Sandro Rusconi (09.03.52) UNIFR Rusconi 2004 Tools for genetic modification Oct. 10, 2004 SUK education Program 'genetic regulation of cardiovascular functions' 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 1995-today Director Swiss National Research Program 37 'Somatic Gene Therapy' 2002-03 Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department
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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)1987-93 Principal Investigator, UNI Zuerich, PD1994-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
Gene therapy: A 14-years hailstorm of highly emotionalised good and bad news
Gene therapy: A 14-years hailstorm of highly emotionalised good and bad news
UNIFRRusconi
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UNIFRRusconi
2004
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
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UNIFRRusconi
2004
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
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UNIFRRusconi
2004
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
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UNIFRRusconi
2004
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
- 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
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.
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
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UNIFRRusconi
2004
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 were so far documentedly saved by GT in european trials (x-SCID, ADA, CGD) (France, UK, Italy) (all in phase I)~200 life qualities improved in several other phase I and II trial
Two persisting major SGT frustration casesTwo persisting major SGT frustration cases
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
Although genes discovered in the 90ties:
lacking suitable vector no satisfactory delivery
method
UNIFRRusconi
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UNIFRRusconi
2004
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)
ErgoThe more effective is a drug the more side effects it
can produce. SGT enjoyed a side-effect-free illusion during its
10-year 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 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: earlier cases: acute and long term SAEs: from Gelsinger to Paris Leukaemiaa
SAEs1: earlier cases: acute and long term SAEs: from Gelsinger to Paris Leukaemiaa
NY May 5, 1995, R. Crystal: adenovirus, cystic fibrosis (lung) one patient mild pneumonia-like conditionTrial interrupted and many others on hold. !! Most Recent Paris' Trial News
In the Discussion or at:www.unifr.ch/nfp37/adverse.html
it is now rather established (2004) that the leukaemia events were caused by treatment specific circumstances (Type of transferred gene, dosing, type of vector, predisposition)
UNIFRRusconi
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UNIFRRusconi
2004
Ergogene therapy can produce short term and long term effetcs
Not all gene therapy approaches are 'random shooting'Not all gene therapy approaches are 'random shooting'
- 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.
- 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.
Blood, 1 May 2004, Vol. 103, No. 9, pp. 3248-3249Autoimmunity in EPO gene transfer (macaques)Els Verhoeyen and François-Loïc Cosset
UNIFRRusconi
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UNIFRRusconi
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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
Non-science factors that have negatively influenced the public perception and progress of gene therapy
Non-science factors that have negatively influenced the public perception and progress of gene therapy
'Naive' statements in the early 90ties
Excess of speculative funds in mid-late 90ties.
Concomitance with stock-market euphoria (little attention to realism)
Reckless statements/promises or misreporting in late 90ties
Tendency by the media to spectacularise good and/or bad news
Ergo An explosive cocktail, just like for sports or arts,... the field tends to degenerate as soon as excessive amounts
of money are involved and when the mass media become overly interested in it.
The error: we pretended making a business issue out of a scientific issue
UNIFRRusconi
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UNIFRRusconi
2004
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
V.Dzau
ParisLeukaemias
J. Gelsinger
90 91 92 93 94 95 96 97 98 99 00 01 02 03 04
UNIFRRusconi
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UNIFRRusconi
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05
C Bordignon
promisingresults
2003-2004
Auto-immunityEpoFactor IX
lentivectorsin clinics?
??
?
Conclusions 1: in spite of the many hurdles, GT has already saved >20 otherwise condemned lives and keeps producing positive signals
Conclusions 1: in spite of the many hurdles, GT has already saved >20 otherwise condemned lives and keeps producing positive signals
X- SCID trials France: 9/10 patients permanently cured of the lethal
disease X-SCID UK: 8/8 patients cured of X-SCID lethal condition
Ergo gene therapy's principle works
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 First gene medicine product registered in China by
Sibiono Inc. (see www.unifr.ch/sibiono.html
UNIFRRusconi
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UNIFRRusconi
2004
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
Ergo we are somewhat ahead but still in
the pioneering phase !
«failure of evidence» does not mean «evidence of failure» !
UNIFRRusconi
2004
UNIFRRusconi
2004
Perspectives: somatic gene therapy will progress in spite of all past, present and future adverse events
Perspectives: somatic gene therapy will progress in spite of all past, present and future adverse events
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
Ergo many adverse events were due more
to human errors than to intrinsic dangers
other undesired effects are due to primitive state of tools
hurdles can be overcome the genuine potential of SGT is intact