Sandro Rusconi

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UNIFR Rusconi 2003. Sandro Rusconi. 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) - PowerPoint PPT Presentation

Text of Sandro Rusconi

  • Sandro RusconiaaaaaaUNIFRRusconi20032003: Gene therapy turning teenage, what have we learned?Feb 19, 2003ECPM Basel 1972-75School teacher (Locarno, Switzerland)1975-79Graduation in Biology UNI Zuerich, Switzerland1979-82PhD curriculum UNI Zuerich, molecular biology1982-84Research assistant UNI Zuerich1984-86Postdoc UCSF, K Yamamoto, (San Francisco)1987-91Principal Investigator, UNI Zuerich1994-todayProfessor Biochemistry UNI Fribourg1995-todayDirector Swiss National Research Program 37'Somatic Gene Therapy'2002-03Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department2002-05President Union of Swiss Societies forExperimental Biology (USGEB)

  • Genetics has been used since millennia,Molecular Biology, only since 30 yearsaaaaaaUNIFRRusconi2003100000 b.C. Empirical genetics10000 b.C.Biotechnology2000 a.d.Molecular biology2001 a.d, Genomics

  • 1 Gene -> 1 or more functionsaaaaaaUNIFRRusconi2003

  • Recap: what is a gene?:a regulated machine for RNA productionaaaaaaUNIFRRusconi2003codingspacerspacerregulatoryTo fulfil its role, a transferred gene must include:regulatory sequences for Tx initiationproper signals for RNA maturation/transportproper signals for mRNA translation

  • 1 Organism -> more than 105 genetically-controlled FunctionsaaaaaaUNIFRRusconi20031 Cm3 of tissue1'000'000'000 cells!

  • Reductionistic molecular biology paradigm(gene defects and gene transfer)aaaaaaUNIFRRusconi2003aaaaaaGene transfer implies either:transfer of new function, ortransfer of restoring function, ortransfer of interfering function

  • Examples of inheritable gene defectsaaaaaaUNIFRRusconi2003Polygenic defectsTypeestimated(frequent)min-max

    Diabetes poly1-4 %HyperurikemiaMulti2-15 %Glaucomapoly1-2 %DisplasiaMulti1-3 %HypercolesterolemiaMulti1-5 %Syn-& Polydactylypoly0.1 -1 %Congenital cardiac defectsMulti0.5-0.8 %Manic-depressive psychosisMulti0.4 -3 %Miopypoly3 -4 %Polycystic kidneypoly0.1-1 %PsoriasisMulti2 -3 %SchizofreniaMulti0.5 -1 %ScoliosisMulti3 -5 %

    Monogenic defectsestimated(rare) min-maxCystic fibrosis, muscular dystrophyimmodeficiencies, metabolic diseases, all togetherHemophilia...0.4 -0.7%

    PredispositionsTypeestimatedmin-max(*) AlzheimerMulti7 -27 %(*) ParkinsonMulti1-3 %(*) Breast cancerMulti4 -8 %(*) Colon CarcinomaMulti 0.1 -1 %(*) ObesityMulti0.5-2 %(*) Alcolholism/ drug addictionMulti0.5-3%

    Sum of incidences min-max (all defects)32-83%Ergo:every person bears one or more latent genetic defectsmany defects are not manifest but lead to predispositionsthere are also protective predispositions

  • Not only the genome determines the health status...aaaaaaUNIFRRusconi2003also acquired conditions may have a genetic component that modulates their healingtraumafracturesburnsinfections

  • The major disease of the 21st century: AgeingaaaaaaUNIFRRusconi2003This major challenge means:higher investmentsmore financial returnslong term treatmentcustomised treatmentsocial security dilemma

  • The THREE missions of medicineaaaaaaUNIFRRusconi2003




    'Molecular Medicine'Application of the know-how in molecular geneticsto medicine


  • The FOUR eras of molecular

  • Now, let's talk about Somatic Gene Therapy (SGT)aaaaaaUNIFRRusconi 2003Definition of SGT:'Use genes as drugs':Correcting disorders by somatic gene transferChronic treatment

    Acute treatment

    Preventive treatment

    Hereditary disorders

    Acquired disorders



    NFP37 somatic gene

  • The SGT principle is simple Yes,...but the devil is often in the detailsaaaaaaUNIFRRusconi 2003There 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 frontparking 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...1990First clinical trial of a monogenic diseaseF. Anderson & Co: ADA deficiency2002Same protocol as Anderson's for ADAgene therapy (C. Bordignon) works!...does not workGene therapy turns teenage in 2003, but: has it really grown up?

  • Why 'somatic'?aaaaaaUNIFRRusconi 2003Germ Line Cells: the cells (spermatocytes and oocytes and their precursors) that upon fertilisation can give rise to a descendant organismSomatic Cells: all the other cells of the bodyi.e. somatic gene therapyis a treatment aiming atsomatic cells and conse- quently does not lead to a hereditary transmission of the genetic alterationErgotransformation of germ line cells is avoided, to exclude risk of erratic mutations due to insertional mutagenesis

  • When/where/ may be SGT indicated?aaaaaaUNIFRRusconi 2003No existing cure or treatmentmost monogenic diseases Side effects and limitations of protein injectioninterleukin 12 (cancer) -> toxic effects and rapid degradationVEGF (ischemias) -> angiomasFactor VIII or IV (hemophilia) -> insufficient basal level Complement to conventionalincrease specificity of conventional therapy (cancer)increase efficacly of conventional therapy (hemophilia) Life quality burden of patientcosts 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

  • SGT's four fundamental questions & playersaaaaaaUNIFRRusconi 2003Efficiency of gene transfer Specificity of gene transfer

    Persistence of gene transfer

    Toxicity of gene transfer

    The variableswhich disease?which gene?which vector?which target organ?which type of delivery?

  • The SGT acrobatics: matching vectors / delivery system / diseaseaaaaaaUNIFRRusconi 2003Chronic ConditionsSlow onset of expression acceptableInitiation 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 therapeuticAcute ConditionsRapid onset of expression necessaryInitiation of the treatment minutes/hours/days before 'point of no return' (ex. brain ischemia)persisting expression of the transgene not required, occasional re-administration (exampleUsually 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 Ergomany divergent variables must be matched for each casean advantage for one purpose becomes a disadvantage for another (viceversa)

  • Pharmacological considerations for DNA transferaaaaaaUNIFRRusconi2003OHOHOOHOHOOOHOHOOMw 50- 500 DaltonsSynthetically preparedRapid diffusion/actionOral delivery possibleCellular delivery: - act at cell surface - permeate cell membrane - imported through channelsCan be delivered as soluble molecules ngstrom/nm sizerapidly reversible treatmentClassical DrugsMw 20000- 100000 DaBiologically preparedSlower diffusion/actionOral delivery not possibleCellular delivery: - act extracellularly

    Can be delivered as soluble molecules nm sizerapidly reversible treatmentProtein DrugsMw N x 1000000 DaBiologically preparedSlow diffusionOral delivery inconceivableCellular delivery: - no membrane translocation - no nuclear translocation - no biological importMust be delivered as complex carrier particles 50-200 nm sizeslowly or not reversibleNucleic AcidsTherapy with nucleic acidsrequires particulated formulationis much more complex than previous drug deliverieshas a different degree of reversibility (dosage problem)

  • THREE classes of anatomical gene deliveryaaaaaaUNIFRRusconi2003Ex-vivoIn-vivotopical deliveryIn-vivosystemic deliveryExamples:- bone marrow- liver cells- skin cellsExamples:- brain- muscle- eye- joints- tumorsExamples:- intravenous- intra-arterial- intra-peritoneal

  • TWO classes of gene transfer vectors: non-viral & viral deliveryaaaaaaUNIFRRusconi2003abNon-viral transfer(transfection of plasmids)Viral gene transfer(Infection by r-vectors)Nuclear envelope barrier! see, Nature BiotechDecember 2001

  • Transfection versus InfectionaaaaaaUNIFRRusconi2003TransfectionInfectionexposed to106 particles/cell12 hoursexposed to 1 particle/cell30 minErgovirally mediated gene transfer is millions of times more efficent than nonviral transfer (when calculated in terms of transfer/particle)

  • Most relevant issues in the two main 'vectorology' sectors (viral versus nonviral)aaaaaaUNIFRRusconi 2003Viral vectorsPackaging 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 1/100strong immunogenicity: capsid and envelope proteins, residual viral genescontaminants: 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 vectorsPackaging 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 materialsmoderate immunogenicity: methylation status of DNA (example CpG motifs)contaminants: adventitious pathogens