Presentation on theme: "Gene Therapy Research 徐国彤 MD， PhD Tongji University School of Medicine"— Presentation transcript:
1 Gene Therapy Research 徐国彤 MD， PhD Tongji University School of Medicine
2 Infectious Disease AIDS Severe Acute Respiratory Syndromes (SRAS) Human immunodeficiency virus (HIV)AIDSSevere Acute Respiratory Syndromes (SRAS)InfluenzaSwine Flu (H1N1)Avian influenza (bird flu)Hand, foot and mouth disease (HFMD)……A young Bangladeshi girl infected with smallpox (1973).smallpox
3 Drug-resistant bacteria NMD-1 "Superbug" Poses a "Cause for Worldwide Concern"Tuberculosis (TB): A fell plague
4 Cancer Lung cancer: Liver cancer: Stomach cancer: Cross section of a human lung. The white area in the upper lobe is cancer; the black areas are discoloration due to smoking.Liver cancer:Left lobe liver tumor in a 50 year old maleStomach cancer:A suspicious stomach ulcer that was diagnosed as cancer on biopsy.
9 What’s gene therapy?Imagine that you accidentally broke one of your neighbor's windows.Many medical conditions result from flaws, or mutations, in one or more of a person's genes. So, if a flawed gene caused our "broken window," can you "fix" it? What are your options?Stay silent: ignore the genetic disorder and nothing gets fixed.Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution.Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem!Stay silent: no one will ever find out that you are guilty, but the window doesn't get fixed.Repair it with some tape: not the best long-term solution.Put in a new window: not only do you solve the problem, but also you do the honorable thing.
10 Content The definition of gene therapy strategies of gene therapy Types and routes of gene therapyVectors of gene therapyGene therapy for clinical useProblems and Perspective
11 What is gene therapy?Gene therapy is the treatment of diseases based on the introduction of genetic material into target cells of the body.Although the technology is still in its infancy, it has been used with some success. Scientific breakthroughs continue to move gene therapy toward mainstream medicine.“ Gens” as medicineThe therapeutic drug is produced in small factory “cells”.
12 Content The definition of gene therapy strategies of gene therapy Types and routes of gene therapyVectors of gene therapyGene therapy for clinical useProblems and Perspective
13 The strategies of gene therapy Gene replacement/correctionReplacing a mutated gene that causes disease with a healthy copy of the geneGene silencing/gene interferenceInactivating, or “knocking out,” a mutated gene that is functioning improperlyGene augmentation/gene addition (modification)The addition of a functional copy of a gene to the genome of an organism, or introducing a new gene into the body to help fight a disease“Suicide gene”Insert genes whose products metabolize normal drugs and ↑ their toxicity to proliferating–ie tumor cells……
14 Gene replacementDelivery of a gene whose function is absent due to loss-of-function mutations in the affected gene. This can be used in autosomal recessive diseases (RP or LCA) or in those that are autosomal dominant due to haploinsufficiency or dominant-negative mutations (RP).Gene replacement by using gene homologous recombination (gene targeting).
15 Gene silencingDelivery of a gene and/or nucleic acid to inhibit the expression of a gene or a gene product with abnormal function. This approach is useful in autosomal dominant diseases (RP) arising from gain-of-function mutations.RNA interference (RNAi)Triple helix-forming oligonucleotides (TFO)Ribozyme……
16 Gene addition/gene augmentation Delivery of a gene whose product provides beneficial effects independently of the primary defective gene; without actually substituting that gene for the flawed or absent gene in the DNA (AAV2-RPE65 for LCA).NATURE BIOTECHNOLOGY VOLUME 24 NUMBER 8 AUGUST 2006
17 Assisted killing of disease cells by immune system cells (gene augmentation) Gene vaccine: IL-2, 4, TNFa, INFr into tumor cellsAdaptive immonotherapy: Cytokines into TILImmunoenhancement: MHC-I into tumor cellIn situ modification of immunogenity of : CLT
18 “Suicide gene”Suicide gene: A gene whose expression in a cell is lethal for that cell under specific conditions.Suicide genes form the basis of a strategy for making cancer cells more vulnerable, more sensitive to chemotherapy.The approach has been to attach parts of genes expressed in cancer cells to other genes for enzymes not found in mammals that can convert a harmless substance (pro-drug) into one that is toxic to the tumor.
20 In vivo gene therapy for brain tumors TKgcvgcv-ppp
21 Content The definition of gene therapy Basic process (strategies) of gene therapyTypes and routes of gene therapyVectors of gene therapyGene therapy for clinical useProblems and Perspective
22 Types of Gene Therapy Germ line gene therapy Somatic gene therapy Target cellex vivo gene therapyin vivo gene therapyin situsystematicalDelivery method
23 Germ line gene therapy Host cells: sperm or eggs Methods: Features: Types of Gene TherapyGerm line gene therapyHost cells: sperm or eggsMethods:----To treat a pre-embryo before implantation in the mother, with theuse of IVF----To treat the germ cells (sperm or egg cells) of afflicted adultFeatures:---functional genes are ordinarily integrated into their genomes---effects would be heritable and would be passed on to latergenerations.---highly effective---technical and ethical reasons
24 Somatic gene therapy Host cell: somatic cells of a patient Methods: Types of Gene TherapySomatic gene therapyHost cell: somatic cells of a patientMethods:----Inserting the gene into any location within the genome to replace a nonfunctional gene, which is the most commonly used----Switching the abnormal gene for a normal gene through homologous recombination. ----Fixing through selective reverse mutation, which returns the gene to its normal functionFeatures---Any modifications and effects will be restricted to the individual patient onlyand will not be inherited by the patient's offspring or later generations---widely used
25 Types of Gene Therapy Germ line gene therapy Somatic gene therapy Target cellex vivo gene therapyin vivo gene therapyin situsystematicalDelivery method
26 Types of Gene Therapyex vivo:---incorporate gene into cells outside the body and then deliver altered cells to patient
27 in vivo: Types of Gene Therapy ---Systematically: vector carrying the gene is introduced directly into the body, often through a blood vessel---in situ: vector carrying the gene is injected into localized and accessible body part
28 In-vivo In-vivo systematic delivery Ex-vivo ex vivo or in situ delivery are currently preferred over systematic deliveryIn-vivoIn situ deliveryIn-vivosystematic deliveryEx-vivoExamples:- brain- muscle- eye- joints- tumorsExamples:- intravenous- intra-arterial- intra-peritonealVExamples:- bone marrow- liver cells- skin cells
31 Target Sites for Gene Therapy Cell /tissueadvantageexamplesEndotheliumCan form capillaries to secrete gene product into bloodstreamclotting factor for hemophiliaskinCan take small piece of skin from patient to grow into large graftSkin graphs can deliver therapeutic proteinslungcells lining the passageways are easily accessibleaerosol spray to treat Cystic FibrosisNerve systemability to treat common illnesses and injuries Challenge: neurons do not dividegene therapy on fibroblasts to allow them to produce neurotransmittersMuscleEasily accessible, near bloodstreamTreatment for Duchenne Muscular DystrophyLiverMultiple functions, capacity to regenerateTreatment for familial hypercholesterolemia
32 Remember! Efficiency Specificity Persistence Toxicity The four technical basic questions in somatic gene therapyRemember!EfficiencyPersistenceSpecificityToxicityEfficiency of gene transferSpecificity of gene transferPersistence of gene transferToxicity of gene transferThe variableswhich disease?which gene?which vector?which target organ?which type of delivery?
33 Content The definition of gene therapy Basic process (strategies) of gene therapyTypes and routes of gene therapyVectors of gene therapyGene therapy for clinical useProblems and Perspective
34 The Ideal Vector for Gene Transfer High concentration of virus allowing many cells to be infected or transducedConvenience and reproducibility of productionAbility to transduce dividing and non-dividing cellsAbility to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episomeA transcriptional unit that can respond to manipulation of its regulatory elementsAbility to target the desired type of cellNo components that elicit an immune response
36 Vectors for gene transfer Transduction of somatic cells can be obtained both by both viral and non-viral nucleic acid transfer.Viral vectors:Gene delivery can be accomplished with high efficiency by using viruses modified as follows: the viral genome is partially or completely deleted of viral genes, which are generally substituted in the vector by an expression cassette containing the desired promoter–transgene combination.Non-viral vectors:Nucleic acids can be additionally delivered as naked DNA or as a complex with lipids or cationic polymers. These compounds usually improve the efficacy of DNA delivery to the target cells. Doublestranded short interfering RNA sequences (siRNAs), used to induce RNA interference of a target transcript, are usually delivered via nonviral methods.
37 Delivery System Viral Vector Non-viral Techniques Retrovirus Nake DNA LentivirusOligonucleotideAdenovirusLipoplexes and polyplexesAdeno-Associated Virus (AAV)Ballistic DNA InjectionHerpes simplex virus (HSV)MicroinjectionAlphavirusesElectroporationPox viruses (Vaccinia virus)Calcium phosphate transfection……
38 viral Vs non-viral vectors (transfection)AWhy are viruses 'better'?viral transfer is much more efficientnonviral transfer must solve a number of hurdles - serum protection/stability - target docking - endosomal escape - nuclear trafficking - genomic integration - immunological camouflageBNuclear envelope barrier!viral transfer(Infection)direct nuclear shuttling!
39 1 Retroviral vectors Viral vector Retrovirus virions contain a protein capsid that is lipid encapsulated. Virions range in diameter from 80 to 130 nm.The viral genome is encased within the capsid along with the proteins integrase and reverse transcriptase.The genome consists of two identical positive (sense) single-stranded RNA molecules ranging in size from 3.5 to 10kb.
40 Retrovirus vectors Advantages Disadvantages High transduction efficiencyRequires dividing cells for infectivityInsert size up to 8kBLow titers ( )Integrates into host genome resulting in sustained expression of vectorIntegration is random, insertional mutagenesisExtremely well studied systemIn vivo delivery remains poor. Effective only when infecting helper cell linesVector proteins not expressed in host /
41 2 lentiviral vectors Advantages : Disadvantages: Belongs to retrovirus family, Lenti, in latin, means slowAdvantages :High-efficiency infection of dividing and non-dividing cells;Long-term stable expression of a transgene;Low immunogenicity.Disadvantages:Random integration: insertional mutations; loss of function of important genes; cancer, etc
42 3 adenoviral vector Advantages Disadvantages High transduction efficiencyExpression is transient (viral DNA does not integrate)Insert size up to 8kbCytopathogenicity (viral protiens can be expressed in host)High viral titer ( )Immunogenicity (In vivo delivery hampered by host immune responseInfects both dividing and non-dividing cells/
43 4 recombinant AAV (rAAV) vectors Recombinant vectors are generated by deleting the rep and cap sequences from the genome and by inserting the therapeutic gene of interest between the ITRs.Hybrid vectors have been generated by including the same AAV vector genome (usually derived from AAV2) in external surface proteins (capsids) from other AAV serotypes; the resulting recombinant vectors (rAAVs) are indicated as ‘rAAV 2/1, 2/2, 2/3, 2/4, 2/ /n’, with the first number indicating the genome (i.e. AAV2 in this case) and the second the capsid.Different rAAV serotypes have different capsids, tropism and transduction characteristics.
47 rAAV vector Advantages: All virus genes removed, Lack of initiating an immune response;Stable expression and safe;Ability to infect a variety of dividing and non-dividing cellsNon-pathogenicDisadvantages:Small genome limits size of foreign DNA larger than 5 kb;Must be closely screened for adenoviral or HSV contaminationLabor intensive productionexcept Hematopoietic cells
48 5 Herpes simplex viruses I Advantages:Preferential neuronsLarge insert sizeCould provide long- term CNS gene expressionHigh titerNo integrationdisadvantages:Hard to prepare, cell toxicitytransient expression in non-neuron cellsLow transduction efficiency
50 Cationic Lipids, Liposomes Non-Viral VectorsNon-Viral VectorsAdvantages:Simple large scale productionLow host immunogenicityMethodsdisadvantagesCalcium PhosphateLow EfficiencyDEAE DextranCationic Lipids, LiposomesDirect DNA InjectionsElectroporationTransient expression
51 DNA injection withElectroporationDNA injection aloneElectroporation
52 Limitations of current vectors r-Adenovirus- no persistence- limited packaging- toxicity, immunogenicityBiolistic bombardmentor local direct injection- limited arear-AAV- no integration in host g.- very limited packaging- autoimmunity?Electroporation- limited organ accessLiposomes, gene correction & Co.- rather inefficient transferr-Retrovirus (incl. HIV)- limited packaging- random insertion- unstable genomeGeneral- low transfer efficiency- no or little genomic integrationGeneral- antibody response- limited packaging- gene silencing- Manufacturing limitationsThe future will probably see an increasing interest in viral-like, but artificial particlesSolutions:- synthetic viruses(“Virosomes”)
53 Content The definition of gene therapy Basic process (strategies) of gene therapyTypes and routes of gene therapyVectors of gene therapyGene therapy for clinical useProblems and perspective
54 Bubble boy diseaseDavid Vetter, who was born with a genetic disorder leaving him no natural immunities against disease, became famous for living behind plastic barriers to protect him from germs. He died at age 12 of 1984
55 Why ADA deficiency is ideal target for the first gene therapy??? 1.The pathological effects are reversible2.The gene defect：the loss of function of a single gene3.Tight control ：not important (ADA levels vary widely in the normal population)4. Target gene：very small and easy to manipulate5. Target cells: lymphocytes, which are accessible, easy to culture and easy toput back into the body of the patient6.The alternative treatments are expensive and/or hazardous
56 ADA deficiency: The First clinical Trial September 14, NIH, French Anderson and R. Michael Blaese perform the first gene therapy trial.Ashanti (4 year old girl)Her lymphocytes were gene-altered (~109) ex vivo used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene (billions of retroviruses used).Cynthia (9 year old girl) treated in same yearProblem: WBC are short-lived, therefore treatment must be repeated regularly.Ashanti DaSilvaAndrew GobeaEffective method: treatment of stem cells fromumbilical cord blood in infantsLack of ADA blocks a biochemical pathway that normally breaks down a metabolic toxin into uric acid, which is then excreted. Without ADA, the substance that ADA normally acts upon (Deoxyadenosine) builds up and destroys T cells. Without helper T cells to stimulate them, B cells cannot mature into the plasma cells that produce antibodies Both branches of the adaptive immune system fail. The child becomes very prone to infections and cancer, and despite medical treatment, usually does not live beyond a year in the outside environment.Culver, Anderson, and Blaese with gene therapy patients.
57 #2: Visual behavior in the children-as assessed by the ability to walk-showed substantial improvements after treatment (Y. 2007) (Video)
58 Before treatment (Video 1) After treatment (Video 2)
59 Diseases were approved for gene therapy 26109105109
61 Died from a massive immune response against viral vector Ornithine Transcarbamylase (OTC) deficiencyJesse GelsingerOTC is an X-linked recessive disorder where one of five enzymes required to break down amino acids liberated from dietary proteins is absent. The nitrogen from the amino acids combines with hydrogens to form ammonia (NH3), which rapidly accumulates in the blood stream and travels to the brain, causing coma and death. Symptoms can be somewhat controlled by following a low-protein diet and taking drugs that bind ammonia.
62 Gene therapy in China Hemaphilla B:X-linked recessivie F IX First disease with gene therapyRetroviral vector and AAV (2 non-specific, 1 muscle, 5 liver)Autologous skin fibrolast transplantationSubcutanous graft薛京伦教授
63 Content The definition of gene therapy Basic process (strategies) of gene therapyTypes and routes of gene therapyVectors of gene therapyGene therapy for clinical useProblems and Perspective
64 Problems with Gene Therapy Short LivedHard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long timeWould have to have multiple rounds of therapyImmune Responsenew things introduced leads to immune responseincreased response when a repeat offender entersViral Vectorspatient could have toxic, immune, inflammatory responsealso may cause disease once insideMultigene DisordersHeart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because you need to introduce more than one geneMay induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis
65 Regulated target gene expression in gene therapy Intrinsic regulatory elements of genesNormal cells-specific :DAT—dopaminergic neuronNSE---all neuronsDiseased cell-specific: AFP, CEAExtrinsic regulatory elemmnts of geneHSP response elementDiseased microenviromentHIF and HREInducible elementTet on/tet off (tetracyclin-resisitance operon)Inducible, regulatedexpression
66 Inducible cell-specific expression Ex vivo, somatic gene therapyDisease mechanismSuffered cell/tissueChoice of vectorInducible cell-specific expression
68 What impact is gene therapy likely to have on medicine in the future? "Someday people will look back on the era before gene therapy in the same way we look back on the era before antibiotics and vaccines. It is now possible to think about treating a whole series of diseases with a one-shot therapy that would last a lifetime.“Dr. Rochelle Hirschhorn (Professor of Medicine, New York University)