Sandro Rusconi (09.03.52)Sandro Rusconi (09.03.52)UNIFRRusconi

2004

UNIFRRusconi

2004

Tools for genetic modification

Oct. 10, 2004SUK education Program

'genetic regulation of cardiovascular functions'

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

Experimental Biology (USGEB) 2003-06 Euregenethy Network

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

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UNIFRRusconi

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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?)

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UNIFRRusconi

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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

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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!

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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

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UNIFRRusconi

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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

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UNIFRRusconi

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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...

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UNIFRRusconi

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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

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UNIFRRusconi

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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 doping performance amelioration cosmetics

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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?

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UNIFRRusconi

2004Pharmacological considerations for DNA transferPharmacological considerations for DNA transfer

OHOH

O

OH

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

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UNIFRRusconi

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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

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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)

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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

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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

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Recombinant AdenovirusesRecombinant AdenovirusesUNIFR

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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)UNIFR

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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

Recombinant retroviruses (incl. HIV)Recombinant retroviruses (incl. HIV)UUNIFR

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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

Naked or complexed DNANaked or 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 vectors

r-Adenovirus- no persistence- limited packaging- toxicity, immunogenicity

Biolistic bombardmentor local direct injection- limited area

r-Retrovirus (incl. HIV) - limited packaging- random insertion- unstable genome

General- antibody response- limited packaging- gene silencing- Manufacturing limitations

Solutions:- synthetic viruses (“Virosomes”)

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”)

Ergo the future will see increasing interest in viral-like, but artificial particles

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UNIFRRusconi

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r-AAV- no integration in host g.- very limited packaging- promotes autoimmunity?

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

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Genuine gene therapy oligos chimeroplasts (*gene correction induction)

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

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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

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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 April 2004:918 cumulative protocols (90-04)4500 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

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! 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

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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

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

Although genes discovered in the 90ties:

lacking suitable vector no satisfactory delivery

method

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UNIFRRusconi

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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

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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

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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'

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 integrase

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- 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.

SAEs 2:mid-term effects: Recent Autoimmunity Reports

SAEs 2: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

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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

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UNIFRRusconi

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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

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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

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UNIFRRusconi

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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

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UNIFRRusconi

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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

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UNIFRRusconi

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Thank you all for the attention,

sandro.rusconi@unifr.chor visit:

www.unifr.ch/nfp37/

...thanks, and let's remain optimistic...thanks, and let's remain optimistic

Swiss National Research Foundation

Zhihong Yang

SUK education program

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That's all, folks!That's all, folks!UNIFRRusconi

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www.unifr.ch/nfp37

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Discussion: Recap: what is a virus ? -> A superbly efficient replicating nanomachine

Discussion: Recap: what is a virus ? -> A superbly efficient replicating nanomachine

UUNIFR

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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)

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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