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Sandro Rusconi (09.03.52) a aa a aa UNIFR Rusconi 2003 2003: wohin führt uns Gentherapie? 20 Oktober 2003 Liestal Biovalley 1972-75School teacher (Locarno,

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Presentation on theme: "Sandro Rusconi (09.03.52) a aa a aa UNIFR Rusconi 2003 2003: wohin führt uns Gentherapie? 20 Oktober 2003 Liestal Biovalley 1972-75School teacher (Locarno,"— Presentation transcript:

1 Sandro Rusconi ( ) a aa a aa UNIFR Rusconi : wohin führt uns Gentherapie? 20 Oktober 2003 Liestal Biovalley School teacher (Locarno, Switzerland) Graduation in Biology UNI Zuerich, Switzerland PhD curriculum UNI Zuerich, molecular biology Research assistant UNI Zuerich Postdoc UCSF, K Yamamoto, (San Francisco) Principal Investigator, UNI Zuerich, PD 1994-todayProfessor Biochemistry UNI Fribourg 1995-todayDirector Swiss National Research Program 37 'Somatic Gene Therapy' Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department President Union of Swiss Societies for Experimental Biology (USGEB) Courmayeur, March 2003 *essentielle wiederholung in Genetik* *grundkonzepte der Gentherapie* *Klinische experimentiering in SGT* *Hohe und Tiefe in der SGT* *Schlussfolgerungen und Perspektiven*

2 *essentielle Wiederholungen in Genetik* a aa a aa UNIFR Rusconi 2003

3 1 Gen -> 1 oder mehrere Funktionen 1 Genom-> über Nx100'000 Funktionen a aa a aa UNIFR Rusconi 2003 RNA DNA GENE Protein 2-5 FUNCTIONS Gene expression Transcription / translation > functions (> functions) genes ( genes?)

4 1 Organismus -> Zellen, verteilt und spezialisiert in Organe und Gewebe a aa a aa UNIFR Rusconi m 2 mm0.2mm 0.02mm DNA RNAProtein 0.001mm 1 Cm 3 Gewebe 1'000'000'000 Zellen

5 Aber was istr eigentlich 'ein Gen'?: eine regulierbare Nano-machine zur Herstellung von RNA a aa a aa UNIFR Rusconi 2003 RNADNAProtein GENEFUNCTION Transcription / translation codingspacer regulatory DNA RNA Um wirksam zu sein sollte ein transferierter Gen beinhalten: Sequenzen fuer Genregulation Signale fuer reifung/transport der RNA Signale fuer Uebersetzung in proteinen

6 Das reduktionistische Paradigma des Molekularbiologes a aa a aa UNIFR Rusconi 2003 a aa a aa GENE transferFUNCTION transfer GENE KOFUNCTION KO GENE OKFUNCTION OK DNA GENE Protein FUNCTION(s) Gentransfer kann beinhalte: transfer einer neuen Funktion, oder transfer einer kompensierenden F., oder transfer einer interferierenden Funktion

7 Beispiele von Vererbbare Defekte a aa a aa UNIFR Rusconi 2003 Polygenic defectsTypeestimated ( frequent )min-max Diabetes poly1-4 % HyperurikemiaMulti2-15 % Glaucomapoly1-2 % DisplasiaMulti1-3 % HypercolesterolemiaMulti1-5 % Syn-& Polydactylypoly % Congenital cardiac defectsMulti % Manic-depressive psychosisMulti % Miopypoly3 -4 % Polycystic kidneypoly0.1-1 % PsoriasisMulti2 -3 % SchizofreniaMulti % ScoliosisMulti3 -5 % Monogenic defectsestimated ( rare ) min-max Cystic fibrosis, muscular dystrophy immodeficiencies, metabolic diseases, all together Hemophilia % PredispositionsTypeestimated min-max (*) AlzheimerMulti7 -27 % (*) ParkinsonMulti1-3 % (*) Breast cancerMulti4 -8 % (*) Colon CarcinomaMulti % (*) ObesityMulti0.5-2 % (*) Alcolholism/ drug addictionMulti0.5-3% Sum of incidences min-max (all defects)32-83% geneticsbehaviourenvironment Ergo: Jedermann ist Träger von mindestens einen Defekt Viele Defekte manifestieren sich erst spät im Leben (Anfälligkeiten) Einige Anfälligkeiten sind positiv (langlebigkeit, Infektionsresistenz etc...)

8 Das genom ist nicht das einzige determinant des gesund-krang gleichgewichtes a aa a aa UNIFR Rusconi 2003 geneticsbehaviourenvironment Muscle distrophy Obesity Artherosclerosis Alzheimer Parkinson s Drug Abuse Homosexuality Familial Breast Cancer Lung Cancer Sporadic Breast Cancer Sogar die heilung von 'erworbene' krankheiten kann genetisch bedingt sein: Trauma, Wunde Brüche Verbrennungen, Infektionen Vergiftungen

9 *grundkonzepte in der Somatischen Gentherapie (SGT)* a aa a aa UNIFR Rusconi 2003

10 Die 4 Aeren der molekularen Medizin a aa a aa UNIFR Rusconi 2003 Eighties Genes as probes ok ** ok Nineties Genes as factories Y2K Genes as drugs Y2K+n Post-genomic improvements of former technologies

11 Somatische Gentherapie (SGT) Definierung und Anwendungsbereich a aa a aa UNIFR Rusconi 2003 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 therapy

12 Das Prinzip darf einfach sein, aber der Teufel liegt häufig in den Details... a aa a aa UNIFR Rusconi 2003 There are many things that are simple in principle, like... getting a train ticket... ! try this 5 min before departure and with a group of Chinese tourists in front parking your car... ! try this at noon, any given day in Zuerich or Paris... counting votes... ! ask Florida's officials... gene therapy... look at progress in 13 years...

13 Pharmakologische Betrachtungen a aa a aa UNIFR Rusconi 2003 OH OH O OH OH O O OH OH O O Mw 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 molecules Ångstrom/nm size rapidly reversible treatment Classical Drugs Mw Da Biologically prepared Slower diffusion/action Oral delivery not possible Cellular delivery: - act extracellularly Can be delivered as soluble molecules nm size rapidly reversible treatment Protein Drugs Mw N x 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 particles nm size slowly or not reversible Nucleic Acids Thérapies avec acides nucléiques nécessitent de formulation en micro-particules bien plus complexes que la pharmacologie conventionnelle différent niveau de reversibilité (problème de dosage et de maitrise des effets indésirables

14 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 therapy is a treatment aiming at somatic 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 Wieso 'somatisch'? a aa a aa UNIFR Rusconi 2003

15 Die vier Grundfragen bei der SGT a aa a aa UNIFR Rusconi 2003 a aa a aa Efficiency of gene transfer Specificity of gene transfer Persistence of gene transfer Toxicity of gene transfer Remember! Le variables welche Krankheit? Welches Gen? Welches Vektor? Welches Organ / Gewebe? Welche Transfermethode?

16 Die drei Transfer-wege bei der SGT: a aa a aa UNIFR Rusconi 2003 Ex-vivoIn-vivo topical delivery In-vivo systemic delivery V Examples: - bone marrow - liver cells - skin cells Examples: - brain - muscle - eye - joints - tumors Examples: - intravenous - intra-arterial - intra-peritoneal

17 Die zwei klassen von 'Vektoren': virale / nicht-virale a aa a aa UNIFR Rusconi 2003 A B Transfert non viral (transfection) viral transfer (Infection) Nuclear envelope barrier! direct nuclear shuttling!

18 Effizienz der Transfektion mit rekombinante DNA im vergleich zur Infektion mit rekombinante Viren a aa a aa UNIFR Rusconi 2003 Transfection Infection cells exposed to 1'000'000 particles/cell 12 hours cells exposed to 3 particle/cell 30 min Ergo das gentransfer mittels rekombinante Viren ist ueber 1'000'000-fach effizienter als jene nicht-viral transfer methode

19 kleine Parade von Genransfervektoren a aa a aa UNIFR Rusconi 2003 Adenovirus Adeno-associated V. Retrovirus (incl. HIV) Naked DNA Liposomes & Co. Oligonucleotides

20 rekombinante Adenoviren a aa a aa UNIFR Rusconi 2003 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 III Adeno can be grown at very high titers, However Do not integrate Can contain RCAs Are toxic /immunogenic

21 r4ekombinante Adeno-associated-virus (AAV) a aa a aa UNIFR Rusconi 2003 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 easily obtained, immunogenicity is very low, However the major problems are: insertional mutagenesis 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

22 Rekombinante Retroviren (inkl. HIV) a aa a aa UUNIFR Rusconi 2003 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 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

23 Reine oder komplexierte DNS a aa a aa UNIFR Rusconi 2003 Approaches Naked DNA injection /biolistic Naked DNA + pressure Naked DNA + electroporation Liposomal 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. +/-)

24 Oligonuklotide a aa a aa UNIFR Rusconi 2003 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, +++)

25 Recap: Limitierungen der heutigen Genvektoren a aa a aa UNIFR Rusconi 2003 Adenovirus - no persistence - limited packaging - toxicity, immunogenicity Biolistic bombardment or local direct injection - limited area Retrovirus (incl. HIV) & AAV - limited packaging - random insertion - unstable genome General - antibody response - limited packaging - gene silencing - random insertion Solutions: - synthetic viruses (Virosomes) Electroporation - limited organ access Liposomes, gene correction & Co. - very inefficient transfer General - low transfer efficiency - no or little genomic integration Solutions: - improved liposomes with viral properties (Virosomes)

26 *klinische Versuche in der SGT* a aa a aa UNIFR Rusconi 2003

27 Der klassische klinische Weg: viel Zeit und Geld a aa a aa UNIFR Rusconi Idea0 2Cell culture assays0.5 Mio 5Pre-clinical tests animal models 2 Mio 7Clinical phase I 5-20 patients verify side effects6 Mio 10Clinical phase II patients dosis escalation12 Mio 15Clinical Phase III > patients multicentric double blind80 Mio 16>>Registration / Availability yeareventcosts U$D This means: assuming 20% of new developments makes it to final registration, the average investment is Mio U$D for each approved drug/procedure

28 Vernichten wir mindestens vier Mythen bei der Gentherapie a aa a aa UNIFR Rusconi 2003 Classical Gene Therapy Image Hereditary disease culprit gene must be known requires 100% efficiency of transfer/expr. gene transfer/expression must persist Reality Many acquired diseases can be treated (ex. infections, traumatic lesions, tumors,...) 'Short circuit' or symptomatic treatments (ex. neurodeg. conditions with trophic factors) Few % sufficient for many diseases (ex. hemophilia, limb ischaemia...) No persistence required in many cases (ex. vaccination, cytotoxic antitumoral factors, restenosis prevention, acute rejection prevention...)

29 Trends bei der klinischen SGT experimentierung a aa a aa UNIFR Rusconi 2003 cancer hered. Infect. vasc trials patients % overall still pending or not yet Initiated ! 66% phase I 21% phase I-II 11% phase II 0.8% phase II-III 0.7% phase III As of August 2003: 660 registered protocols 3672 treated patients Ergo en dépit de son age la TGS peut compter couramment seulement 1% d'essais en phase III I I-II II

30 Einige Meilensteine a aa a aa UNIFR Rusconi , 1993, 2000 // ADA deficiency F Anderson, M Blaese // C Bordignon Anderson, 1990 Bordignon, 2000 (ESGT, Stockholm) 2002, science 296, 2410 ff) 1997, 2000, Critical limb ischemia J Isner ( ), I Baumgartner, Circulation 1998 Isner, , Restenosis V Dzau, HGT 1998 Dzau, , Crigler Njiar (animal) C Steer, PNAS 1999 Kmiec, , Hemophilia M Kay, K High 2000, SCID A Fischer, Science April 2000 Fischer, , correction Apo E4 (animal model) G. Dickson, 2000 esgt, 2002 BBA Dickson, , correction Parkinson (animal model) P Aebischer, Science, Nov 2000 Aebischer, , ONYX oncolytic Viruses D Kirn (Cancer Gene Ther 9, p ) Kirn, 2000, Intravascular adenoviral agents in cancer patients: Lessons from clinical trials (review)

31 *Die hoch- und tief-punkte... * a aa a aa UNIFR Rusconi 2003

32 Zwei besonders frustrierende Faelle: Muskelschwund und Mucoviszidose a aa a aa UNIFR Rusconi 2003 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) 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 Trotz Isolierung der entspre- chenden Genen in 1984 kein geeignetes Vektor keine geeignete Lieferungsmethode

33 Die mesit befuerchtete Nebeneffekte der Gentherapie a aa a aa UNIFR Rusconi 2003 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) Ergo die Nebeneffekte waren nicht so scvheinbar wenn SGT ineffizient war Heute muessen wir diese Nebeneffekte seriös betrachten

34 Paris, Jan 14, 2003, A Fischer: a second patient of the cohort of 9 comes up with a similar disease than the one reported in october trials in USA are temporarily suspended 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. 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). 4 bittere Feststellungen aber nur einen Patient bis jetzt direkt an SGT gestorben a aa a aa UNIFR Rusconi 2003 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 interrupted and many others on hold.

35 Der klinische Versuch in Paris X-SCID (A. Fischer, Hôpital Necker) a aa a aa UNIFR Rusconi 2003 Disease deficiency of the receptor gamma(c) incapacity of maturing lymphocytes severe combined immunodeficiency lethal at 4 months if untreated survival 10 years under sterile conditions Gene Therapeutical approach explant BM (3-6 month old) select CD34+ transduce with retroviral vector encoding gamma(c) re-infusion, follow-up Conventional treatments maintenance under sterile condition treatment with antibiotics transplant of matching bone marrow

36 Die Odyssee des klinischen Versuchs in Paris a aa a aa UNIFR Rusconi 2003 Chronology 1998 start 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 gene, notification authorities October 2003, public disclosure, chemotherapy, good response, report at ESGT congress. Adverse 2 december 2002, T cell hyper-proliferation in a second, 36 months patient hyper-proliferatory cells also contain insertion of transgene close to LMO2 gene January 2003, notification to authorities, public disclosure, treatment chemotherapy

37 Die Fragen a aa a aa UNIFR Rusconi 2003 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 Perspectives if the answers are 'YES''NO' 'UNK' goodbadnot good badgoodnot good 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?

38 Anhäufung von hoch- und tief-Punkte: ein Rollercoaster! a aa a aa UNIFR Rusconi 2003 high Low mood NIH Motulski report Lentivectors in pre-clinic Adeno III J. Isner ADA R. Crystal Adeno I AAV germline in mice? V.Dzau A. Fischer M. Kay lentivectors in clinics? NFP37 C Bordignon Ergo whenever a reasonable cruise speed was achieved, a major adverse event has brought us back square one 03 Adverse events in Paris J. Wilson J. Gelsinger

39 Weitere Faktoren die zum schlechten 'Image' der Gentherapie beigetragen haben a aa a aa UNIFR Rusconi 2003 Naive statements by some good-willing scientists in the early 90ties Not-so-naive statements by not-so-naive scientists in search of fame Huge amount of money that flowed into the research and development that attracted many incompetent researchers. Concomitance with stock-market euphoria (little attention to realism) Reckless statements or misreporting by greedy scientists or company managers to increase the value of their stock options (memorandum by the ASGT on conflict of interest 2000, Tendency by the media to spectacularise good news and/or bad news Ergo zuviel geld und spekulation: ein explosiver cocktail, wie beim Sport oder Kunst...

40 *Conclusions & Perspectives* a aa a aa UNIFR Rusconi 2003

41 Schlussfolgerungen a aa a aa UNIFR Rusconi 2003 Grundkonzepte The therapeutic gene transfer in somatic cells must cope with: efficiency, specificity, persistence and toxicity many genes with potential therapeutic value have been identified, and essentially all types of diseases can be treated by gene transfer Vektoren und Modelle There is the choice of a certain number of viral and non viral vectors, none of them being generally applicable Viral vectors have the advantage of efficiency and nonviral vector the advantage of lower toxicity/danger. Viral vectors have the disadvantage of limited packaging and some toxicity, while nonviral vector have the major disadvantage of low efficiency of transfer Klinische Versuche over 600 trials and 3500 patients in 12 years only a handful of trials is now reaching phase III Progress further slowed down by periodical pitfalls

42 Perspectiven: SGT wird weiter fortschreiten trotz schwieruigkeiten und unvermeidbare Unfälle a aa a aa UNIFR Rusconi 2003 Grundlage Forschung und 'Vektorologie' the better understanding of gene interactions and networking (functional genomics) could improve the utilisation of gene-based or gene targeted strategies novel paradigms can become available (Si RNA, PNA triplex etc...) specifically integrating gene constructs or artificial chromosomes become more realistic Praeklinische Forschung scaling up to larger animal models (dog and monkey) permits better appreciation of dosage requirements new transgenic models may give improved similarities to human diseases Klinische Forschung Use of recombinant lentiviruses may be imminent 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 der grösste teil der fehler waren menschliche Fehler Die Huerde koennen bewältigt werden.

43 Danke fuer die aufmerksamkeit und fuer spezielle Fragen, bitte schreiben sie an: oder besuchen sie die WEB seite: und bleiben wir optimistisch a aa a aa UNIFR Rusconi 2003 Swiss National Research Foundation My collaborators at UNIFR Biovalley Program Gymnasium Liestal

44 END, let's open the Discussion a aa a aa UNIFR Rusconi 2003

45 ***Diskussions-slides... a aa a aa UNIFR Rusconi 2002 text ttt

46 Gene therapy in Switzerland: the 30 projects financed by the NFP37 programme ( ) a aa a aa UNIFR Rusconi 2003 NFP37phase Aphase B (96-99) (99-01) Submissions3026 Granted1918 Total requested 32 Mio 9 Mio Granted 7.6 Mio 6 Mio DISEASE ORIENTATION Cancer 8 10 Acquired disorders 2 7 Vector development 5 3 Hereditary disorders 2 4 Infectious diseases 1 2 RESEARCH LEVEL Fundamental 10 7 Preclinical (animal models) 5 9 Clinical phase I 2 3 Clinical Phase II 0 1 Clinical Phase III 0 0 Ethical/social aspects 1 1 Nationales Forschungsprogramm 37 NFP37 « somatic gene therapy » Please Note the NFP37 represented at most 30% of the Swiss- based experimentation in SGT during

47 The SGT acrobatics: matching vectors / delivery system / disease a aa a aa UNIFR Rusconi 2003 Chronic Conditions Slow onset of expression acceptable Initiation of the treatment weeks/months/years before 'point of no return' (ex. cystic fibrosis) persisting expression of the transgene or re-administration required (example hemophilia) Usually based on compensation of 'genetic loss-of-function' (permanent re- gain of function; ex. ADA) Regulation of gene expression often necessary (because of persistence) For some diseases even a small % of tissue transformation is already therapeutic Acute Conditions Rapid onset of expression necessary Initiation of the treatment minutes/hours/days before 'point of no return' (ex. brain ischemia) persisting expression of the transgene not required, occasional re-administration (example ischemia) Usually based on augmentation of resident function (transient gain of function; ex. VEGF) Regulation of gene expression not necessary (because of transiency) For most diseases even a small % of transformation is already therapeutic Ergo many divergent variables must be matched for each case an advantage for one purpose becomes a disadvantage for another (viceversa)

48 Die Hauptkrankheit des 21. Jahrhunderts: Veralterung a aa a aa UNIFR Rusconi Life expectancy (CH) cancer incidence % M E2/E E3/E4 E4/E4 Alzheimers free % aa getting

49 Pas toutes les stratégies de transfert se basent sur un ancrage au hasard a aa a aa UNIFR Rusconi 2003 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

50 Mais un virus c'est quoi? Une machine auto-réplicative extrèmement efficace a aa a aa UUNIFR Rusconi 2003ß E L1L2 standard viral genome 100 nm replication entrydisassemblydockinggenome replication late genes exp assembly capsid E L1L2 Spread Etc... early genes exp

51 Comment peut-on construire des virus récombinants? a aa a aa UNIFR Rusconi 2002 EL1L2 rp Wild type genome Normal target cells Virions Recombinant genome R-Virions E E E E E E E Packaging cells Normal target cells X Packaging

52 D'autres tecnolgies émergentes entrent en compétition brutale avec la thérapie génique a aa a aa 1. Cell Therapy (Stem cells, SC) identified in many tissues cell transfer could be combined with gene transfer there would be no anatomical barriers for gene transfer Selection /amplification of desired transformants Current limitations of SC Lack of control on differentiation and trans-determination Difficulties in complex organ-reconstruction Future of SC: Increasing number of SC types will be characterised culturing conditions will be perfectioned May replace in vivo gene transfer for treatment of chronic conditions? UNIFR Rusconi 2003 V 2. Challengers from the small/medium molecules STI571 (Glivec) anti HER2 (Herceptin) Si RNA? Challengers from the biomechanics world bone reconstruction intelligent protheses (stents) micropumps artificial organs

53 La génétique est practiquée depuis de millénaires, la biologie moléculaire seulement depuis 30 ans a aa a aa UNIFR Rusconi b.C. Empirical genetics b.C. Biotechnology 2000 a.d. Molecular biology 2001 a.d, Genomics

54 When/where/ may be SGT indicated? a aa a aa UNIFR Rusconi 2003 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

55 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 category a aa a aa UNIFR Rusconi 2003 Disease Type

56 Comparing relevant issues in the two main 'vectorology' sectors (viral versus nonviral) a aa a aa UNIFR Rusconi 2003 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 production (existence or not of packaging cell systems) 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 industrial production are different between viral and non-viral vectors when ignoring thir low efficiency, nonviral vectors appears largely superior

57 The THREE missions of medicine a aa a aa UNIFR Rusconi 2003 Prevention Diagnosis Therapy 'Molecular Medicine' Application of the know-how in molecular genetics to medicine + + +

58 Ideal properties of a systemically delivered non-viral formulation a aa a aa UNIFR Rusconi 2003 Stability particle should resist serum inactivation particle should be inert to immune inactivation Addressability particle should possess a vascular addressing signature particle should bear a tissue-docking specificity DNA construct should include tissue-specific regulatory elements Efficiency cargo should be protected from cytoplasmic inactivation (ex. lysosomes) cargo should contain nuclear-translocating signals DNA cargo should include genome-integration functions DNA element must be guaranteed to function after genomic integration (no silencing) Other properties Particle should not include immunogenic/toxic surfaces Cargo should not encode immunogenic/toxic products Cargo should include anti-apoptotic functions Ergo several independent problems must be solved for a nonviral formulation to be suitable for clinical treatment and for industrial production most viral vectors include many, if not all those properties

59 Public perception problems a aa a aa UNIFR Rusconi 2003 Negative perception of manipulative genetics general aversion of genetic manipulation fear of catastrophic scenarios Deception after excessive promises hopes reinforced by media spectacularisation and over-simplification deception after non-complied deadline Confusion with other gene-based and non-gene-based technologies stem cell technology human cloning procedures genetically modified food

60 *d Why so many cancer trials? a aa a aa UNIFR Rusconi 2002 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

61 *d Ethical dilemmas can sometimes slow down progress and hamper objective appreciations a aa a aa UNIFR Rusconi 2002 ADA gene therapy « it does not work » Feeling: It s time we face reality, my friends… We re not exactly rocket scientists ADA gene therapy 2000 « it always worked but we couldn t know » The PEG-ADA ethical dilemma has prevented earlier results recognition (Bordignon, ESGT meeting Stockholm, , Science July 2002 ) vector

62 a aa a aa *d Cancer molecular treatment Example: Oncolytic viruses on the example of ONYX-015 A) Normal Adenovirus can propagate in virtually all cells B) ONYX-015 deleted E1B function can propagate efficiently only in P53 -deficient cells (e.g. most cancer cells) Clinical success Head & Neck Cancer Awaiting for further successes (currently in Phase II and III) expected to be useful in combination with conventional therapy ADVANTAGE: the 'drug' has its own dynamics DISADVANTAGE: danger of evolving viruses unclear if it works in adeno-immune patients unclear if if works in immuno- compromised patients (chemotherapy) UNIFR Rusconi 2002

63 *d Recap: what is a virus ? -> A superbly efficient replicating machine a aa a aa UUNIFR Rusconi 2002 E L1L2 standard viral genome 100 nm replication entrydisassemblydockinggenome replication late genes exp assembly capsid E L1L2 Spread Etc... early genes exp

64 *d Examples of gene transfer treatments against cancer a aa a aa UNIFR Rusconi 2002 percentage of trials (therapeutic potential) Directly cytotoxic Prodrug activation Tumor-specific cytotoxic expression 38% of protocols (strong complementary effect, possible relapse) Immunostimulatory Transfer of Immuno-attracting functions Instruction of immune cells 31% of protocols (very strong potential, low relapse chances) Immunoprotective Transfer chemoresistance genes in immune cells 5 % of protocols (very limited application, laborious, not widely explored) Tumor modulation Restore tumor-suppressor functions down-regulate pro-oncogenic functions 26 % of protocols (good effect but low bystander and likely relapse) Type of treatment examples

65 *d Gene correction strategies (independent of delivery vectors) a aa a aa UUNIFR Rusconi 2002 Approaches Trans-splicing mRNA Ribozymic splicing of RNA Chimeroplasts-induced repair Triple-helix-guided repair Homologous recombination Advantages / Limitations All extremely promising approaches which could permit the treatment of dominant defects. They do not require specificity of delivery, and are not subject to gene silencing. Also they would permit from the biosafety point of view germ line correction, However the major issue today is: Controversial efficiency of repair Examples Crigler Njiar (animal, %) M. Distrophy (animal, %) Correction Apo E 4 (animal, %) Correction Albino (animal, +/- <1%)

66 *d Gene therapy FAM (Frequently Asserted Myths…) a aa a aa UNIFR Rusconi is withdrawing huge public funding Gene therapy... Wrong: 1998: USA < 2% of NIH budget, CH less than 1% of NF budget...will be invariably expensive Wrong: example DNA based vaccination Isner, is solely targeting hereditary diseases Wrong: Cancer, Cardiovascular, Restenosis, Ischemia,... Fischer, has not yet proven its therapeutic value Kmiec, 1999 Wrong: J. Isner/ I. Baumgartner (ischemia), A. Fischer (SCID), C Bordignon (ADA); M. Kay (Haemophilia, F McCormick (cancer), … based only on a shotgun insertion' Wrong: Gene correction strategies, a dangerous procedure Wrong: 1 death out of >3000 patients...will be imediately boosted by genomics Wrong: it will take long time to benefit from genomics knowledge

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