Sandro RusconiSandro RusconiUNIFRRusconi
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UNIFRRusconi
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2003: Gene therapy turning teenage, what
have we learned?
Feb 19, 2003ECPM Basel
1972-75 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 Zuerich1994-today Professor Biochemistry UNI Fribourg1995-today Director Swiss National Research Program 37
'Somatic Gene Therapy'2002-03 Sabbatical, Tufts Med. School Boston and
Univ. Milano, Pharmacology Department2002-05 President Union of Swiss Societies for
Experimental Biology (USGEB)
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
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UNIFRRusconi
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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
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UNIFRRusconi
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RNADNA
GENE
Protein
2-5 FUNCTIONS
Gene expression
Transcription / translation
>300 ’000 functions(>150 ’000 functions)
100 ’000 genes(50 ’000 genes?)
Recap: what is a gene?:a regulated machine for RNA production
Recap: what is a gene?:a regulated machine for RNA production
UNIFRRusconi
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UNIFRRusconi
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RNADNA Protein
GENE FUNCTIONTranscription / translation
codingspacer spacerregulatoryDNA
RNA
To fulfil its role, a transferred gene must include: regulatory sequences for Tx initiation proper signals for RNA maturation/transport proper signals for mRNA translation
1 Organism -> more than 105 genetically-controlled Functions
1 Organism -> more than 105 genetically-controlled Functions
UNIFRRusconi
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UNIFRRusconi
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2m 2 mm 0.2mm
0.02mm
DNA RNA Protein
0.001mm1 Cm3 of tissue 1'000'000'000 cells!
Reductionistic molecular biology paradigm(gene defects and gene transfer)
Reductionistic molecular biology paradigm(gene defects and gene transfer)
UNIFRRusconi
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UNIFRRusconi
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GENE transfer FUNCTION transfer
GENE KO FUNCTION KO
GENE OK FUNCTION OK
DNA
GENE
Protein
FUNCTION(s)
Gene transfer implies either: transfer of new function, or transfer of restoring function, or transfer of interfering function
Examples of inheritable gene defectsExamples of inheritable gene defectsUNIFRRusconi
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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
Ergo: every person bears one or more
latent genetic defects many defects are not manifest
but lead to predispositions there are also protective predispositions
Not only the genome determines the health status...Not only the genome determines the health status...UNIFRRusconi
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UNIFRRusconi
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genetics behaviour environment
Muscle distrophy
Obesity
Artherosclerosis
Alzheimer
Parkinson ’s
Drug AbuseHomosexuality
Familial Breast Cancer
Lung Cancer
Sporadic Breast Cancer
also acquired conditions may have a genetic component that modulates their healing
trauma fractures burns infections
The major disease of the 21st century: AgeingThe major disease of the 21st century: AgeingUNIFR
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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
This major challenge means: higher investments more financial returns long term treatment customised treatment social security dilemma
The THREE missions of medicineThe THREE missions of medicineUNIFR
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UNIFR
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Prevention
Diagnosis
Therapy
'Molecular Medicine'Application of the
know-how in molecular genetics
to medicine
+
+
+
The FOUR eras of molecular medicineThe FOUR eras of molecular medicineUNIFRRusconi
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UNIFRRusconi
2003EightiesGenes 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
Now, let's talk about Somatic Gene Therapy (SGT)Now, let's talk about Somatic Gene Therapy (SGT)UNIFRRusconi2003
UNIFRRusconi2003
Definition of SGT:'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
The SGT principle is simple Yes,...but the devil is often in the details
The SGT principle is simple Yes,...but the devil is often in the details
UNIFRRusconi2003
UNIFRRusconi2003
There are many things that are simple in principle, like...
getting a train ticket... ! try this 5 min before departureand with a group of Chinese tourists in front
parking your car... ! try this at noon, any given day in Zuerich or Geneva ...
counting votes... ! ask Florida's officials ...
gene therapy... look at progress in 13 years...
1990 First clinical trial of a monogenic diseaseF. Anderson & Co: ADA deficiency
2002 Same protocol as Anderson's for ADAgene therapy (C. Bordignon)
...it works!
...does not work
Gene therapy turns teenage in 2003, but:has it really grown up?
Why 'somatic'?Why 'somatic'?UNIFRRusconi2003
UNIFRRusconi2003
Germ Line Cells: the cells (spermatocytes and oocytes 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 therapyis a treatment aiming atsomatic cells and conse-quently does not lead to a hereditary transmission of the genetic alteration
Ergo transformation of
germ line cells is avoided, to exclude risk of erratic mutations due to insertional mutagenesis
When/where/ may be SGT indicated?When/where/ may be SGT indicated?UNIFRRusconi2003
UNIFRRusconi2003
No existing cure or treatment most monogenic diseases
Side effects and limitations of protein injection interleukin 12 (cancer)
-> toxic effects and rapid degradation VEGF (ischemias)
-> angiomas Factor VIII or IV (hemophilia)
-> insufficient basal level
Complement to conventional increase specificity of conventional therapy (cancer) increase efficacly of conventional therapy (hemophilia)
Life quality burden of patient costs of enzyme therapy (ex. ADA) burden of daily injections (ex. Insulin)
Ergo: there are many indications
for SGT as stand-alone or as complementary therapy
SGT's four fundamental questions & playersSGT's four fundamental questions & playersUNIFRRusconi2003
UNIFRRusconi2003
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
Remember!
The variables which disease? which gene? which vector? which target organ? which type of delivery?
The SGT acrobatics: matching vectors / delivery system / disease
The SGT acrobatics: matching vectors / delivery system / disease
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UNIFRRusconi2003
Chronic Conditions Slow onset of expression acceptable Initiation of the treatment
weeks/months/years before 'point of no return' (ex. cystic fibrosis)
persisting expression of the transgene or re-administration required (example hemophilia)
Usually based on compensation of 'genetic loss-of-function' (permanent re-gain of function; ex. ADA)
Regulation of gene expression often necessary (because of persistence)
For some diseases even a small % of tissue transformation is already therapeutic
Acute Conditions Rapid onset of expression necessary Initiation of the treatment
minutes/hours/days before 'point of no return' (ex. brain ischemia)
persisting expression of the transgene not required, occasional re-administration (example
Usually based on augmentation of resident function (transient gain of function; ex. VEGF)
Regulation of gene expression not necessary (because of transiency)
For most diseases even a small % of transformation is already therapeutic
Ergo many divergent variables must be matched for each case an advantage for one purpose becomes a disadvantage for another (viceversa)
Pharmacological considerations for DNA transferPharmacological considerations for DNA transferUNIFR
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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
rapidly reversible treatment
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
rapidly reversible treatment
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
slowly or not reversible
Nucleic Acids
Therapy with nucleic acids requires particulated formulation is much more complex than previous drug deliveries has a different degree of reversibility (dosage problem)
THREE classes of anatomical gene deliveryTHREE classes of anatomical gene deliveryUNIFR
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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 vectors: non-viral & viral delivery
TWO classes of gene transfer vectors: non-viral & viral delivery
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a
b
Non-viral transfer(transfection of plasmids)
Viral gene transfer(Infection by r-vectors)
Nuclear envelope barrier! see, Nature BiotechDecember 2001
Transfection versus InfectionTransfection versus InfectionUNIFR
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Transfection
Infection
exposed to106 particles/cell12 hours
exposed to 1 particle/cell30 min
Ergo virally mediated gene transfer is millions of times more efficent than nonviral
transfer (when calculated in terms of transfer/particle)
Most relevant issues in the two main 'vectorology' sectors (viral versus nonviral)
Most relevant issues in the two main 'vectorology' sectors (viral versus nonviral)
UNIFRRusconi2003
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Viral vectors Packaging capacity from 4 to 30 kb problem for
some large genes (ex. dystrophin gene or CFTR gene)
important toxic load: ratio infectious/non-infectious particles from 1/10 to 1/100
strong immunogenicity: capsid and envelope proteins, residual viral genes
contaminants: replication-competent viruses (ex. wild type revertant viruses)
Viral amount (titre) obtainable with recombinants (ex. 10exp5 = poor, 10exp10=excellent)
Complexity of production (existence or not of packaging cell systems)
Emotional problems linked to pathogenicity of donor vectors (ex. lentiviruses)
Nonviral vectors Packaging capacity not an issue, even very large
constructs can be used (example entire loci up to 150 kb)
minor toxic load: small percentage of non relevant adventitious materials
moderate immunogenicity: methylation status of DNA (example CpG motifs)
contaminants: adventitious pathogens from poor DNA purification (ex endotoxins)
Amount of DNA molecules is usually not a problem, the other components depends on chemical synthesis
No particular complexity, except for specially formulated liposomes
no particular emotional problems linked to the nature of the reagents
Ergo problems that must be solved to be suitable for clinical treatment and for industrial
production are different between viral and non-viral vectors when ignoring thir low efficiency, nonviral vectors appears largely superior
Ideal properties of a systemically delivered non-viral formulation
Ideal properties of a systemically delivered non-viral formulation
UNIFRRusconi2003
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Stability particle should resist serum inactivation particle should be inert to immune inactivation
Addressability particle should possess a vascular addressing signature particle should bear a tissue-docking specificity DNA construct should include tissue-specific regulatory elements
Efficiency cargo should be protected from cytoplasmic inactivation (ex. lysosomes) cargo should contain nuclear-translocating signals DNA cargo should include genome-integration functions DNA element must be guaranteed to function after genomic integration
(no silencing)Other properties Particle should not include immunogenic/toxic surfaces cargo should not encode immunogenic/toxic products Cargo should include anti-apoptotic functions
Ergo several independent
problems must be solved for a nonviral formulation to be suitable for clinical treatment and for industrial production
most viral vectors include many, if not all those properties
Small parade of popular vectors/methodsSmall parade of popular vectors/methodsUNIFR
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Adenovirus
Adeno-associated V.
Retrovirus (incl. HIV)
Naked DNA
Liposomes & Co.
Oligonucleotides
Recombinant AdenovirusesRecombinant AdenovirusesUNIFR
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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 adeno-associated-virus (AAV)Recombinant adeno-associated-virus (AAV)UNIFR
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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
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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
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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. +/-)
OligonucleotidesOligonucleotidesUNIFR
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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 vectorsRecap: current limitations of popular vectorsUNIFR
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Adenovirus- no persistence- limited packaging- toxicity, immunogenicity
Biolistic bombardmentor local direct injection- limited area
Retrovirus (incl. HIV)- limited packaging- 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- no or little genomic integration
Solutions:- improved liposomes with viral properties (“Virosomes”)
Ergo the future will see increasing interest in viral-like, but artificial particles
Not all gene therapy approaches are 'random shooting'Not all gene therapy approaches are 'random shooting'UNIFRRusconi2003
UNIFRRusconi2003
Random integrating vectors r-lentiviruses r-retroviruses r-AAV plasmids (low frequency) plasmids + transposase (eg 'sleeping beauty')
Transient, non integrating vectors adenovirus plasmid RNA virus based oligonucleotides (SiRNA, antisense, ribozymes) artificial chromosomes
Gene correction vectors chimeroplasts (RNA-DNA chimeric oligos) single stranded DNA (homologous recom)
Ergo genotoxic non-genotoxic
Specifically integrating vectors hybrid vectors (HSV-AAV) Phage 31 integrase-based designer integrase
Rapid and transient action required
Adeno II, Plasmid, modulatory oligonucleotides
Trauma or infection(Ischemia, fracture, burn, wound, acute infection, anaphyllaxis)
rapid & transient expression of cytotoxic or immunomodulators
Adeno II, Plasmid, oncolytic recombinant viruses
Solid tumors +/- metastat.(cervical, breast, brain, skin)
No rapid expression necessary, persistence required, low toxicity
AAV, nonviral, LentiLocal chronic or progressive (ex. CNS, joints, eyes)
Justifications /IssuesMost suitable vector
persistence of expression of the transferred gene, minimize readministration
Chronic Metabolic (ex. OTC, Gaucher, Haemophilia, hematopoietic)
AAV, Lenti, Adeno III, r-retroviruses, repair oligo
Which vector for which disease categoryWhich vector for which disease categoryUNIFRRusconi2003
UNIFRRusconi2003
Disease Type
Technologies related to-, but not genuinely definable as 'gene therapy'
Technologies related to-, but not genuinely definable as 'gene therapy'
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UNIFRRusconi2003
Bioactive oligonucleotides antisense decoy dsDNA decoy RNA ribozymes DNAzymes Si RNA
Oncolytic viruses ONYX-15, ONYX-638 (r-adeno) r-HSV r-FSV
Implants of encapsulated cells neurotrophic factor producer cell implants hormone-producing cells
Cardiac ischaemia(Heart)
VEGF gene (vascular growth
factor), plasmid, intracardiac
2000 J. Isner
Limb ischaemia(Hands, Feet)
VEGF gene (vascular growth
factor), plasmid, intramuscular
1998 J. Isner
'Classical' SGT models and strategies'Classical' SGT models and strategiesUNIFRRusconi2003
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SCID(Immunodeficiency)
IL2R gene (gamma-C receptor)
retrov., ex vivo BM
2000 A. Fischer
Haemophilia B(Blood)
Factor IX gene (clotting factor),
aav, adenoIII, intramuscular
1999-2000 M. Kay, K. High
Cystic Fibrosis(Lung, Pancreas)
CFTR gene (chlorine transpor-
ter), retrov., aav, adenoII, local
no significant resultsin spite of several trials
ADA deficiency(Immunodeficiency)
ADA normal gene (enzyme)
retrovirus, ex-vivo BM
1990 F. Anderson, 2002 C. Bordignon
Disease transferred function Clinical Results
additional 'popular' and emerging examples:Morbus Gaucher, Morbus Parkinson, Crigler Njiar, OTC deficiency, Duchenne's MD, Restenosis control
Gene Therapy in the clinic: Trials WordldwideGene Therapy in the clinic: Trials WordldwideUNIFRRusconi2003
UNIFRRusconi2003
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/genetherapy
66% phase I21% phase I-II11% phase II0.8% phase II-III0.7% phase III
As of December 2002:632 registered protocols3472 treated patients
Ergo in spite of 13 year-
research only less than 1% of the trials has reached phase III
II-II
II
Gene therapy in Switzerland: the 30 projects financed by the NFP37 programme (1996-2001)
Gene therapy in Switzerland: the 30 projects financed by the NFP37 programme (1996-2001)
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NFP37 phase A phase B (96-99) (99-01)
Submissions 30 26 Granted 19 18 Total requested 32 Mio 9 Mio Granted 7.6 Mio 6 Mio DISEASE ORIENTATION Cancer 8 10 Acquired disorders 2 7 Vector development 5 3 Hereditary disorders 2 4 Infectious diseases 1 2
RESEARCH LEVEL Fundamental 10 7 Preclinical (animal models) 5 9 Clinical phase I 2 3 Clinical Phase II 0 1 Clinical Phase III 0 0 Ethical/social aspects 1 1
NationalesForschungsprogramm 37
NFP37« somatic gene therapy »
www.unifr.ch/nfp37
Please Note the NFP37 represented at
most 30% of the Swiss-based experimentation in SGT during 1996-2001
Gene Therapy Clinical and Preclinical MilestonesGene Therapy Clinical and Preclinical MilestonesUNIFRRusconi2003
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1990, 1993, 2000 // ADA deficiencyF Anderson, M Blaese // C Bordignon
Anderson, 1990
Bordignon, 2000 (ESGT, Stockholm)2002, science 296, 2410 ff)
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, 20002002
2000, correction Apo E4 (animal model)G. Dickson, 2000 esgt, 2002 BBA
Dickson, 2000
2000, correction Parkinson (animal model)P Aebischer, Science, Nov 2000
Aebischer, 2000
2001, ONYX oncolytic VirusesD Kirn (Cancer Gene Ther 9, p 979-86)
Kirn, 2000,20012002
Intravascular adenoviral agents in cancer patients:
Lessons from clinical trials(review)
Two major SGT frustration casesTwo major SGT frustration casesUNIFRRusconi2003
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Muscular dystrophy (incidence 1: 3000 newborn males)
requires persistence of expression extremely large gene (14 kb transcript, 2 megaBP gene unclear whether regulation necessary unclear at which point disease is irreversible
Cystic fibrosis (incidence 1: 2500 newborns)
luminal attempts failed because of anatomical / biochemical barrier: no receptors, mucus layer
large gene that requires probably regulation requires long term regulation unclear at which point disease becomes irreversible
Although genes discovered in the 90ties:
no suitable vector no satisfactory delivery
method
The most feared potential side-effects of gene transferThe most feared potential side-effects of gene transferUNIFRRusconi2003
UNIFRRusconi2003
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)
Ergo Most side effects are still related to the rather
primitive state of the vectorology/delivery
Paris, Jan 14, 2003, A Fischer: a second patient of the cohort of 9 comes up with a similar disease than the one reported in october 2002. 30 trials in USA are temporarily suspended
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.
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).
Three (four) bitter lessons, but only one treatment-related death so far
Three (four) bitter lessons, but only one treatment-related death so far
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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 interrupted and many others on hold.
Public perception problemsPublic perception problemsUNIFRRusconi2003
UNIFRRusconi2003
Negative perception of manipulative genetics general aversion of genetic manipulation fear of catastrophic scenarios
Deception after excessive promises hopes reinforced by media spectacularisation and
over-simplification deception after non-complied deadline
Confusion with other gene-based and non-gene-based technologies
stem cell technology human cloning procedures genetically modified food
Other factors that have negatively influenced the public perception and progress of gene therapy
Other factors that have negatively influenced the public perception and progress of gene therapy
UNIFRRusconi2003
UNIFRRusconi2003
Naive statements by some good-willing scientists in the early 90ties Not-so-naive statements by not-so-naive scientists in search of fame Huge amount of money that flowed into the research and development
that attracted many incompetent researchers. Concomitance with stock-market euphoria (little attention to realism) Reckless statements or misreporting by greedy scientists or company managers
to increase the value of their stock options (memorandum by the ASGT on conflict of interest 2000, www.asgt..org)
Tendency by the media to spectacularise good news and/or bad news
Ergo An explosive cocktail, just like for sports or arts,... the field tends to degenerate as soon as huge amounts of money are involved
and when the mass media become interested in it.
Ups and Downs of Gene Therapy: a true roller coaster ride!
Ups and Downs of Gene Therapy: a true roller coaster ride!
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high
Low
moo
d
NIHMotulskireport
Lentivectorsin pre-clinic
Adeno III
J. Isner
ADA
R. Crystal
Adeno 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
C Bordignon
Ergo whenever a reasonable cruise
speed was achieved, a major adverse event has brought us back square one
03
Adverseevents inParis
J. WilsonJ. Gelsinger
Genes, cells, tissue transplants...some people fear possible negative developments
Genes, cells, tissue transplants...some people fear possible negative developments
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Amelioration instead of therapy?
Too High-techtoo expensive
Bioweapons?
aa beauty woman.mov
robot woman.mov2
Somatic Gene Therapy is facing fierce competitionSomatic Gene Therapy is facing fierce competition
1. Cell Therapy (Stem cells (SC)) identified in many tissues cell transfer could be combined with gene transfer there would be no anatomical barriers for gene transfer Selection /amplification of desired transformants
Current limitations of SC Lack of control on differentiation and trans-determination Difficulties in complex organ-reconstruction
Future of SC: Increasing number of SC types will be characterised culturing conditions will be perfectioned May replace in vivo gene transfer for treatment of chronic
conditions?
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V
2. Breakthroughs from the small/medium molecules
STI571 (Glivec) anti HER2 (Herceptin) Si RNA? ...
3. Challengers from the biomechanics world
bone reconstruction intelligent protheses (stents) micropumps artificial organs
ConclusionsConclusionsUNIFRRusconi2003
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Fundamentally a gene encodes usually more than one function The therapeutic gene transfer in somatic cells must cope
with: efficiency, specificity, persistence and toxicity many genes with potential therapeutic value have been
identified, and essentially all types of diseases can be treated by gene transfer
Vectors and models There is the choice of a certain number of viral and non
viral vectors, none of them being generally applicable viral vectors have the advantage of efficiency and nonviral
vector the advantage of lower toxicity/danger. viral vectors have the disadvantage of limited packaging
and some toxicity, while nonviral vector have the major disadvantage of low efficiency of transfer
Clinically over 600 trials and 3500 patients in 12 years only a handful of trials is now reaching phase III Progress further slowed down by periodical pitfalls
PerspectivesPerspectivesUNIFRRusconi2003
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Fundamental level & vectorology the better understanding of gene interactions and
networking (functional genomics) could improve the utilisation of gene-based or gene targeted strategies
novel paradigms can become available (Si RNA, PNA triplex etc...)
specifically integrating gene constructs or artificial chromosomes becoime more realistic
Preclinically scaling up to larger animal models (dog and monkey)
permits better appreciation of dosage requirements new transgenic models may give improved similarities to
human diseases
Clinically Use of recombinant lentiviruses may be imminent Increase of Phase III procedures over the next 5 years First therapeutical applications may be registered within
3-5 years challenge by other emerging therapies
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 ! UNIFRRusconi2003
UNIFRRusconi2003
Swiss National Research Foundation
My collaborators at UNIFR
ECPM
Discussion: The Paris' trial (see also www.unifr.ch/nfp37/adverse.html)
Discussion: The Paris' trial (see also www.unifr.ch/nfp37/adverse.html)
UNIFRRusconi2003
UNIFRRusconi2003
Disease deficiency of the receptor gamma(c) incapacity of maturing lymphocytes severe combined immunodeficiency lethal at 4 months if untreated survival 10 years under sterile conditions
Gene Therapeutical approach explant BM (3-6 month old) select CD34+ transduce with retroviral vector encoding gamma(c) re-infusion, follow-up
Conventional treatments maintenance under sterile condition treatment with antibiotics transplant of matching bone marrow
Discussion: The Paris' odyssey(see also www.unifr.ch/nfp37/adverse.html)
Discussion: The Paris' odyssey(see also www.unifr.ch/nfp37/adverse.html)
UNIFRRusconi2003
UNIFRRusconi2003
Chronology 1998 start treatment of patients 2000 publication results first 2 patients 2001/2002 publication further 8 patients 9 out of 10 responded well, back home, normal life
Adverse 1 summer 2002, high WBC in a 36 months patient september 2002, hyper-proliferatory cells with insertion in proximity of LMO2 gene, notification authorities October 2003, public disclosure, chemotherapy, good response, report at ESGT congress. October 2003 3 US and 3 EU trials on hold
Adverse 2 december 2002, T cell hyper-proliferation in a second, 36 months patient hyper-proliferatory cells also contain insertion of transgene close to LMO2 gene January 2003, notification to authorities, public disclosure, treatment chemotherapy January 2003, 27 US and 5 EU trials on hold
Discussion: Questions & hypotheses from the Paris' Trial(see also www.unifr.ch/nfp37/adverse.html)
Discussion: Questions & hypotheses from the Paris' Trial(see also www.unifr.ch/nfp37/adverse.html)
UNIFRRusconi2003
UNIFRRusconi2003
Facts in both patients insertion of the transgene in proximity of LMO2 this type of insertion not found in CD34+ cells in these patients LMO2 expression is apparently increased in these patients LMO2 gene already known as proto-oncogene involved in
some chromosomal-translocations found in some leukaemias gamma(c) receptor can respond to IL-2, IL-5, IL-7, IL-9, IL-15,
Il-21 and ... gamma(c) receptor is therefore itself a pro-proliferatory and
anti-apoptotic signaling molecule
Perspectives if the answers are'YES' 'NO' 'UNK'good bad not goodgood bad not goodgood bad not goodbad good not good
Questions/hypotheses
is this adverse event specific for the disease status? is the transgene contributing to the hyper-proliferatory potential? is the gamma(c) synergising with LMO2? Has there been such an adverse event in the over 20 retrovirally
transduced patients treated so far for other diseases?
Discussion: Recap: what is a virus ? -> A superbly efficient replicating machine
Discussion: 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
Discussion: Engineering of replication-defective, recombinant viruses (Principle)
Discussion: 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
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