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UNIFR
Rusconi
2002
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iceage.sub.mov
aa zoom-inplanetsDNA.mov
aa secret bioweaponlab.mov
aa robotwoman.mov
aa beautywoman.mov
aa balance-world.mov
aa damocles.mov
bb DNArepairModel.mov
bb DNAerror+replcells.mov
affy_tech.mov
aa drugsmanufact.mov
sequencing.swf
Timeline.swf
bb icelandic pedigree.mov
aa getting oldcomp2.mov
aa genetomato.mov
bb pedigree iceland.mov
genet walk on DNA.mov
intro.swf
sruSki2.mov
UNIFR
Sandro Rusconi
Rusconi
2002
1972-75
1975-79
1979-82
1982-84
1984-86
1987-91
1994-today
1996-today
2001
2002
2002
Primary school teacher (Locarno, Switzerland)
Graduation in Biology UNI Zuerich, Switzerland
PhD curriculum UNI Zuerich, molecular biology
Research assistant UNI Zuerich
Postdoc UCSF, K Yamamoto, (San Francisco)
Principal Investigator, UNI Zuerich (mol. bio.)
Professor Biochemistry UNI Fribourg
Director Swiss National Research Program 37
'Somatic Gene Therapy'
Participant Swiss Natl. Res Program 50
'Endocrine disruptors'
Sabbatical, Tufts Med. School Boston and
Univ. Milano, Pharmacology Department
President Union of Swiss Societies for
Bormio,
Experimental Biology (USGEB)
March 2002
Milano, Nov 29, 2002
Scienze Farmacologiche
Progetto Genoma Umano
Analisi del genoma
umano e applicazioni
per la medicina:
impatto a corto, medio
e lungo termine
Schedule
Basic understanding of 'genes and genomes':
what is a gene, how many genes, molecular
biology dogma
genetic diseases, environmental factors,
Essential concepts on 'molecular medicine'
applications and problems, ageing as the major
disease
Genomics and other 'omics'
essential technologies
Applications and Impacts
diagnosis, prevention, therapy
Conclusions
concerns, plausibile developments
UNIFR
Rusconi
2002
Did I talk about ... 'Future' ... ?
UNIFR
Rusconi
2002
There are many different ways
to guess the future, not all of
them being equally reliable...
Genetics has been used since millennia,
Molecular Biology, only since 30 years
100’000 b.C.
Empirical genetics
10’000 b.C.
Biotechnology
2000 a.d.
Molecular biology
2001 a.d, Genomics
UNIFR
Rusconi
2001
UNIFR
1 Gene -> 1 or more functions
Rusconi
2002
DNA
RNA(s)
Protein(s)
Transcription / translation
Gene expression
GENE
2-5 FUNCTIONS
100 ’000 genes
(50 ’000 genes?)
>300 ’000 functions
(>150 ’000 functions)
UNIFR
1 Organism -> more than 105
genetically-controlled Functions
Rusconi
2002
2 mm
2m
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
But...how many genes exactly?
The debate is still hot
UNIFR
Rusconi
2002
Human genome consortium 30'000 identified
Celera 30'000 identified
HGC x Celera -> 45'000 (only 15'000 overlap)
How many gene products are
we still missing?
Over 150'00 unique EST cDNAs have been identified
My own personal experience: 50% of genes of my research
interest yet unannotated
What is in fact a gene?: a segment of DNA acting as a
regulated machine for RNA production
DNA
RNA(s)
Protein(s)
GENE
Transcription / translation
FUNCTION(s)
protein(s)
RNA(s)
DNA
spacer
regulatory
coding
spacer
UNIFR
Rusconi
2002
Reductionistic molecular biology paradigm
(gene defects and gene transfer)
DNA
Protein
GENE
FUNCTION(s)
GENE OK
FUNCTION(s) OK
GENE KO
FUNCTION(s) KO
GENE transfer
FUNCTION(s) transfer
UNIFR
Rusconi
2002
Gene amplification / manipulation techniques
(genetic engineering, recombinant DNA)
segments of genomic DNA can be specifically cut and isolated
Basic manipulations can be essentially
isolated segment can be recombined with a plasmid vector
performed in your house cellar
additional
sophistications:
plasmid vector
is transferred
into bacteria where it can multiply
- Polymerase chain reaction (PCR)
- customDNA
synthetic
oligonucleotides
isolated recombinant
can be further
recombined to obtain
- labeling
and hybridisation techniques
the final desired
molecule
- custom sequencing
Final molecule
is transferred intogene
cells or
organisms
- pre-formulated
transfer
cocktails
- recombinant viral vectors
UNIFR
Rusconi
2002
UNIFR
Rusconi
2002
The THREE missions of medicine
Prevention
+
'Molecular Medicine'
Diagnosis
Application of the
know-how in
molecular genetics
to medicine
+
+
Therapy
UNIFR
The FOUR eras of molecular medicine
Rusconi
2002
Eighties
Genes as probes
Nineties
Genes as factories
Y2K
Genes as drugs
1 2 3 4 5
ok ** ok ** **
50
10
3000
80 85 90 95 99
1000technologies
Y2K+n Post-genomic improvements
of former
80 85 90 95 00
UNIFR
Rusconi
2002
100%
10
1
E2/E
Ergo:
Modern medicine faces a major challenge: M
4
understanding and treatment
E3/E4
of age-related conditions
20 40 60 80
E4/E4
80
70
60
1900
2000
20 40 60
50
1900
100
Alzheimer’s free %
Life expectancy (CH)
cancer incidence
The major disease of the 21st century: Ageing
1920
1940
1960
1980
199
1900
80
2000
UNIFR
Not only the genome determines the health status...
Rusconi
2002
genetics
Muscle distrophy
Familial Breast Cancer
Sporadic Breast Cancer
Lung Cancer
Obesity
Artherosclerosis
Alzheimer
Parkinson ’s
Drug Abuse
Homosexuality
behaviour
environment
Genomics: the importance of population
homogeneity for functiuonal-locus detection
UNIFR
Rusconi
2002
Fact
Several genome mining companies
are analysing genetically coherent
ethnic groups:
example icelandic population
Example
Millenium (owned by Roche) has apparently
already identified over twenty genetic loci
linked to delayed ageing
Problem
The major question: 'who owns which
fraction of the patent rights'?
UNIFR
The gen(OME) (the ensemble of genes)
and other characters of the 'omics' saga
TCACCATGCGT
Rusconi
2002
CCTAGG
Structural
genomics
ACGGATCA
AAAGTA
TTTAGAACAGGT
Functional g. 1:
(transcriptomics)
Functional g. 2:
(Proteomics)
you said...comics?
Functomics
Morphomics
further
???
glycomics?
spliceomics
metabolomics
lipidomics
Genomics milestones
The adventure started quite long time ago,
but took speed only recently
UNIFR
Rusconi
2002
Part 1: structural genomics...
UNIFR
Rusconi
2002
Structural
Genomics
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Determination of genome sequence
Determination of genome variability
Determination of genomic evolution
virus, bacteria, yeast, plants, mammals
intra-species polymorphism,
inter-species polymorphism
Structural genomics aims and techniques
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Automated, high throughput
fluorescent sequencing
(size-dependent time)
micro-array-based resequencing
(size independent time)
single nucleotide polymorphism
detection techniques
ultra-microscope-based sequencing (SNP)
direct polymerase output sequencing
Relevant criteria:

precision (new sequencing)

rapidity & convenience (re-sequencing)

annotation of sequence

electronic storage/retrieval
UNIFR
Rusconi
2002
Structural genomics:
current impact on research
UNIFR
Rusconi
2002
Good:

Paradigmatic shift: from gene hunters to function hunters
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Cross comparison: human, mouse, drosophila, C elegans, yeast,
Arabidopsis

Rapid contextualisation of newly identified sequences

Availability of cloned genomic segments
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Easy design of analytical or preparative PCR amplicons
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In silico search for paralogues and homologues and gene families
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Rapid assessment of inter-species conserved elements

Excellent source of working hypotheses about functionally
relevant sequences
Bad:
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Limited access to Celera and other privately-owned project data

Limited a priori-gene recognition

mistakes or mis-annotation can slow down some specific projects

too many teams turn to genomics without proper knowledge
Structural genomics current impact on clinics
UNIFR
Rusconi
2002
Good:

More thorough assessment of cancer-linked predispositions

Discovery of genes linked to neuro-degenerative conditions

microsatellite polymorphism markers

chromosomal painting

Identification of age-linked gene loci

amelioration and speeding up of molecular diagnostics
Bad:

possible funds withdrawal from other productive research

too much faith combined with too much determinism could
lead to major initial failures
UNIFR
Molecular diagnostics: three levels
Rusconi
2002
Prenatal
 hereditary disorders
 severe predispositions
Real time
(monitoring)
 pathogens profile
 Immune status
 cancer status
Pre-symptomatic
 Predispositions to:
 cancer
 autoimmune d.
 degenerative d.
 behavioural d.
 pharmacogenetics
UNIFR
Molecular diagnosis I: prenatal
Rusconi
2002
Hematological defects
- Thalassemias
- Hemophilias
Conventional
Metabolic disorders
most of these assays are
- Phenylketonuria
- Ornithine TC already available, but are currently
adopted only when there is a familial
Immunodeficiencies
- ADA
history
- SCID
Neurological defects
- fragile X
- Ataxia
Genomically-driven
Muscolo-skeletalcould
defects
improve and extend diagnosis
- muscular dystrophy
by rapid re-sequencing technologies
- osteogenesis imperfecta
that detect subtle mutations
Cardiovascular defects
...
even if they are spontaneous
-> mass screening
UNIFR
Impact on molecular diagnosis II: pre-symptomatic
Rusconi
2002
Predispositions to:
Convenional
- colon
many of these diseases can be
- breast
efficiently treated if precociously
Autoimmune diseases
- asthma
diagnosed (currentlky limited to
- rheumatoid A.
familial cases)
Degenerative diseases
Cancer
?
- Alzheimer
- Parkinson
Genomically-driven:
higher predictive value and massBehavioural diseases
- Drug addiction screening through molecular
Pharmaco-sensitivity
- hyper reactivity structural and functional genomic
- toxicity
analyses
- hypo reactivity
Molecular diagnosis III:
'real time' molecular diagnosis
UNIFR
Rusconi
2002
better monitoring of:
Conventional
pathogens' profile many of these conditions can be
- differentiation
efficiently treated only if correctly
- antibiotica resistance
- virulence
monitored and distinguished
- biofilm
(example antibiotic resistance)
Immune status
- antigens load
Genomically-driven
- antibody diversity
more rapid and precise verification
cancer follow-up of the steady-state status of the
- clonal status
disease through molecular profiling
- tumor markers
(example leukemias)
- evolution
- MRD
-> better prognosis / treatment
Structural genomics mid/long-term impact
UNIFR
Rusconi
2002
Mid term:
 Differential diagnosis of neoplastic conditions
 Complete panel of pathogen's genomes
 Bedside, precise analysis of infectious diseases
(Pocket analysers)
Angelo Nicolin (this meeting)

the genome and new
pharmacological therapies
Long term:
 New anti-microbial drugs through bacterial
genomics
 Identification of novel drug targets
 Understanding of single nucleotide polymorphism
 Comprehension of polygenic diseases
Part 2: Functional genomics...
UNIFR
Rusconi
2002
Functional
Genomics
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Collection of expressed cDNA

Measurement of mRNA expression differentials 
Determination of differential Protein profiles

Knock-out knock-in conditional KO of genes

EST collections
Transcriptomics
Proteomics
functomics
Conventional, 'brute force' genomics
(analysis of steady-state RNA)
stimulus
UNIFR
Rusconi
2002
No stimulus
Uridine

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several hours, or days
RNA1
RNA2
Very complex mixture of:
transcriptional + post-tr. effects
primary + secondary events
functional genomics
(display or array)
Label
cDNA1
cDNA2
UNIFR
Molecular diagnosis : microarrays
Rusconi
2001
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mg tissue
Extract RNA
Label fluorescently
Hybridise to microarray
Computer-assisted inspection
UNIFR
Principle of microarrays
Rusconi
2002
differential analyses:
SideAlternative
view
Top view
 SAGE
Silicon display
 Differential mRNA
Nylon
 Subtractive cDNA cloning
Glass
Micro-spotting
baits collection
criteria
Relevant
 probes / surface (dots/mm2)
Hybridize apparatuses, time)
 Costs (materials,
labelled mixture
 Sensitivity (low/high expressors)
NO HYB
 Selectivity (gene families, alternative splicing etc)
A>B
 Representativity
Rinse/detect
A<B
A=B
Principle of proteomics

Tissues/cells from different status

Cell/ tissue lysis

2D gel electrophoresis separation

Already known proteins: Spot identification / intensity

Novel proteins/modificatins: Spot elution / microsequencing
UNIFR
Rusconi
2002
Functional genomics milestones
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UNIFR
Rusconi
2002
1975 Britten and Davidson hybridisation kinetics mRNA-genomic DNA
1980 transgenic mice
1985 Subtractive cDNA libraries (tissue specific genes)
1998 knockout transgenics
1990 mRNA differential display
1995 oligonucleotide microarrays
1999 MALDI TOF, proteomics
2000 wide commercialisation of:
arrays, dedicated bait filters, spotters, readers, custom analyses
transgenic custom companies
2002 proteomic arrays with antibodies
Functional genomics:
current impact on research
UNIFR
Rusconi
2002
Good:
 Financial availability of microarray services & tools
 Possibility to tackle functional gene patterns
alterations
 Novel hypotheses on primary and subsequent
responders (infections, stresses, stimuli, drugs,
development)
Bad:
 too deterministic interpretation blocks understanding
 too many research teams join in the 'fashion field'
 insufficient attention and understanding on the
mechanistic aspects (real time kinetics)
Functional genomics current impact on clinics
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UNIFR
Rusconi
2002
clinical experimental leukemia profiling
early clinical assessment of pharmacogenomics principles
strong interest of pharmaceutical companies
increasing interest of practicting clinicians
UNIFR
Impact on prevention: vaccination
Rusconi
2002
Recent & prospected
Conventional
Vaccines

a posteriori identification of
antigens
Hepatitis B

preparation of vaccine by
- 1995
inactivated pathogen or single
recombinant gene fermentation
AIDS
- 2008 ?
Cancers
- 2005-2010 ?
Malaria
- 2010 ?
Alzheimer
- 2010 ?
Massimo Di Nicola (this meeting)

immunotherapy of cancer
Genomically driven

a priori or custom identification
of vaccines

vaccine cocktails with
recombinant DNA
Impact on drug administration:
Principles in pharmacogenetics
variant
Side-E
Effects
desired
dosis
Good
Acceptable
Unacceptable
UNIFR
Rusconi
2002
Individualised treatment
through genomic profiling
- Dosing
- Side effects
- Hyper sensitivity
-Diego
HypoFornasari
sensitivity
(this meeting)
-Indicated
/ contraindicated
pharmacogenetics
- Drugs combination
Preventive approaches
- functional food, vaccination
- behavioural medical coaching
UNIFR
Impact on Therapy I: Biopharmaceuticals
Rusconi
2002
Isolate gene of interest (GOI)
Conventional

disease -> gene -> product

single product treatments
Redesign GOI
Transform redesigned GOI
Into micro-organisms
Production through large-scale
Fermentation processes
Genomically driven

product 1990
-> gene ->disease
1980
2000

multiple product treatments

improvements in gene therapy
Impact on Therapy II:
improvements on GENE THERAPY?
IMPROVED REGULATION OF TRANSGENES
Discovery of new promoters / enhancers / LCR / etc
Construction of therapeutical mini-chromosomes
SPECIFICALLY INTEGRATING TRANSGENES
Identify target sites for integrases
Identify target sites for triple-stranded inhibitors
PROVIDING NEW MOLECULAR TARGETS
Clarify disease-causing pathways
Use of gene combination cocktails
UNIFR
Rusconi
2001
Functional genomics mid/long term influence
UNIFR
Rusconi
2002
Mid term

gene patterns triggered by basic signal transduction
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gene patterns involved in major pathologies

gene patterns in pathogenic micro-organisms

knock-outs of all known genetic function

fundamental genetic response to nutrients/drugs
Long term
Gaetano Finocchiaro (this meeting)

making sense of polymorphic response to nutrients
and

nervous
system therapy
drugs

clearer picture on complex pathologies (cancer,
alzheimer, parkinson, rheumatoid arthritis,
atherosclerosis)

understanding of genetic background/modifiers

follow-up / prediction of genetic drift of microbial
pathogens (eg influenza)

identification of new emerging infectious pathogens
Genomics:
public understanding and public concern
Major current public opinion issues

different concerns in different cultural backgrounds

general fear of 'gene manipulation' technology

concerns about unsolicited genetic practices (screening)

confusion of genetics/ reproductive technology

privacy / data protection (insurances, employers)
More hypothetical but still plausible issues

eugenic practices

novel epidemics through artificial gene shuttling

ethnically-targeted poisons

novel forms of behavioural screening/control
UNIFR
Rusconi
2002
Genes, cells, transplants, ... some may ask:
«where are we going»?
UNIFR
Rusconi2
2002
Amelioration instead of
therapy?
Too sophisticated
too expensive?
Bioweapons?
Genomics conclusion
UNIFR
Rusconi
2002
Facts

The genomics era is a logical consequence of gene
technology progress

There is a good basic understanding of involved
molecules

There is a wealth of sequence data
However:

There is a deluge of functional data (transcriptomics)

We still dont know how many genes/genome

The post- genomics era has already started

We still dont know how many functions/gene

The first impact has been at the research level

Clinical impact is currently minor

There are social / ethical / legal concerns
Nevertheless:

Genomics will keep advancing exponentially

there will be applications in diagnosis within few years

There may be improvements of therapeutical
applications within about a decade
...Thanks !
UNIFR
Rusconi
2002
University of Milano
CEND
Our personal project/goal may indeed
appear very small and harmless...
My collaborators at UNIFR
This does not necessarily apply
to its consequences...
Swiss National Research Foundation
Thank you all for the attention,
and... if you are too shy to ask
send an e-mail to:
[email protected]
or visit:
www.unifr.ch/nfp37