Sandro Rusconi 09'03'52

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Sandro Rusconi (09.03.52)
UNIFR Rusconi 2005
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 and Univ. 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)
March 1, 2005 ECPM course
Gene Therapy growing teenage, what have we
learned?
2005-xx Head of governmental division for
culture and university affairs of Canton Ticino
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Gene therapy A 15-years hailstorm of highly
emotionalised good and bad news
UNIFR Rusconi 2005
BBC, NBC, CNN,...
Jesse Gelsinger Oct 1999
New York Times Washington Post Times Le
Monde Frankfurter Allgemeine ...
Feb 1990 First trial ADA deficiency
A Fischer, E Thrasher Paris UK Dec 2000
Dec 1988 IL-2 cancer treatment trial
AAV germline Sept 2000
No previous medical procedure generated so many
discussions so long before being ever clinically
applicable
Mar 1994 SAE cystic fibrosis
C Bordignon, Milano trial May 2002
Jun 1995 Motulsky NIH report
First SAE Paris Sep 2002
Feb 1996 r-lentiviruses
Nature Science NEJM ...
Oct 1998 VEGF ischemia
second SAE Paris Feb 2003
Internet
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1 Gene -gt 1 or more functions
UNIFR Rusconi 2005

• Ergo
• to say 'one gene-gtone function' is like
  pretending'one disease -gt one drug'

• Multifunctional character implies
• cross talk with different pathways
• unclarified hyerarchical position
• unclarified side-effects potential

gt300 000 functions (gt150 000 functions)
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Recap what is a gene?a regulated nanodevice
for RNA production
UNIFR Rusconi 2005

• 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

coding
spacer
spacer
regulatory
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1 Organism -gt more than 105 developmentally and
genetically-controlled functions
UNIFR Rusconi 2005

• Remember
• 1 Cm3 of tissue
• 1'000'000'000 cells!

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Reductionistic molecular biology paradigm(gene
defects and gene transfer)
UNIFR Rusconi 2005

• Gene transfer implies either
• transfer of new function, or
• transfer of restoring function, or
• transfer of interfering function

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Examples of inheritable gene defects
UNIFR Rusconi 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 Mul
ti 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 - max Cystic fibrosis, muscular
dystrophy immodeficiencies, metabolic diseases,
all together Hemophilia... 0.4 - 0.7
Predispositions Type estimated min - 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

• Ergo
• every person bears one or more latent genetic
  defects
• many defects are not manifest but lead to
  predispositions
• there are also protective predispositions

Sum of incidences min - max (all
defects) 32 - 83
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Not only the genome determines the health
status...
UNIFR Rusconi 2005

• also acquired conditions may have a genetic
  component that modulates their healing
• trauma
• fractures
• burns
• infections

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The major disease of the 21st century Ageing
UNIFR Rusconi 2005

• This major challenge means
• higher investments
• more financial returns
• long term treatment
• customised treatment
• social security dilemmawill molecular therapy
  boost the efficacy of treatment of age-related
  diseases?

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The THREE missions of medicine
UNIFR Rusconi 2005
Prevention

'Molecular Medicine' Application of the
know-how in molecular genetics to medicine
Diagnosis


Therapy
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The FOUR eras of molecular medicine
UNIFR Rusconi 2003
genomeABC.mov
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Somatic Gene Therapy (SGT)
UNIFR Rusconi 2005
Chronic treatment
Definition of SGT 'Use genes as
drugs' Correcting disorders by somatic gene
transfer
Acute treatment
Preventive treatment
NFP37 somatic gene therapy www.unifr.ch/nfp37
Hereditary disorders
Acquired disorders
Loss-of-function
Gain-of-function
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The SGT principle is simple Yes,...but the
devil is often in the details
UNIFR Rusconi 2005
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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Why 'somatic'?
UNIFR Rusconi 2005

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

• Ergo
• transformation of germ line cells is avoided, to
  exclude risk of erratic mutations due to
  insertional mutagenesis

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

• Somatic Cells all the other cells of the body

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When/where/ may be SGT (currently) indicated?
UNIFR Rusconi 2005

• No existing cure or treatment
• most monogenic diseases

• Side effects and limitations of protein injection
• interleukin 12 (cancer)-gt toxic effects and
  rapid degradation
• VEGF (ischemias)-gt angiomas
• Factor VIII or IV (hemophilia)-gt insufficient
  basal level

• Ergo
• there are many indications for SGT as stand-alone
  or as complementary therapy

• Complement to conventional
• increases specificity of conventional therapy
  (cancer)
• increases efficacy of conventional therapy
  (hemophilia)

• Perfid deviation dreams (with current
  technologyI
• gene-based sports doping
• performance amelioration
• cosmetics

• Life quality burden of patient
• costs of enzyme therapy (ex. ADA)
• burden of daily injections (ex. Insulin)

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SGT's four fundamental questions players
UNIFR Rusconi 2005
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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Pharmacological considerations for DNA transfer
UNIFR Rusconi 2005
Nucleic Acids
Classical Drugs
Protein Drugs

• Mw N x 1000000 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

• 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

• 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

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• 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)

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THREE classes of anatomical gene delivery
UNIFR Rusconi 2005
Ex-vivo
In-vivo topical delivery
In-vivo systemic delivery
Examples - bone marrow - liver cells - skin cells
Examples - brain - muscle - eye - joints - tumors
Examples - intravenous - intra-arterial -
intra-peritoneal
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TWO classes of gene transfer vectors non-viral
viral delivery
UNIFR Rusconi 2005
Non-viral transfer (transfection of plasmids)
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Viral gene transfer (Infection by r-vectors)
b
Nuclear envelope barrier! see, Nature
Biotech December 2001
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Transfection versus Infection
UNIFR Rusconi 2005
Transfection
exposed to 106 particles/cell 12 hours
Infection
exposed to 1 particle/cell 30 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)
UNIFR Rusconi 2005

• 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-infecti
  ous 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, 10exp10excellent)
• 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/methods
UNIFR Rusconi 2005
Naked DNA Liposomes Co. Oligonucleotides
r-Adenovirus r-Adeno-associated
V. r-Retrovirus (incl. HIV)
but remember... "Nobody's perfect "!
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Recombinant Adenoviruses
UNIFR Rusconi 2005

• Manufacturing
• Generation I/ II
• Generation III
• Hybrid adenos
• Adeno-RV
• Adeno-AAV
• Adeno-Transposase

• Advantages / Limitations
• 8 Kb capacity Generation I / IIgt30 Kb capacity
  Generation IIIAdeno can be grown at very high
  titers,However
• Do not integrate in host genome
• Can contain RCAs
• Are toxic /immunogenic

• Examples
• OTC deficiency (clin, ---)
• Cystic Fibrosis (clin, --- )
• Oncolytic viruses (clin, )

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Recombiant Adeno-associated-virus (AAV)
UNIFR Rusconi 2005

• Advantages / Limitations
• Persistence in the genome permits long-
• term expression, high titers are easily
• obtained, immunogenicity is very low,
• However the major problems are
• insertional mutagenesis
• Promotes autoimmunity?
• Small capacity (lt4.5 kb) which does not allow to
  accommodate large genes or gene clusters.

Manufacturing Helper-dependent production Helper
independent production Cis-complementing
vectors Co-infection

• Examples
• Hemophilia A (clin, animal, (autoimm?)
• Gaucher (clin, animal, )
• Brain Ischemia (animal, )
• Cystic fibrosis (animal, /-)
• retinopathy (animal (/-)

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Recombinant retroviruses (incl. HIV)
UNIFR Rusconi 2005
Manufacturing Murine Retroviruses VSV-pseudotyped
RV Lentiviruses ! Self-inactivating
RV Combination viruses

• Advantages / Limitations
• 9 Kb capacity integration through
• transposition also in quiescent cells
• (HIV), permit in principle long-term
• treatments, however disturbed by
• Insertional mutagenesis
• Gene silencing
• High mutation rate
• Low titer of production

• Examples
• SCID (IL2R defect, Paris) (clin, )
• Adenosine Deaminase deficiency (clin, !!!)
• Parkinson (preclin, )
• Anti cancer (clin /-)

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Naked or complexed DNA
UNIFR Rusconi 2005
Approaches Naked DNA injection /biolistic Naked
DNA pressure Naked DNA electroporation Liposom
al formulations Combinations

• Advantages / Limitations
• Unlimited size capacity lower
• immunogenicity and lower bio-risk
• of non viral formulations is
• disturbed by
• Low efficiency of gene transfer
• Even lower stable integration

• Examples
• Critical limb Ischemia (clin, )
• Cardiac Ischemia (clin, /-)
• Vaccination (clin, /-)
• Anti restenosis (preclin. /-)

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Oligonucleotides
UNIFR Rusconi 2005
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, )

v !
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Recap current limitations of popular vectors
UNIFR Rusconi 2004
r-Adenovirus - no persistence - limited
packaging - toxicity, immunogenicity
Biolistic bombardment or local direct injection -
limited area
Electroporation - limited organ access
r-AAV - no integration in host g. - very limited
packaging - autoimmunity?
Liposomes, gene correction Co. - rather
inefficient transfer
r-Retrovirus (incl. HIV) - limited packaging -
random insertion - unstable genome

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

General - low transfer efficiency - no or little
genomic integration
General - antibody response - limited packaging -
gene silencing - Manufacturing limitations
Solutions - improved liposomes with viral
properties (Virosomes)
Solutions - synthetic viruses (Virosomes)
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Which vector for which disease category
UNIFR Rusconi 2005
Justifications /Issues
Most 'suitable' vector
Disease Type
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Technologies related to-, but not all genuinely
definable as 'gene therapy'
UNIFR Rusconi 2005

• Transiently bioactive oligonucleotides
• antisense
• decoy dsDNA, decoy RNA
• ribozymes DNAzymes
• Si RNA oligonucleotides

• Genuine gene therapy oligos
• chimeroplasts (gene correction induction)

• Oncolytic viruses
• ONYX-15, ONYX-638 (r-adeno)
• r-HSV
• r-FSV

from www.nature.com

• Ergo
• among all these, SiRNA seems to be the most
  promising inhibitor factor, and can be
  permanently expressed from DNA vectors

• Implants of encapsulated cells
• neurotrophic factor producer cell implants
• hormone-producing cells

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'Classical' SGT models and strategies
UNIFR Rusconi 2005
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)
UNIFR Rusconi 2005

• 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

As of Jan 2005938 cumulative protocols
(90-2004) 4600 treated /enrolled patients
66 phase I 19 phase I-II 13 phase II 0.8
phase II-III 1.7 phase III
! As of Jan 1, 2004 1 approved product in China
(Gendicine, by Sibiono Inc. 2004
21 overall still pending or not yet Initiated
! www.wiley.com/genetherapy
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Gene Therapy Clinical and Preclinical Milestones
UNIFR Rusconi 2005
1990, 1993, 2000, 2004 // ADA deficiency F
Anderson, M Blaese // C Bordignon
1997, 2000, Critical limb ischemia J Isner (
4.11.2001), I Baumgartner, Circulation 1998
1998, Restenosis V Dzau, HGT 1998
2000, Hemophilia M Kay, K High
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)
2000, 2002, X-SCID A Fischer, Science April 2000,
UK trials 2003
2001, 2003 ONYX oncolytic Viruses D Kirn (Cancer
Gene Ther 9, p 979-86)
2004, Chronic Granulomatous Disease M Grez
Frankfurt R Seger Zürich
2004, Gendicine (adeno-p53 vector) L Peng,
Sibiono Inc, Shenzen, China
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Two persisting major SGT frustration cases
UNIFR Rusconi 2005

• 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

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The most feared potential side-effects of gene
transfer
UNIFR Rusconi 2004

• 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-gt insertional
  mutagenesis (-gt cancer risk)
• Contamination of germ line cells

• immune response or long term side effects from
  new or foreign gene product -gt autoimmunity

• Random integration in genome-gt insertional
  mutagenesis (-gt 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

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SAEs1 best documented cases acute and long term
SAEs from Gelsingers' death to Paris'
Leukaemias caused by insertional mutagenesis
UNIFR Rusconi 2005
NY May 5, 1995, R. Crystal adenovirus, cystic
fibrosis (lung) one patient mild pneumonia-like
condition Trial interrupted and many others on
hold.
Most Recent Paris' Trial News discussed
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
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).
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.
Paris, Jan 14, 2003, A Fischer retrovirus
X-SCID (bone marrow) same cohort a second patient
developed a similar leukemia 30 trials in USA
were temporarily suspended
Ergo gene therapy can produce both short-term and
long-term severe side effects through acute
immunogenicity or insertional mutagenesis (cancer
risk)
Paris, Jan 24, 2005, A Fischer retrovirus
X-SCID (bone marrow) same cohort a third patient
developed a similar leukemia what will happen?
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Future solutions to insertional mutagenesis
targeted gene transfer approaches
UNIFR Rusconi 2005

• Ergo
• genotoxic
• non-genotoxic

• Random integrating vectors
• r-retroviruses
• r-lentiviruses
• r-AAV
• plasmids (low frequency)
• plasmids transposase (eg 'sleeping beauty')

• Specifically integrating vectors
• hybrid vectors (HSV-AAV)
• Phage 31 integrase-based
• designer integrases (ZnFinger proteins)

• Transient, non integrating vectors
• adenovirus
• plasmid
• RNA virus based
• oligonucleotides (SiRNA, antisense, ribozymes)
• artificial chromosomes

Ergo vector systems that allow specific or at
least better location-controlled gene delivery
are experimentally well advanced (see
accompanying text)

• Gene correction vectors
• chimeroplasts (RNA-DNA chimeric oligos)
• single stranded DNA (homologous recom)

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SAEs2 mid-term effects Recent Autoimmunity
Reports
UNIFR Rusconi 2005
Blood, 1 May 2004, Vol. 103, No. 9, pp.
3248-3249 Autoimmunity in EPO gene transfer
(macaques) Els Verhoeyen and François-Loïc Cosset
- 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
Ergo somatic gene transfer can generate mid-term
self immunity under inappropriate circumstances
K High, ASGT June meeting 2004 Abstract1002
Immune Responses to AAV and to Factor IX in a
Phase I Study of AAV-Mediated, Liver-Directed Gene
Transfer for Hemophilia B
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SAEs3 Non-science factors that have disturbed
the public perception and progress of gene therapy
UNIFR Rusconi 2005

• '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

• 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

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Ups and Downs of Gene Therapy a true
roller-coaster ride!
UNIFR Rusconi 2005
A. Fischer M. Kay
high
R. Crystal
V.Dzau
C Bordignon
lentivectors in clinics?
Adeno I
J. Isner
promising results 2003-2004
F Anderson
AAV germline in mice?
NIH Motulski report
Adeno III
mood

• Ergo
• whenever a reasonable cruise speed was achieved,
  a major adverse event has brought us back square
  one or even below

Lentivectors
Auto-immunity
Low
Paris I and II Leukaemias
J. Gelsinger
Paris III
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Conclusions 1 in spite of the many hurdles, GT
has already saved gt20 condemned lives and keeps
producing positive signals
UNIFR Rusconi 2004

• 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

• 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)

• Ergo
• gene therapy's principle works
• we better know limitations and potential of
  individualvectors

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Conclusions 2 GT has proven several concepts,
has several tools, but is still in the pioneering
phase
UNIFR Rusconi 2005

• 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

• Ergo
• we are somewhat ahead but still in the pioneering
  phase !
• failure of evidence does not mean evidence of
  failure !

• Clinically
• over 900 trials and gt4000 patients in 14 years
• only a handful of trials is now reaching phase
  III
• Progress further slowed down by periodical
  pitfalls

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Perspectives somatic gene therapy will progress
in spite of all past, present and future
incidents/accidents
UNIFR Rusconi 2005

• 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

• 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

• 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

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...Thanks, and let's remain optimistic
UNIFR Rusconi 2005
ECPM education program

Fritz Bühler, Annette Mollet
Swiss National Research Foundation
Thank you all for the attention,
sandro.rusconi_at_unifr.ch or visit www.unifr.ch
/nfp37/
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That's all, folks!
UNIFR Rusconi 2005
www.unifr.ch/nfp37
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UNIFR Rusconi 2004
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Discussion Recap what is a virus ? -gt A
superbly efficient replicating nanomachine
UUNIFR Rusconi 2002
entry
disassembly
docking
genome replication
early genes exp
replication
late genes exp
assembly
standard viral genome
Spread
Etc...
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Discussion Engineering of replication-defective
, recombinant viruses (Principle)
UNIFR Rusconi 2002
E
Wild type genome
Virions
R-Virions
Packaging
Packaging
Packaging
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Discussion The Paris' trial (see also
www.unifr.ch/nfp37/adverse.html)
UNIFR Rusconi2003

• 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

• Conventional treatments
• maintenance under sterile condition
• treatment with antibiotics
• transplant of HLA-matching bone marrow

• Gene Therapeutical approach
• explant BM (3-6 month old)
• select CD34/CD38-
• transduce with retroviral vector encoding
  gamma(c)
• re-infusion, follow-up

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Discussion The Paris' odyssey(see also
www.unifr.ch/nfp37/adverse.html)
UNIFR Rusconi2005

• 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 patienthyper-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

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Discussion Questions hypotheses from the
Paris' Trial(see also www.unifr.ch/nfp37/adverse.
html)
UNIFR Rusconi2005

• 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 2005
• yes
• yes
• yes
• no

• 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?

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Discussion Examples of gene transfer treatments
against cancer
UNIFR Rusconi2002

• percentage of trials(therapeutic potential)

• Type of treatment
• examples

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Discussion Why so many cancer trials?
UNIFR Rusconi2002
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
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Discussion An effective cancer molecular
treatmentOncolytic viruses on the example of
ONYX-015
UNIFR Rusconi2002

• 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 dynamicsDISADVANTAGE
• danger of evolving viruses
• unclear if it works in adeno-immune patients
• unclear if if works in immuno-compromised
  patients (chemotherapy)

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Title
UNIFR Rusconi 2004

• text
• text

• Ergo
• txt

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