technical remarks
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UNIFR Rusconi 2002. Technical Remarks. This slide show was prepared with Power Point 98 for Macintosh therefore, 'thank' to the limited portability it may halt or improperly display on windows-based machines. - PowerPoint PPT PresentationTRANSCRIPT
Technical RemarksTechnical RemarksUNIFRRusconi2002
UNIFRRusconi2002
This slide show was prepared with Power Point 98 for Macintoshtherefore, 'thank' to the limited portability it may halt or improperly display on windows-based machines.
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sruSki2.movgenet walk on DNA.movsport boxe 01.movaa getting oldcomp2.movmolecular_therapycardio1.movsport GIRO98.MOVsport football 01.movsport maradona 01.mov
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S. Rusconi
Sandro RusconiSandro RusconiUNIFRRusconi
2002
UNIFRRusconi
2002
Sports doping:Is there a realistic
application for gene transfer?
Bern, Nov 22, 2002Swiss Olympic
gene doping workshop
1972-75 Primary school teacher (Locarno, Switzerland)1975-79 Graduation in Biology UNI Zuerich, Switzerland1979-82 PhD curriculum UNI Zuerich, molecular biology1982-84 Research assistant UNI Zuerich1984-86 Postdoc UCSF, K Yamamoto, (San Francisco)1987-91 Principal Investigator, UNI Zuerich (mol. bio.)1994-today Professor Biochemistry UNI Fribourg1996-today Director Swiss National Research Program 37
'Somatic Gene Therapy'2001 Participant Swiss Natl. Res Program 50
'Endocrine disruptors'2002 Sabbatical, Tufts Med. School Boston and
Univ. Milano, Pharmacology Department2002 President Union of Swiss Societies for
Experimental Biology (USGEB)
Essential concepts on 'molecular medicine' & molecular doping: applications and problems,
Gene-based dopingapplications, comparison with other doping, detection
Techniques of gene transfer (Gene Therapy)problems and solutions, vectors, clinical achievements
ScheduleScheduleUNIFR
Rusconi
2002
UNIFR
Rusconi
2002
Basic understanding of 'genes': what is a gene, how many genes, molecular biology dogmagenetic diseases, environmental factors, ageing
Conclusionsplausibility table
Genetics has been used since millennia,Molecular Biology, only since 30 years
Genetics has been used since millennia,Molecular Biology, only since 30 years
UNIFRRusconi
2001
UNIFRRusconi
2001
100’000 b.C. Empirical genetics
10’000 b.C.Biotechnology
2000 a.d.Molecular biology
2001 a.d, Genomics
1 Gene -> 1 or more functions1 Gene -> 1 or more functionsUNIFRRusconi
2001
UNIFRRusconi
2001
RNADNA
GENE
Protein
2-5 FUNCTIONS
Gene expression
Transcription / translation
>300 ’000 functions(>150 ’000 functions)
100 ’000 genes(50 ’000 genes?)
What is in fact a gene?: a segment of DNA acting as a regulated machine for RNA production
What is in fact a gene?: a segment of DNA acting as a regulated machine for RNA production
UNIFRRusconi
2002
UNIFRRusconi
2002
RNADNA Protein
GENE FUNCTIONTranscription / translation
codingspacer spacerregulatoryDNA
RNA
1 Organism -> more than 105 genetically-controlled Functions
1 Organism -> more than 105 genetically-controlled Functions
UNIFRRusconi
2002
UNIFRRusconi
2002
2m 2 mm 0.2mm
0.02mm
DNA RNA Protein
0.001mm
Reductionistic molecular biology paradigm(gene defects and gene transfer)
Reductionistic molecular biology paradigm(gene defects and gene transfer)
UNIFRRusconi
2002
UNIFRRusconi
2002
GENE transfer FUNCTION transfer
GENE KO FUNCTION KO
GENE OK FUNCTION OK
DNA
GENE
Protein
FUNCTION(s)
Gene amplification / manipulation techniques(genetic engineering, recombinant DNA)
Gene amplification / manipulation techniques(genetic engineering, recombinant DNA)
UNIFRRusconi
2002
UNIFRRusconi
2002
nucleus & DNA.mov
segments of genomic DNA can be specifically cut and isolated
isolated segment can be recombined with a plasmid vector
plasmid vector is transferred into bacteria where it can multiply
isolated recombinant DNA can be further recombined to obtainthe final desired molecule
Final molecule is transferred into cells or organisms
Science-grade materialcan be essentially prepared in your cellar
...not so clinical-grade material!
The FOUR eras of molecular medicineThe FOUR eras of molecular medicineUNIFRRusconi
2002
UNIFRRusconi
2002EightiesGenes as probes
ok ** ** **ok1 2 4 53
NinetiesGenes as factories
80 85 90 95 99
10
50
Y2KGenes as drugs
80 85 90 95 00
1000
3000
Y2K+n Post-genomic improvements of former technologies
The major disease of the 21st century: AgeingThe major disease of the 21st century: AgeingUNIFR
Rusconi
2002
UNIFR
Rusconi
2002
60
70
80
50
1920 1940 1960 1980 1991900
Life
exp
ecta
ncy
(CH
)
4
20 40 60 80
100
10
1
canc
er in
cide
nce
1900 200020 40 60 80
100%
M
E2/E
E3/E4
E4/E4
Alz
heim
er’s
fre
e %
1900 2000
many treatments that slow down ageingor age-related degenerative diseases are also potential doping treatments
aa getting oldcomp2.mov
Now, let's talk about Somatic Gene Therapy (somatic gene transfer)
Now, let's talk about Somatic Gene Therapy (somatic gene transfer)
UNIFRRusconi2002
UNIFRRusconi2002
Definition of GT:'Use genes as drugs':Correcting disorders by somatic gene transfer
Chronic treatment
Acute treatment
Preventive treatment
Hereditary disorders
Acquired disorders
Loss-of-function
Gain-of-function
NFP37 somatic gene therapywww.unifr.ch/nfp37
Somatic gene therapy’s (gene transfer) four fundamental questions
Somatic gene therapy’s (gene transfer) four fundamental questions
UNIFRRusconi2002
UNIFRRusconi2002
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
Remember!
Why 'somatic'?Why 'somatic'?UNIFRRusconi2001
UNIFRRusconi2001
Germ Line Cells: the cells (and their precursors) that upon fertilisation can give rise to a descendant organism
Somatic Cells: all the other cells of the body
i.e. somatic gene transferis a treatment aiming atsomatic cells and conse-quently does not lead to a hereditary transmission of the genetic alteration
Pharmacological considerationsPharmacological considerationsUNIFR
Rusconi
2001
UNIFR
Rusconi
2001
OHOH
O
OHOH
O
O
OHOH
O
O
Mw 50- 500 Daltons Synthetically prepared Rapid diffusion/action Oral delivery possible Cellular delivery:
- act at cell surface- permeate cell membrane- imported through channels
Can be delivered as soluble moleculesÅngstrom/nm size
Classical Drugs
Mw 20 ’000- 100 ’000 Da Biologically prepared Slower diffusion/action Oral delivery not possible Cellular delivery:
- act extracellularly
Can be delivered as soluble moleculesnm size
Protein Drugs
Mw N x 1’000’000 Da Biologically prepared Slow diffusion Oral delivery inconceivable Cellular delivery:
- no membrane translocation - no nuclear translocation- no biological import
Must be delivered as complex carrier particles50-200 nm size
Nucleic Acids
THREE classes of physiological gene deliveryTHREE classes of physiological gene deliveryUNIFR
Rusconi
2001
UNIFR
Rusconi
2001
Ex-vivo In-vivotopical delivery
In-vivosystemic delivery
V
Examples:- bone marrow- liver cells- skin cells
Examples:- brain- muscle- eye- joints- tumors
Examples:- intravenous- intra-arterial- intra-peritoneal
TWO classes of gene transfer vehicles: non-viral & viralTWO classes of gene transfer vehicles: non-viral & viralUNIFR
Rusconi
2001
UNIFR
Rusconi
2001
a
b
Non-viral transfer(transfection)
Viral gene transfer(Infection)
Nuclear envelope barrier! see, Nature BiotechDecember 2001
Transfection with recombinant DNAVs Infection with recombinant viruses
Transfection with recombinant DNAVs Infection with recombinant viruses
UNIFR
Rusconi
2001
UNIFR
Rusconi
2001
Transfection
Infection
exposed to106 particles/cell12 hours
exposed to 3 particle/cell30 min
Quick parade of popular vectors/methodsQuick parade of popular vectors/methodsUNIFR
Rusconi
2002
UNIFR
Rusconi
2002
Adenovirus
Adeno-associated V.
Retrovirus (incl. HIV)
Naked DNA
Liposomes & Co.
Oligonucleotides
Recombinant AdenovirusesRecombinant AdenovirusesUNIFR
Rusconi
2002
UNIFR
Rusconi
2002
Approaches
Generation I
Generation III
Hybrid adenos: Adeno-RV Adeno-AAV Adeno-Transposase
Examples OTC deficiency (clin, ---) Cystic Fibrosis (clin, --- ) Oncolytic viruses (clin, +++)
Advantages / Limitations
8 Kb capacity Generation I >30 Kb capacity Generation IIIAdeno can be grown at very high titers,However Do not integrate
Can contain RCAs
Are toxic /immunogenic
Recombinant AAV (adeno-associated-virus)Recombinant AAV (adeno-associated-virus)UNIFR
Rusconi
2002
UNIFR
Rusconi
2002
Examples Hemophilia A (clin, animal, +++) Gaucher (clin, animal, +++) Brain Ischemia (animal, +++) Cystic fibrosis (animal, +/-)
Advantages / Limitations
Persistence in the genome permits long-term expression, high titers are easilyobtained, immunogenicity is very low,However the major problem is:
Small capacity (<4.5 kb) which does not allow to accommodate large genes or gene clusters.
Approaches
Helper-dependent production
Helper independent production
Cis-complementing vectors
Co-infection
Recombinant Retroviruses (includes HIV-based)Recombinant Retroviruses (includes HIV-based)UUNIFR
Rusconi
2002
UUNIFR
Rusconi
2002
Approaches
Murine Retroviruses
VSV-pseudotyped RV
Lentiviruses !
Self-inactivating RV
Combination viruses
Examples SCID (IL2R defect, Paris) (clin, +++) Adenosine Deaminase deficiency (clin, +++!!!) Parkinson (preclin, +++) Anti cancer (clin +/-)
Advantages / Limitations
9 Kb capacity + integration throughtransposition also in quiescent cells(HIV), permit in principle long-termtreatments, however disturbed by: Insertional mutagenesis
Gene silencing
High mutation rate
Low titer of production
Naked / complexed DNANaked / complexed DNAUNIFR
Rusconi
2002
UNIFR
Rusconi
2002
Approaches
Naked DNA injection /biolistic
Naked DNA + pressure
Naked DNA + electroporation
Liposomal formulations
Combinations
Advantages / Limitations
Unlimited size capacity + lowerimmunogenicity and lower bio-riskof non viral formulations isdisturbed by
Low efficiency of gene transfer
Even lower stable integration
Examples Critical limb Ischemia (clin, +++) Cardiac Ischemia (clin, +/-) Vaccination (clin, +/-) Anti restenosis (preclin. +/-)
Oligo-nucleotidesOligo-nucleotidesUNIFR
Rusconi
2002
UNIFR
Rusconi
2002
√ !
Approaches
Antisense
Ribozymes/DNAzymes
Triple helix
Decoy / competitors
Gene-correcting oligos
Advantages / Limitations
these procedures may be suitable for :
handling dominant defects
transient treatments (gene modulation)
permanent treatments (gene correction)
Examples Anti cancer (clin,preclin., +/-) Restenosis (clin, +++) Muscular Distrophy (animal, +++)
Recap: current limitations of popular gene transfer vectors
Recap: current limitations of popular gene transfer vectors
UNIFR
Rusconi
2002
UNIFR
Rusconi
2002
Adenovirus- no persistence- limited packaging- toxicity- immunogenicity
Biolistic bombardmentor local direct injection- limited area
Retrovirus (incl. HIV)- limited package- random insertion- unstable genome
General- antibody response- limited packaging- gene silencing
Solutions:- synthetic viruses (“Virosomes”)
Electroporation- limited organ access
Liposomes, gene correction & Co.- very inefficient transfer
General- low transfer efficiency 1/10’000 of viruses’ in vivo
Solutions:- improved liposomes with viral properties (“Virosomes”)
The most feared potential side-effects of gene transferThe most feared potential side-effects of gene transferUNIFRRusconi2002
UNIFRRusconi2002
Immune response to vector
immune response to new or foreign gene product
General toxicity of viral vectors
Adventitious contaminants in recombinant viruses
Random integration in genome
-> insertional mutagenesis (-> cancer risk)
Contamination of germ line cells
Random integration in genome
-> insertional mutagenesis (-> cancer risk)
Gene Therapy in the clinic: Trials WordldwideGene Therapy in the clinic: Trials WordldwideUNIFRRusconi2002
UNIFRRusconi2002
cancer
hered.
Infect.vasc.
40
60
100
20
80
trials
500
1500
1000
patients
1992 1994 1996 19981990 2000
21% overall still pending or not yet Initiated !www.wiley.com
86% phase I13% phase II1 % phase III
As of Sept. 2002:599 registered protocols4000 treated patients
Gene Therapy MilestonesGene Therapy MilestonesUNIFRRusconi2002
UNIFRRusconi2002
1990, 1993, 2000 // ADA deficiencyF Anderson, M Blaese // C Bordignon
Anderson, 1990
Bordignon, 2000 (ESGT, Stockholm)proves efficacy of the same protocol
1997, 2000, Critical limb ischemiaJ Isner († 4.11.2001), I Baumgartner, Circulation 1998
Isner, 1998
1998, RestenosisV Dzau, HGT 1998
Dzau, 1999
1999, Crigler Njiar (animal)C Steer, PNAS 1999
Kmiec, 1999
2000, HemophiliaM Kay, K High
2000, SCIDA Fischer, Science April 2000
Fischer, 2000
2000, correction Apo E4 (animal model)G. Dickson, ESGT congress, 7.10.2000 Stockholm
Dickson, 2000
2000, correction Parkinson (animal model)P Aebischer, Science, Nov 2000
Aebischer, 2000
2001, ONYX oncolytic VirusesD Kirn (Gene Ther 8, p 89-98)
Kirn, 2001
Clinical trials with ONYX-015,what we learned?
(Review)
Gene Therapy Adverse events: NY 1995 // UPenn 1999 // Paris 2002
Gene Therapy Adverse events: NY 1995 // UPenn 1999 // Paris 2002
UNIFRRusconi2002
UNIFRRusconi2002
NY May 5, 1995, R. Crystal: in a trial with adenovirus mediated gene transfer to treat cystic fibrosis (lung) one patient developed a mild pneumonia-like condition and recovered in two weeks. The trial was interrupted and many others were put on hold.
UPenn, Sept. 19, 1999, J. Wilson: in a trial with adenovirus mediated gene transfer to treat OTC deficiency (liver) one patient (Jesse Gelsinger) died of a severe septic shock. Many trials were put on hold for several months (years).
Paris, Oct 2, 2002, A Fischer: in a trial with retrovirus mediated gene transfer to treat SCID (bone marrow) one patient developed a leukemia-like condition. The trial has been suspended to clarify the issue of insertional mutagenesis, and some trials in US and Germany have been put on hold.
Ups and Downs of Gene Therapy: a true roller coaster ride!
Ups and Downs of Gene Therapy: a true roller coaster ride!
UNIFRRusconi2002
UNIFRRusconi2002
high
Low
moo
d
NIHMotulskireport Lentivectors
in pre-clinic
Adeno III
J. Isner
J. WilsonJ. Gelsinger
ADA
R. CrystalAdeno I
90 91 92 93 94 95 96 97 98 99 00 01 02
AAV germline in mice?
V.Dzau
A. FischerM. Kay
lentivectorsin clinics?
NFP37
Paris
C Bordignon
Ergo:in spite of its respectable age,gene transfer is still in its infancyand still produces more controversiesthan clinical results
The THREE levels of dopingThe THREE levels of dopingUNIFR
Rusconi
2002
UNIFR
Rusconi
2002
Before thecompetition
(anabolic enhancers)
During the competition(perfomance enhancers)
After thecompetition
(repair enhancers)
'Molecular treatmentsApplication of the
know-how in molecular genetics
to doping
+
+
+
Which gene transfer approaches would be compatible with doping strategies
Which gene transfer approaches would be compatible with doping strategies
UNIFRRusconi2002
UNIFRRusconi2002
ex vivo, hematopoietic tissue:erythropoietin?
in vivo local (example muscle):metabolic enhancers, growth factors, muscular fiber changers
in vivo local (example joints):pain reducers, inflammation inhibitors, recovery and repair factors
in vivo systemic:anabolic factors, endocrine factors, pain killers
Which are the objective current limitations ingene-based doping strategies
Which are the objective current limitations ingene-based doping strategies
UNIFRRusconi2002
UNIFRRusconi2002
Viral gene transfer immune problems limited readministration general toxicity, genotoxicity
Nonviral gene transfer generally inefficient lack of persistence, requires readministration
Strategy-independent problems laborious, not readily available long term gene expression difficult to control irreversible effects or permanent tagging
Which side effects could be feared ingene-based doping strategies
Which side effects could be feared ingene-based doping strategies
UNIFRRusconi2002
UNIFRRusconi2002
Short -mid term Autoimmunity Hyperimmunity Toxic shock
Long term Fibrosis Cancer Conventional effects of
administered factors Inaccessibility to future gene
therapy interventions (immunity)
Intrinsic to reckless application(probably the biggest danger) malpractice (unsuitable
vector/administration route) non-clinical grade material
(adventitious pathogens or allergens)
lack of follow-up
Which detection methods would be (or not) evisageable for gene-based doping strategies
Which detection methods would be (or not) evisageable for gene-based doping strategies
UNIFRRusconi2002
UNIFRRusconi2002
Antibody detection (viral antigens or other epitopes) recombinant-nucleic acids detection (PCR) recombinant protein detection
(MALDI-TOF / proteomics)
Gene transfer may be anatomically difficult to detect (if locally administered) but leaves permanent genetic marking
the detection of nucleic acids cannot be performed in body fluids (except for systemically administered treatments) and might require specific tissue biopsy
Final side-by-side comparison: gene-based doping versus drug- or protein-based doping
Final side-by-side comparison: gene-based doping versus drug- or protein-based doping
UNIFRRusconi2002
UNIFRRusconi2002
Category Drug/protein Gene-based
Rapidity of effects rapid slow
Reversibility rapid slow/none
Complexity of treatm. simple complex
Associated risks depends high
Detectability arduous
'straightforward'
Dosage straightforward difficultErgo:The odds speak currently rather against the adoption ofgene-based doping, but this applies to common-sense clinical practice, and this aspect is not guaranteed in the doping field
Thank you all for the attention, and... if you are too shy to asksend an e-mail to:[email protected] visit:www.unifr.ch/nfp37
...Thanks ! ...Thanks ! UNIFRRusconi2002
UNIFRRusconi2002
Swiss National Research Foundation
Our own project/goal may indeed appear very small and harmless...
This does not necessarily apply to its consequences...
My collaborators at UNIFR
Swissolympics
discussion slidesdiscussion slidesUNIFRRusconi2002
UNIFRRusconi2002
Examples of inheritable gene defectsExamples of inheritable gene defectsUNIFRRusconi
2002
UNIFRRusconi
2002
Polygenic defects Type estimated(‘ frequent ’) min - max
Diabetes poly 1 - 4 %Hyperurikemia Multi 2 - 15 %Glaucoma poly 1 - 2 %Displasia Multi 1 - 3 %Hypercolesterolemia Multi 1 - 5 %Syn-& Polydactyly poly 0.1 - 1 %Congenital cardiac defects Multi 0.5 - 0.8 %Manic-depressive psychosis Multi 0.4 - 3 %Miopy poly 3 - 4 %Polycystic kidney poly 0.1 - 1 %Psoriasis Multi 2 - 3 %Schizofrenia Multi 0.5 - 1 %Scoliosis Multi 3 - 5 %
Monogenic defects estimated(‘ rare ’) min - maxCystic fibrosis, muscular dystrophyimmodeficiencies, metabolic diseases, all togetherHemophilia... 0.4 - 0.7%
Predispositions Type estimatedmin - max
(*) Alzheimer Multi 7 - 27 %(*) Parkinson Multi 1 - 3 %(*) Breast cancer Multi 4 - 8 %(*) Colon Carcinoma Multi 0.1 - 1 %(*) Obesity Multi 0.5 - 2 %(*) Alcolholism/ drug addiction Multi 0.5 - 3%
Sum of incidences min -max (all defects) 32 - 83%
genetics behaviour environment
The long way to drug/procedure registration is the principal cause of financial burden, but we cannot avoid it
The long way to drug/procedure registration is the principal cause of financial burden, but we cannot avoid it
UNIFR
Rusconi
2002
UNIFR
Rusconi
2002
0 Idea 0
2 Cell culture assays 0.5 Mio
5 Pre-clinical testsanimal models 2 Mio
7 Clinical phase I5-20 patientsverify side effects 6 Mio
10 Clinical phase II30-100 patientsdosis escalation 12 Mio
15 Clinical Phase III>300- 1000 patientsmulticentricdouble blind 80 Mio
16>> Registration / Availability
year event costs U$D
This means:assuming 20% of new developmentsmakes it to final registration,the average investment is 300-500 Mio U$Dfor each approved drug/procedure
Not only the genome determines the health status...Not only the genome determines the health status...UNIFRRusconi
2002
UNIFRRusconi
2002
genetics behaviour environment
Muscle distrophy
Familial Breast Cancer
Lung Cancer
Obesity
Artherosclerosis
Alzheimer
Parkinson ’s
Drug AbuseHomosexuality
Sporadic Breast Cancer
Recap: what is a virus ? -> A superbly efficient replicating machine
Recap: what is a virus ? -> A superbly efficient replicating machine
UUNIFR
Rusconi
2002
UUNIFR
Rusconi
2002
E L1 L2
standard viral genome
100 nm
replication
entry disassemblydocking genome replication
late genes exp
assembly
capsid
E L1 L2
Spread
Etc...
early genes exp
Engineering of replication-defective, recombinant viruses (Principle)
Engineering of replication-defective, recombinant viruses (Principle)
UNIFR
Rusconi
2002
UNIFR
Rusconi
2002
E L1 L2 rprp
Wild type genome Normal target cells Virions
Recombinant genome R-Virions
E E E
EE
EE
Packaging cells
Normal target cells
X
PackagingPackagingPackaging
Cardiac ischaemia(Heart)
VEGF gene(vascular promoter)
2000 J. Isner
Limb ischaemia(Hands, Feet)
VEGF gene(vascular promoter)
1998 J. Isner
'Classical' GT models and strategies'Classical' GT models and strategiesUNIFRRusconi2002
UNIFRRusconi2002
Disease transferred function Clinical Results
SCID(Immunodeficiency)
IL2R gene(gamma-C receptor)
2000 A. Fischer
Haemophilia B(Blood)
Factor IX gene(blood clotting factor
1999-2000 M. Kay and K. Horwitz
Cystic Fibrosis(Lung, Pancreas)
CFTR gene(chlorine transporter)
no significant resultsin spite of several trials
ADA deficiency(Immunodeficiency)
ADA normal gene(enzyme)
1990 F. Anderson, 2002 C. Bordignon