 142

Role of metabolomics in biomarker identification and pattern recognition .......... 143

Validation of biomarkers in large-scale human metabolomics studies................ 143

Pharmacometabonomics............... 143

Metabonomic technologies for toxicology studies.................. 144

Metabonomics/metabolomics and personalized nutrition...... 144

7. Personalized Biological Therapies.............. 146

Introduction............. 146

Recombinant human proteins ....... 146

Therapeutic monoclonal antibodies 146

Cell therapy............... 147

Autologous tissue and cell transplants.................. 147

Stem cells ............... 147

Role of stem cells derived from unfertilized embryos............. 147

Cloning and personalized cell therapy .................. 148

Use of stem cells for drug testing ... 148

Gene therapy ............ 148

Personalized cancer vaccines........ 149

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Patient-specific cancer vaccines ..... 149

Personalized cancer vaccines .... 149

Antigen-specific vaccines............... 150

Autologous cell vaccines................ 150

Individual cancers are antigenically distinct .......... 150

Autologous HSP70-peptide vaccine................. 151

Personalized melanoma vaccines.... 151

Antisense therapy ..... 152

RNA interference...... 152

8. Personalized Medicine in Major Therapeutic Areas ......... 154

Introduction............. 154

Management of viral infections ..... 155

Management of HIV.. 155

Differences in response of the body to HIV ...... 155

Variations in action of drugs on HIV................ 155

Role of diagnostic testing in HIV 155

CD4 counts as a guide to drug therapy for AIDS .................. 156

Drug-resistance in HIV............. 156

Measurement of Repl