Technical RemarksTechnical RemarksUNIFRRusconi2002

UNIFRRusconi2002

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S. Rusconi

Sandro RusconiSandro RusconiUNIFRRusconi

2002

UNIFRRusconi

2002

Sports doping:Is there a realistic

application for gene transfer?

Bern, Nov 22, 2002Swiss Olympic

gene doping workshop

1972-75 Primary school teacher (Locarno, Switzerland)1975-79 Graduation in Biology UNI Zuerich, Switzerland1979-82 PhD curriculum UNI Zuerich, molecular biology1982-84 Research assistant UNI Zuerich1984-86 Postdoc UCSF, K Yamamoto, (San Francisco)1987-91 Principal Investigator, UNI Zuerich (mol. bio.)1994-today Professor Biochemistry UNI Fribourg1996-today Director Swiss National Research Program 37

'Somatic Gene Therapy'2001 Participant Swiss Natl. Res Program 50

'Endocrine disruptors'2002 Sabbatical, Tufts Med. School Boston and

Univ. Milano, Pharmacology Department2002 President Union of Swiss Societies for

Experimental Biology (USGEB)

Essential concepts on 'molecular medicine' & molecular doping: applications and problems,

Gene-based dopingapplications, comparison with other doping, detection

Techniques of gene transfer (Gene Therapy)problems and solutions, vectors, clinical achievements

ScheduleScheduleUNIFR

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UNIFR

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Basic understanding of 'genes': what is a gene, how many genes, molecular biology dogmagenetic diseases, environmental factors, ageing

Conclusionsplausibility table

Genetics has been used since millennia,Molecular Biology, only since 30 years

Genetics has been used since millennia,Molecular Biology, only since 30 years

UNIFRRusconi

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UNIFRRusconi

2001

100’000 b.C. Empirical genetics

10’000 b.C.Biotechnology

2000 a.d.Molecular biology

2001 a.d, Genomics

1 Gene -> 1 or more functions1 Gene -> 1 or more functionsUNIFRRusconi

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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?)

What is in fact a gene?: a segment of DNA acting as a regulated machine for RNA production

What is in fact a gene?: a segment of DNA acting as a regulated machine for RNA production

UNIFRRusconi

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UNIFRRusconi

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RNADNA Protein

GENE FUNCTIONTranscription / translation

codingspacer spacerregulatoryDNA

RNA

1 Organism -> more than 105 genetically-controlled Functions

1 Organism -> more than 105 genetically-controlled Functions

UNIFRRusconi

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UNIFRRusconi

2002

2m 2 mm 0.2mm

0.02mm

DNA RNA Protein

0.001mm

Reductionistic molecular biology paradigm(gene defects and gene transfer)

Reductionistic molecular biology paradigm(gene defects and gene transfer)

UNIFRRusconi

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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 amplification / manipulation techniques(genetic engineering, recombinant DNA)

Gene amplification / manipulation techniques(genetic engineering, recombinant DNA)

UNIFRRusconi

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UNIFRRusconi

2002

nucleus & DNA.mov

segments of genomic DNA can be specifically cut and isolated

isolated segment can be recombined with a plasmid vector

plasmid vector is transferred into bacteria where it can multiply

isolated recombinant DNA can be further recombined to obtainthe final desired molecule

Final molecule is transferred into cells or organisms

Science-grade materialcan be essentially prepared in your cellar

...not so clinical-grade material!

The FOUR eras of molecular medicineThe FOUR eras of molecular medicineUNIFRRusconi

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UNIFRRusconi

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ok ** ** **ok1 2 4 53

NinetiesGenes as factories

80 85 90 95 99

10

50

Y2KGenes as drugs

80 85 90 95 00

1000

3000

Y2K+n Post-genomic improvements of former technologies

The major disease of the 21st century: AgeingThe major disease of the 21st century: AgeingUNIFR

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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

many treatments that slow down ageingor age-related degenerative diseases are also potential doping treatments

aa getting oldcomp2.mov

Now, let's talk about Somatic Gene Therapy (somatic gene transfer)

Now, let's talk about Somatic Gene Therapy (somatic gene transfer)

UNIFRRusconi2002

UNIFRRusconi2002

Definition of GT:'Use genes as drugs':Correcting disorders by somatic gene transfer

Chronic treatment

Acute treatment

Preventive treatment

Hereditary disorders

Acquired disorders

Loss-of-function

Gain-of-function

NFP37 somatic gene therapywww.unifr.ch/nfp37

Somatic gene therapy’s (gene transfer) four fundamental questions

Somatic gene therapy’s (gene transfer) four fundamental questions

UNIFRRusconi2002

UNIFRRusconi2002

Efficiency of gene transfer

Specificity of gene transfer

Persistence of gene transfer

Toxicity of gene transfer

Remember!

Why 'somatic'?Why 'somatic'?UNIFRRusconi2001

UNIFRRusconi2001

Germ Line Cells: the cells (and their precursors) that upon fertilisation can give rise to a descendant organism

Somatic Cells: all the other cells of the body

i.e. somatic gene transferis a treatment aiming atsomatic cells and conse-quently does not lead to a hereditary transmission of the genetic alteration

Pharmacological considerationsPharmacological considerationsUNIFR

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UNIFR

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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

Classical Drugs

Mw 20 ’000- 100 ’000 Da Biologically prepared Slower diffusion/action Oral delivery not possible Cellular delivery:

- act extracellularly

Can be delivered as soluble moleculesnm size

Protein Drugs

Mw N x 1’000’000 Da Biologically prepared Slow diffusion Oral delivery inconceivable Cellular delivery:

- no membrane translocation - no nuclear translocation- no biological import

Must be delivered as complex carrier particles50-200 nm size

Nucleic Acids

THREE classes of physiological gene deliveryTHREE classes of physiological gene deliveryUNIFR

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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 vehicles: non-viral & viralTWO classes of gene transfer vehicles: non-viral & viralUNIFR

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UNIFR

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a

b

Non-viral transfer(transfection)

Viral gene transfer(Infection)

Nuclear envelope barrier! see, Nature BiotechDecember 2001

Transfection with recombinant DNAVs Infection with recombinant viruses

Transfection with recombinant DNAVs Infection with recombinant viruses

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UNIFR

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Transfection

Infection

exposed to106 particles/cell12 hours

exposed to 3 particle/cell30 min

Quick parade of popular vectors/methodsQuick parade of popular vectors/methodsUNIFR

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UNIFR

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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 AAV (adeno-associated-virus)Recombinant AAV (adeno-associated-virus)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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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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UNIFR

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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. +/-)

Oligo-nucleotidesOligo-nucleotidesUNIFR

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UNIFR

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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 gene transfer vectors

Recap: current limitations of popular gene transfer vectors

UNIFR

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UNIFR

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Adenovirus- no persistence- limited packaging- toxicity- immunogenicity

Biolistic bombardmentor local direct injection- limited area

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

General- antibody response- limited packaging- gene silencing

Solutions:- synthetic viruses (“Virosomes”)

Electroporation- limited organ access

Liposomes, gene correction & Co.- very inefficient transfer

General- low transfer efficiency 1/10’000 of viruses’ in vivo

Solutions:- improved liposomes with viral properties (“Virosomes”)

The most feared potential side-effects of gene transferThe most feared potential side-effects of gene transferUNIFRRusconi2002

UNIFRRusconi2002

Immune response to vector

immune response to new or foreign gene product

General toxicity of viral vectors

Adventitious contaminants in recombinant viruses

Random integration in genome

-> insertional mutagenesis (-> cancer risk)

Contamination of germ line cells

Random integration in genome

-> insertional mutagenesis (-> cancer risk)

Gene Therapy in the clinic: Trials WordldwideGene Therapy in the clinic: Trials WordldwideUNIFRRusconi2002

UNIFRRusconi2002

cancer

hered.

Infect.vasc.

40

60

100

20

80

trials

500

1500

1000

patients

1992 1994 1996 19981990 2000

21% overall still pending or not yet Initiated !www.wiley.com

86% phase I13% phase II1 % phase III

As of Sept. 2002:599 registered protocols4000 treated patients

Gene Therapy MilestonesGene Therapy MilestonesUNIFRRusconi2002

UNIFRRusconi2002

1990, 1993, 2000 // ADA deficiencyF Anderson, M Blaese // C Bordignon

Anderson, 1990

Bordignon, 2000 (ESGT, Stockholm)proves efficacy of the same protocol

1997, 2000, Critical limb ischemiaJ Isner († 4.11.2001), I Baumgartner, Circulation 1998

Isner, 1998

1998, RestenosisV Dzau, HGT 1998

Dzau, 1999

1999, Crigler Njiar (animal)C Steer, PNAS 1999

Kmiec, 1999

2000, HemophiliaM Kay, K High

2000, SCIDA Fischer, Science April 2000

Fischer, 2000

2000, correction Apo E4 (animal model)G. Dickson, ESGT congress, 7.10.2000 Stockholm

Dickson, 2000

2000, correction Parkinson (animal model)P Aebischer, Science, Nov 2000

Aebischer, 2000

2001, ONYX oncolytic VirusesD Kirn (Gene Ther 8, p 89-98)

Kirn, 2001

Clinical trials with ONYX-015,what we learned?

(Review)

Gene Therapy Adverse events: NY 1995 // UPenn 1999 // Paris 2002

Gene Therapy Adverse events: NY 1995 // UPenn 1999 // Paris 2002

UNIFRRusconi2002

UNIFRRusconi2002

NY May 5, 1995, R. Crystal: in a trial with adenovirus mediated gene transfer to treat cystic fibrosis (lung) one patient developed a mild pneumonia-like condition and recovered in two weeks. The trial was interrupted and many others were put on hold.

UPenn, Sept. 19, 1999, J. Wilson: in a trial with adenovirus mediated gene transfer to treat OTC deficiency (liver) one patient (Jesse Gelsinger) died of a severe septic shock. Many trials were put on hold for several months (years).

Paris, Oct 2, 2002, A Fischer: in a trial with retrovirus mediated gene transfer to treat SCID (bone marrow) one patient developed a leukemia-like condition. The trial has been suspended to clarify the issue of insertional mutagenesis, and some trials in US and Germany have been put on hold.

Ups and Downs of Gene Therapy: a true roller coaster ride!

Ups and Downs of Gene Therapy: a true roller coaster ride!

UNIFRRusconi2002

UNIFRRusconi2002

high

Low

moo

d

NIHMotulskireport Lentivectors

in pre-clinic

Adeno III

J. Isner

J. WilsonJ. Gelsinger

ADA

R. CrystalAdeno I

90 91 92 93 94 95 96 97 98 99 00 01 02

AAV germline in mice?

V.Dzau

A. FischerM. Kay

lentivectorsin clinics?

NFP37

Paris

C Bordignon

Ergo:in spite of its respectable age,gene transfer is still in its infancyand still produces more controversiesthan clinical results

The THREE levels of dopingThe THREE levels of dopingUNIFR

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UNIFR

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Before thecompetition

(anabolic enhancers)

During the competition(perfomance enhancers)

After thecompetition

(repair enhancers)

'Molecular treatmentsApplication of the

know-how in molecular genetics

to doping

+

+

+

Which gene transfer approaches would be compatible with doping strategies

Which gene transfer approaches would be compatible with doping strategies

UNIFRRusconi2002

UNIFRRusconi2002

ex vivo, hematopoietic tissue:erythropoietin?

in vivo local (example muscle):metabolic enhancers, growth factors, muscular fiber changers

in vivo local (example joints):pain reducers, inflammation inhibitors, recovery and repair factors

in vivo systemic:anabolic factors, endocrine factors, pain killers

Which are the objective current limitations ingene-based doping strategies

Which are the objective current limitations ingene-based doping strategies

UNIFRRusconi2002

UNIFRRusconi2002

Viral gene transfer immune problems limited readministration general toxicity, genotoxicity

Nonviral gene transfer generally inefficient lack of persistence, requires readministration

Strategy-independent problems laborious, not readily available long term gene expression difficult to control irreversible effects or permanent tagging

Which side effects could be feared ingene-based doping strategies

Which side effects could be feared ingene-based doping strategies

UNIFRRusconi2002

UNIFRRusconi2002

Short -mid term Autoimmunity Hyperimmunity Toxic shock

Long term Fibrosis Cancer Conventional effects of

administered factors Inaccessibility to future gene

therapy interventions (immunity)

Intrinsic to reckless application(probably the biggest danger) malpractice (unsuitable

vector/administration route) non-clinical grade material

(adventitious pathogens or allergens)

lack of follow-up

Which detection methods would be (or not) evisageable for gene-based doping strategies

Which detection methods would be (or not) evisageable for gene-based doping strategies

UNIFRRusconi2002

UNIFRRusconi2002

Antibody detection (viral antigens or other epitopes) recombinant-nucleic acids detection (PCR) recombinant protein detection

(MALDI-TOF / proteomics)

Gene transfer may be anatomically difficult to detect (if locally administered) but leaves permanent genetic marking

the detection of nucleic acids cannot be performed in body fluids (except for systemically administered treatments) and might require specific tissue biopsy

Final side-by-side comparison: gene-based doping versus drug- or protein-based doping

Final side-by-side comparison: gene-based doping versus drug- or protein-based doping

UNIFRRusconi2002

UNIFRRusconi2002

Category Drug/protein Gene-based

Rapidity of effects rapid slow

Reversibility rapid slow/none

Complexity of treatm. simple complex

Associated risks depends high

Detectability arduous

'straightforward'

Dosage straightforward difficultErgo:The odds speak currently rather against the adoption ofgene-based doping, but this applies to common-sense clinical practice, and this aspect is not guaranteed in the doping field

Thank you all for the attention, and... if you are too shy to asksend an e-mail to:sandro.rusconi@unifr.chor visit:www.unifr.ch/nfp37

...Thanks ! ...Thanks ! UNIFRRusconi2002

UNIFRRusconi2002

Swiss National Research Foundation

Our own project/goal may indeed appear very small and harmless...

This does not necessarily apply to its consequences...

My collaborators at UNIFR

Swissolympics

discussion slidesdiscussion slidesUNIFRRusconi2002

UNIFRRusconi2002

Examples of inheritable gene defectsExamples of inheritable gene defectsUNIFRRusconi

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UNIFRRusconi

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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

The long way to drug/procedure registration is the principal cause of financial burden, but we cannot avoid it

The long way to drug/procedure registration is the principal cause of financial burden, but we cannot avoid it

UNIFR

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UNIFR

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0 Idea 0

2 Cell culture assays 0.5 Mio

5 Pre-clinical testsanimal models 2 Mio

7 Clinical phase I5-20 patientsverify side effects 6 Mio

10 Clinical phase II30-100 patientsdosis escalation 12 Mio

15 Clinical Phase III>300- 1000 patientsmulticentricdouble blind 80 Mio

16>> Registration / Availability

year event costs U$D

This means:assuming 20% of new developmentsmakes it to final registration,the average investment is 300-500 Mio U$Dfor each approved drug/procedure

Not only the genome determines the health status...Not only the genome determines the health status...UNIFRRusconi

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UNIFRRusconi

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genetics behaviour environment

Muscle distrophy

Familial Breast Cancer

Lung Cancer

Obesity

Artherosclerosis

Alzheimer

Parkinson ’s

Drug AbuseHomosexuality

Sporadic Breast Cancer

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

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

UUNIFR

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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

Engineering of replication-defective, recombinant viruses (Principle)

Engineering of replication-defective, recombinant viruses (Principle)

UNIFR

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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

Cardiac ischaemia(Heart)

VEGF gene(vascular promoter)

2000 J. Isner

Limb ischaemia(Hands, Feet)

VEGF gene(vascular promoter)

1998 J. Isner

'Classical' GT models and strategies'Classical' GT models and strategiesUNIFRRusconi2002

UNIFRRusconi2002

Disease transferred function Clinical Results

SCID(Immunodeficiency)

IL2R gene(gamma-C receptor)

2000 A. Fischer

Haemophilia B(Blood)

Factor IX gene(blood clotting factor

1999-2000 M. Kay and K. Horwitz

Cystic Fibrosis(Lung, Pancreas)

CFTR gene(chlorine transporter)

no significant resultsin spite of several trials

ADA deficiency(Immunodeficiency)

ADA normal gene(enzyme)

1990 F. Anderson, 2002 C. Bordignon