sandro rusconi (09.03.52)
DESCRIPTION
UNIFR Rusconi 2005. Sandro Rusconi (09.03.52). 1972-75 School teacher (Locarno, Switzerland) 1975-79 Graduation in Biology UNI Zuerich, Switzerland 1979-82 PhD curriculum UNI Zuerich, molecular biology 1982-84 Research assistant UNI Zuerich - PowerPoint PPT PresentationTRANSCRIPT
Sandro Rusconi (09.03.52)Sandro Rusconi (09.03.52)UNIFRRusconi
2005
UNIFRRusconi
2005
Gene Therapy growing teenage,
what have we learned?
March 1, 2005ECPM course
1972-75 School teacher (Locarno, Switzerland)
1975-79 Graduation in Biology UNI Zuerich, Switzerland
1979-82 PhD curriculum UNI Zuerich, molecular biology
1982-84 Research assistant UNI Zuerich
1984-86 Postdoc UCSF, K Yamamoto, (San Francisco)
1987-93 Principal Investigator, UNI Zuerich, PD
1994-today Professor Biochemistry UNI Fribourg
1996-2002 Director Swiss National Research Program 37
'Somatic Gene Therapy'
2002-03 Sabbatical, Tufts Med. School Boston andUniv. Milano, Pharmacology Department
2002-05 President Union of Swiss Societies for
Experimental Biology (USGEB)
2002-06 Euregenethy Network (EU-harmonsation of
biosafety and ethical aspects in gene therapy)
2005-xx Head of governmental division for cultureand university affairs of Canton Ticino
Gene therapy: A 15-years hailstorm of highly emotionalised good and bad news
Gene therapy: A 15-years hailstorm of highly emotionalised good and bad news
UNIFRRusconi
2005
UNIFRRusconi
2005
BBC, NBC, CNN,...
New York TimesWashington PostTimesLe MondeFrankfurter Allgemeine...
Feb 1990 First trial ADA deficiency
Dec 1988 IL-2 cancer treatment trial
Mar 1994 SAE cystic fibrosis
NatureScienceNEJM...
Jun 1995 Motulsky NIH report
Feb 1996 r-lentiviruses
Oct 1998 VEGF ischemia
Jess
e Gelsi
nger Oct
1999
A Fischer, E Thrasher Paris & UK Dec 2000
AAV germline Sept 2000
C Bordignon, Milano trial May 2002
First SAE Paris Sep 2002 second SAE Paris Feb 2003
Internet
No previous medical procedure generated so many discussions
so long before being ever clinically applicable
1 Gene -> 1 or more functions1 Gene -> 1 or more functions
RNA(s)DNA
GENE
Protein(s)
2-5 FUNCTIONS
Gene expression
Transcription / translation
>300 ’000 functions(>150 ’000 functions)
100 ’000 genes(50 ’000 genes?)
UNIFRRusconi
2005
UNIFRRusconi
2005
Multifunctional character implies: cross talk with different pathways unclarified hyerarchical position unclarified side-effects potential
Ergo to say
'one gene->one function' is like pretending'one disease -> one drug'
Recap: what is a gene?:a regulated nanodevice for RNA production
Recap: what is a gene?:a regulated nanodevice for RNA production
RNA(s)DNA Protein(s)
GENE FUNCTIONTranscription / translation
codingspacer spacerregulatoryDNA
RNA
Therefore, to fulfil its role, a transferred gene must include:
regulatory sequences for Transcription proper signals for RNA maturation/transport proper signals for mRNA translation proper signals for mRNA degradation
UNIFRRusconi
2005
UNIFRRusconi
2005
1 Organism -> more than 105 developmentally and genetically-controlled functions
1 Organism -> more than 105 developmentally and genetically-controlled functions
2m 2 mm 0.2mm
0.02mm
DNA RNA Protein
0.001mmRemember1 Cm3 of tissue 1'000'000'000 cells!
UNIFRRusconi
2005
UNIFRRusconi
2005
Reductionistic molecular biology paradigm(gene defects and gene transfer)
Reductionistic molecular biology paradigm(gene defects and gene transfer)
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
UNIFRRusconi
2005
UNIFRRusconi
2005
Examples of inheritable gene defectsExamples of inheritable gene defectsUNIFRRusconi
2005
UNIFRRusconi
2005
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
2005
UNIFRRusconi
2005
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: Ageing
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
aa getting oldcomp2.mov
This major challenge means: higher investments more financial returns long term treatment customised treatment social security dilemma
will molecular therapy boost the efficacy of treatment of age-related diseases?
UNIFRRusconi
2005
UNIFRRusconi
2005
The THREE missions of medicineThe THREE missions of medicine
Prevention
Diagnosis
Therapy
'Molecular Medicine'Application of the
know-how in molecular genetics
to medicine
+
+
+
UNIFRRusconi
2005
UNIFRRusconi
2005
The FOUR eras of molecular medicineThe FOUR eras of molecular medicineUNIFRRusconi
2003
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
Somatic Gene Therapy (SGT)Somatic Gene Therapy (SGT)
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
UNIFRRusconi
2005
UNIFRRusconi
2005
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
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...
UNIFRRusconi
2005
UNIFRRusconi
2005
Why 'somatic'?Why 'somatic'?
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
UNIFRRusconi
2005
UNIFRRusconi
2005
When/where/ may be SGT (currently) indicated?When/where/ may be SGT (currently) indicated?
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 increases specificity of conventional therapy (cancer) increases efficacy 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
Perfid deviation dreams (with current technologyI:
gene-based sports doping performance amelioration cosmetics
UNIFRRusconi
2005
UNIFRRusconi
2005
SGT's four fundamental questions & playersSGT's four fundamental questions & players
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
The variables which disease? which gene? which vector? which target organ? which type of delivery?
UNIFRRusconi
2005
UNIFRRusconi
2005
UNIFRRusconi
2005
UNIFRRusconi
2005Pharmacological considerations for DNA transferPharmacological considerations for DNA transfer
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 (intrinsic dosage / titration problem)
THREE classes of anatomical gene deliveryTHREE classes of anatomical gene delivery
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
UNIFRRusconi
2005
UNIFRRusconi
2005
TWO classes of gene transfer vectors: non-viral & viral delivery
TWO classes of gene transfer vectors: non-viral & viral delivery
a
b
Non-viral transfer(transfection of plasmids)
Viral gene transfer(Infection by r-vectors)
Nuclear envelope barrier! see, Nature BiotechDecember 2001
UNIFRRusconi
2005
UNIFRRusconi
2005
Transfection versus InfectionTransfection versus Infection
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)
UNIFRRusconi
2005
UNIFRRusconi
2005
Comparing relevant issues in the two main 'vectorology' sectors (viral versus nonviral)
Comparing relevant issues in the two main 'vectorology' sectors (viral versus nonviral)
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 manufacturing (existence or not of packaging cell systems) ('MAD' !)
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
manufacturing are different between viral and non-viral vectors when ignoring thir low efficiency, nonviral vectors appears largely superior
UNIFRRusconi
2005
UNIFRRusconi
2005
Short list of popular vectors/methodsShort list of popular vectors/methods
r-Adenovirus
r-Adeno-associated V.
r-Retrovirus (incl. HIV)
Naked DNA
Liposomes & Co.
Oligonucleotides
UNIFRRusconi
2005
UNIFRRusconi
2005
Recombinant AdenovirusesRecombinant AdenovirusesUNIFRRusconi2005
UNIFRRusconi2005
Manufacturing
Generation I/ II
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 / II>30 Kb capacity Generation IIIAdeno can be grown at very high titers,However Do not integrate in host genome
Can contain RCAs
Are toxic /immunogenic
Recombiant Adeno-associated-virus (AAV)Recombiant Adeno-associated-virus (AAV)
Examples Hemophilia A (clin, animal, +++(autoimm?) Gaucher (clin, animal, +++) Brain Ischemia (animal, +++) Cystic fibrosis (animal, +/-) retinopathy (animal (+/-)
Advantages / Limitations
Persistence in the genome permits long-term expression, high titers are easilyobtained, immunogenicity is very low,However the major problems are: insertional mutagenesis Promotes autoimmunity? Small capacity (<4.5 kb) which does
not allow to accommodate large genes or gene clusters.
Manufacturing
Helper-dependent production
Helper independent production
Cis-complementing vectors
Co-infection
UNIFRRusconi2005
UNIFRRusconi2005
Recombinant retroviruses (incl. HIV)Recombinant retroviruses (incl. HIV)
Manufacturing
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
UNIFRRusconi2005
UNIFRRusconi2005
Naked or complexed DNANaked or complexed DNA
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. +/-)
UNIFRRusconi2005
UNIFRRusconi2005
OligonucleotidesOligonucleotides
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, +++)
UNIFRRusconi2005
UNIFRRusconi2005
Recap: current limitations of popular vectorsRecap: current limitations of popular vectors
r-Adenovirus- no persistence- limited packaging- toxicity, immunogenicity
Biolistic bombardmentor local direct injection- limited area
Electroporation- limited organ access
Liposomes, gene correction & Co.- rather inefficient transfer
General- low transfer efficiency- no or little genomic integration
Solutions:- improved liposomes with viral properties (“Virosomes”)
UNIFRRusconi
2004
UNIFRRusconi
2004
r-AAV- no integration in host g.- very limited packaging- autoimmunity?
r-Retrovirus (incl. HIV) - limited packaging- random insertion- unstable genome
General- antibody response- limited packaging- gene silencing- Manufacturing limitations
Solutions:- synthetic viruses (“Virosomes”)
Ergo the future will probably see an
increasing interest in viral-like, but artificial particles
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 category
Disease Type
UNIFRRusconi
2005
UNIFRRusconi
2005
Technologies related to-, but not all genuinely definable as 'gene therapy'
Technologies related to-, but not all genuinely definable as 'gene therapy'
Transiently bioactive oligonucleotides antisense decoy dsDNA, decoy RNA ribozymes DNAzymes Si RNA oligonucleotides
Oncolytic viruses ONYX-15, ONYX-638 (r-adeno) r-HSV r-FSV Implants of encapsulated cells
neurotrophic factor producer cell implants hormone-producing cells
UNIFRRusconi
2005
UNIFRRusconi
2005
from www.nature.com
Genuine gene therapy oligos chimeroplasts (*gene correction induction)
Ergo among all these, SiRNA
seems to be the most promising inhibitor factor, and can be permanently expressed from DNA vectors
Cardiac ischaemia(Heart)
VEGF gee (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 strategies
SCID(Immunodeficiency)
IL2R gene (gamma-C receptor)
retrov., ex vivo BM
2000 A. Fischer2002, UK trials
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
UNIFRRusconi
2005
UNIFRRusconi
2005
Gene Therapy in the clinics: Trials Worldwide (cumulative)
Gene Therapy in the clinics: Trials Worldwide (cumulative)
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 I19% phase I-II13% phase II0.8% phase II-III1.7% phase III
As of Jan 2005:938 cumulative protocols (90-2004)4600 treated /enrolled patients
Ergo in spite of 13 year- research only
less than 2% of the trials has reached phase III
not necessarily due to the «novel»'fail early, fail fast' paradigm
II-II
II
UNIFRRusconi
2005
UNIFRRusconi
2005
! As of Jan 1, 2004:1 approved product in China (Gendicine, by Sibiono Inc. 2004
Gene Therapy Clinical and Preclinical MilestonesGene Therapy Clinical and Preclinical Milestones
1990, 1993, 2000, 2004 // 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
2000, HemophiliaM Kay, K High
2000, 2002, X-SCIDA Fischer, Science April 2000, UK trials 2003
Fischer, 20002002
2001, 2003 ONYX oncolytic VirusesD Kirn (Cancer Gene Ther 9, p 979-86)
Kirn, 2000,200120022003
Intravascular adenoviral agents in cancer patients:
Lessons from clinical trials(review)
2004, Chronic Granulomatous DiseaseM Grez Frankfurt; R Seger Zürich
Manuel GrezHans Peter HossleReinhard Seger2004
very encouraging data from just initiated clinical trial,prospected >10 patients
UNIFRRusconi
2005
UNIFRRusconi
2005
Approved commercialisation of Gendicine (Jan 2004) for cancer treatment in China
SibionoShenzen
2004, Gendicine (adeno-p53 vector)L Peng, Sibiono Inc, Shenzen, China
21 lives saved 21 lives were so far documentedly saved by GT in european trials (x-SCID, ADA, CGD) (France, UK, Italy) (all in phase I)~200 lives quality-improved in several other phase I and II trial~xxx lives saved or quality-improved ?by Gendicine (still undocumented)
Two persisting major SGT frustration casesTwo persisting major SGT frustration cases
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)
most 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
In spite of genes discovered in the 90ties:
lacking suitable vector no satisfactory delivery
method no persistence treatment too late
UNIFRRusconi
2005
UNIFRRusconi
2005
The most feared potential side-effects of gene transferThe most feared potential side-effects of gene transfer
Immune response to vector
immune response or long term side effects from
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«The more effective is a drug, the more side effects
it will generate». SGT enjoyed a side-effect-free illusion during its
first 10-year of non-working early period Many side effects are still related to the rather
primitive state of the vectorology/delivery
UNIFRRusconi
2004
UNIFRRusconi
2004
immune response or long term side effects from
new or foreign gene product -> autoimmunity
Paris, Jan 24, 2005, A Fischer: retrovirus X-SCID (bone marrow) same cohorta third patient developed a similar leukemia what will happen?
Paris, Jan 14, 2003, A Fischer: retrovirus X-SCID (bone marrow) same cohorta second patient developed a similar leukemia 30 trials in USA were temporarily suspended
Paris, Oct 2, 2002, A Fischer: retrovirus , x-SCID (bone marrow) one patient developed a leukemia-like condition.Trial suspended and some trials in US and Germany on hold until 2003.
UPenn, Sept. 19, 1999, J. Wilson: adenovirus , OTC deficiency (liver) one patient (Jesse Gelsinger) died of a severe septic shock. Many trials were put on hold for several months (years).
SAEs1: best documented cases: acute and long term SAEs: from Gelsingers' death to Paris' Leukaemias caused by insertional mutagenesis
SAEs1: best documented cases: acute and long term SAEs: from Gelsingers' death to Paris' Leukaemias caused by insertional mutagenesis
NY May 5, 1995, R. Crystal: adenovirus, cystic fibrosis (lung) one patient mild pneumonia-like conditionTrial interrupted and many others on hold.
UNIFRRusconi
2005
UNIFRRusconi
2005
Ergogene therapy can produce both short-term and long-term severe side effects through acute immunogenicity or insertional mutagenesis (cancer risk)
Most Recent Paris' Trial Newsdiscussed at:
www.unifr.ch/nfp37/adverse03.html
it is now rather established (2004) that the Paris' leukaemia events were caused by
treatment-specific circumstances (type of transferred gene, dosing, type of vector,
predisposition)The third SAE might delay the nextly
planned restart of patients recruitment
Future solutions to insertional mutagenesis: targeted gene transfer approaches
Future solutions to insertional mutagenesis: targeted gene transfer approaches
Random integrating vectors r-retroviruses r-lentiviruses 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 integrases (ZnFinger proteins)
UNIFRRusconi
2005
UNIFRRusconi
2005
Ergovector systems that allow specific or at least better location-controlled gene delivery are experimentally well advanced (see accompanying text)
- Chenuaud and colleagues (page 3303)- Gao and colleagues (page 3300)inadvertent autoimmune response in nonhuman primates resulting from transfer of a gene encoding a self-antigen.- delivered the homologous EPO cDNA driven by ubiquitous and/or regulatable promoters via AAV vectors injected in muscle or aerosolized in lung, resulting in supra-physiologic serum levels of EPO, from 10- to 100 000- fold over the baseline
SAEs2: mid-term effects: Recent Autoimmunity Reports
SAEs2: mid-term effects: Recent Autoimmunity Reports
Blood, 1 May 2004, Vol. 103, No. 9, pp. 3248-3249Autoimmunity in EPO gene transfer (macaques)Els Verhoeyen and François-Loïc Cosset
UNIFRRusconi
2005
UNIFRRusconi
2005
K High, ASGT June meeting 2004[Abstract1002] Immune Responses to AAV and to Factor IX in a Phase I Study of AAV-Mediated, Liver-DirectedGene Transfer for Hemophilia B
Ergosomatic gene transfer can generate mid-term self immunity under inappropriate circumstances
SAEs3: Non-science factors that have disturbed the public perception and progress of gene therapy
SAEs3: Non-science factors that have disturbed the public perception and progress of gene therapy
'Naive' statements in the early 90ties
Excess of speculative financing in mid-late 90ties.
Concomitance with stock-market euphoria
Reckless statements/promises or misreporting in late 90ties
Tendency by the media to spectacularise good and/or bad news
QuickTime™ et un décompresseurVidéo sont requis pour visualiser
cette image.
Ergo Money and media: an explosive cocktail, just like for sports or
arts,... the field tends to degenerate as soon as excessive financial speculations are involved and when the mass media become overly interested in it.
The fundamental error: we pretended making a business issue out of a scientific issue
UNIFRRusconi
2005
UNIFRRusconi
2005
Ups and Downs of Gene Therapy: a true roller-coaster ride!
Ups and Downs of Gene Therapy: a true roller-coaster ride!
high
Low
moo
d
NIHMotulskireport
Lentivectors
Adeno III
J. Isner
F Anderson
R. Crystal
Adeno I
A. FischerM. Kay
AAV germline in mice?
Ergo whenever a reasonable cruise
speed was achieved, a major adverse event has brought us back «square one» or even below
V.Dzau
Paris I and IILeukaemias
J. Gelsinger
90 91 92 93 94 95 96 97 98 99 00 01 02 03 04
UNIFRRusconi
2005
UNIFRRusconi
2005
05
C Bordignon
promisingresults
2003-2004
Auto-immunity
lentivectorsin clinics?
?
?
?
Paris III
Conclusions 1: in spite of the many hurdles, GT has already saved >20 condemned lives and keeps producing positive signals
Conclusions 1: in spite of the many hurdles, GT has already saved >20 condemned lives and keeps producing positive signals
X- SCID trials France: 9/10 patients permanently cured of the lethal
disease X-SCID UK: 6/6 patients cured of X-SCID lethal condition
Ergo gene therapy's principle works we better know limitations and
potential of individualvectors
ADA deficiency C Bordignon trials 4/4 patients permanently corrected +
detoxified
Others significant amelioration of CLI condition in Phase II trials important therapeutic benefit with oncolytic viruses promising amelioration in hemophilia patients promising results from Chronic Granulomatosis treatment First gene medicine product registered in China by
Sibiono Inc. (see www.unifr.ch/sibiono.html)
UNIFRRusconi
2004
UNIFRRusconi
2004
QuickTime™ et un décompresseurVidéo sont requis pour visualiser
cette image.
Conclusions 2: GT has proven several concepts, has several tools, but is still in the pioneering phase
Conclusions 2: GT has proven several concepts, has several tools, but is still in the pioneering phase
Fundamentally many new potentially therapeutic genes identified All types of diseases can be virtually treated by gene
transfer we start to manage efficiency, specificity, persistence and
toxicity
Vectors and models Choice of among a number of viral and non viral vectors Viral vectors have the advantage of efficiency nonviral vector the advantage of lower toxicity/danger. Viral vectors have the disadvantage of limited packaging
and some toxicity nonviral vectors have the major disadvantage of low
efficiency of transfer
Clinically over 900 trials and >4000 patients in 14 years only a handful of trials is now reaching phase III Progress further slowed down by periodical pitfalls
QuickTime™ et un décompresseurSorenson Video 3 sont requis pour visualiser
cette image.
Ergo we are somewhat ahead but still
in the pioneering phase !
«failure of evidence» does not mean «evidence of failure» !
UNIFRRusconi
2005
UNIFRRusconi
2005
Perspectives: somatic gene therapy will progress in spite of all past, present and future incidents/accidents
Perspectives: somatic gene therapy will progress in spite of all past, present and future incidents/accidents
Fundamental level & vectorology
Better understanding of gene interactions and networking Gene inhibition through Si RNA specifically integrating gene constructs artificial chromosomes become more realistic
Preclinically scaling up to larger animal models (dog and monkey) new transgenic models may give improved similarities to
human diseases
Clinically Use of recombinant lentiviruses 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
QuickTime™ et un décompresseurCinepak Codec von Radius[32] sont requis pour visualisercette image.
QuickTime™ et un décompresseurCinepak sont requis pour visualiser
cette image.
Ergo many adverse events were due rather
to human errors than to intrinsic dangers
other undesired effects are due to prototypic state of tools
hurdles can be overcome the genuine potential of SGT is intact
UNIFRRusconi
2005
UNIFRRusconi
2005
Thank you all for the attention,
or visit:
www.unifr.ch/nfp37/
...Thanks, and let's remain optimistic...Thanks, and let's remain optimistic
Swiss National Research Foundation
Fritz Bühler, Annette Mollet
ECPM education program
QuickTime™ et un décompresseurSorenson Video sont requis pour visualisercette image.
QuickTime™ et un décompresseurCinepak sont requis pour visualiser
cette image.
QuickTime™ et un décompresseurCinepak sont requis pour visualiser
cette image.
QuickTime™ et un décompresseurSorenson Video sont requis pour visualiser
cette image.
UNIFRRusconi
2005
UNIFRRusconi
2005
That's all, folks!That's all, folks!
QuickTime™ et un décompresseurSorenson Video sont requis pour visualiser
cette image.
UNIFRRusconi
2005
UNIFRRusconi
2005
www.unifr.ch/nfp37
UNIFRRusconi
2004
UNIFRRusconi
2004
Discussion: Recap: what is a virus ? -> A superbly efficient replicating nanomachine
Discussion: Recap: what is a virus ? -> A superbly efficient replicating nanomachine
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
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+/CD38- transduce with retroviral vector encoding gamma(c) re-infusion, follow-up
Conventional treatments maintenance under sterile condition treatment with antibiotics transplant of HLA-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)
UNIFRRusconi2005
UNIFRRusconi2005
Chronology 1998 A Fischer's team starts 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 oncogene, 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
Adverse 3 January 2005, T cell hyper-proliferation in a third, ~36 months patient Fischer's trial is again 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)
UNIFRRusconi2005
UNIFRRusconi2005
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
answers early 2005yesyesyesno
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: Examples of gene transfer treatments against cancer
Discussion: Examples of gene transfer treatments against cancer
UNIFRRusconi2002
UNIFRRusconi2002
percentage of trials(therapeutic potential)
Directly cytotoxic Prodrug activation Tumor-specific cytotoxic expression
38% of protocols(strong complementary effect, possible relapse)
Immunostimulatory Transfer of Immuno-attracting functions Instruction of immune cells
31% of protocols(very strong potential, low relapse chances)
Immunoprotective Transfer chemoresistance genes in
immune cells
5 % of protocols(very limited application, laborious, not widely explored)
Tumor modulation Restore tumor-suppressor functions down-regulate pro-oncogenic functions
26 % of protocols(good effect but low bystander and likely relapse)
Type of treatment examples
Discussion: Why so many cancer trials?Discussion: Why so many cancer trials?UNIFRRusconi2002
UNIFRRusconi2002
Better benfit/risk balance and high emotional acceptance (terminal patients, ethical committees)
Market potential higher than monogenic diseases (most thereof being orphan diseases)
Many more diversified approaches envisageable than in monogenic diseases
Much higher number of patients/center than in monogenic diseases
Discussion: An effective cancer molecular treatment:Oncolytic viruses on the example of ONYX-015
Discussion: An effective cancer molecular treatment:Oncolytic viruses on the example of ONYX-015
A) Normal Adenovirus can propagate in virtually all cells
B) ONYX-015 deleted E1B function can propagate efficiently only in P53 -deficient cells (e.g. most cancer cells)
Clinical success Head & Neck Cancer
Awaiting for further successes (currently in Phase II and III)
expected to be useful in combination with conventional therapy
ADVANTAGE: the 'drug' has its own dynamics
DISADVANTAGE: danger of evolving viruses unclear if it works in adeno-immune
patients unclear if if works in immuno-
compromised patients (chemotherapy)
UNIFRRusconi2002
UNIFRRusconi2002
TitleTitleUNIFRRusconi
2004
UNIFRRusconi
2004
text text
Ergo txt