1、Gene Therapy第1頁，共31頁。 BackgroundCancerCCVD: Cardio-CerebrovascularDiseasesViral Disease GeneticDisease: more than 2000 diseases CNS: CentralNervousSystemDiseaseImmuneDiseasesDiabetesHuman beings fight against all kind of diseases第2頁，共31頁。 BackgroundChemical drugsSurgery Physical Therapy Transplantat

2、iontherapyImmunotherapyRegenerativemedicineDifferent treatments to the diseasesGenetherapy第3頁，共31頁。 BackgroundDefinition: Gene therapyis the use ofDNAas adrugto treat disease by delivering therapeutic DNA into a patients cells. Genetherapy Using DNA to express functional protein Using siRNA or shRNA

3、 to attenuate abnormal gene expression in mRNA level Genome editing to correct mutant gene sequence: ZFN, TALEN, CRISPR-Cas9 第4頁，共31頁。 BackgroundApplications: Gene therapy can be applied in many diseases, such as cancer, viral disease, genetic disease, et al. And mostly appliable to the single-gened

4、isorders. Genetherapy The first approved gene therapy case in the United States took place on 14 September 1990. There are more than 2000 clinical trials have being launched in the past seven years.第5頁，共31頁。 Background Technicalproblem: Barriers to the targetDNA/RNA is unstable in the bloodstream, c

5、an be immunogenic and does not readily cross membranes to enter cells. Nuclease, renal filtrationPoor selectivity and inefficiency of enrichment in the target cell or tissue.第6頁，共31頁。 BackgroundDifferent delivery toolsVirus Vector: Retrovirus, Lentivirus, Adenovirus, Adeno-associated virus (AAV), He

6、rpes Simplex Virus (HSV-1), et al.Non-virus method: Cyclodextrin Polymers, Lipids, Peptide, Antibodies, Aptamers, and small molecules第7頁，共31頁。 BackgroundVirus based transductionWidely studied RNA virusWide host rangeInfect dividing cell and just infect onceIntegrate into host genomeLow ImmunogenicLo

7、ng expression periodInsertion mutation by random integration and oncogenicityCant infect non-dividing cell and low virus titerRetrovirus: 第8頁，共31頁。 BackgroundVirus based transductionRNA virus derived from HIVCan infect non-dividing cellLow ImmunogenicLong expression periodInsertion mutation by rando

8、m integration and oncogenicityLow virus titerLentivirus: 第9頁，共31頁。 BackgroundVirus based transductionDNA virusHigh virus titer (up to 1014VP/ml) and high infection efficiencyWide host range and large transgene capacity ( 37kb ) Infect dividing and non-dividing cell Low integration level, exist as ep

9、isome in host cellNo insertion mutation by random integration and oncogenicityShort expression period (5-20 days)Complex procedure and manipulationPotential immunogenic and inflammatory response ( Jesse Gelsinger, 18 years old, died from severe immune response caused by adeno-virus based gene therap

10、y in 1999)Adenovirus:第10頁，共31頁。 BackgroundVirus based transductionDNA virus without pathogenicitySpecific host rangeInfect dividing and non-dividing cell Low integration level, exist as episome in host cellNo insertion mutation by random integration and oncogenicity (site-specific integrate into 19

11、chromosome)Long expression periodNo immunogenic and inflammatory response Small transgene capacity ( 3kb ) Low virus titer (1012 VP/ml)Complex procedure and manipulationHost range limitationAdeno-associated virus (AAV) 第11頁，共31頁。 BackgroundVirus based transductionDNA virusHigh infection efficiencyIn

12、fect dividing and non-dividing cell Neurotropic virusLarge transgene capacity ( up to 150kb )Long expression periodHigh immunogenic and inflammatory response and necrosis Herpes Simplex Virus-1 第12頁，共31頁。 BackgroundVirus based transductionHSV/AAV Ad/EBV HSV/EBVAd/AAVAd/retrovirusHybrid virus vector第

13、13頁，共31頁。 BackgroundsiRNA-based gene therapy第14頁，共31頁。 BackgroundNon-virus method Chemical modification Chemical modification can make the RNA be resistant to the nuclease cleavage. 第15頁，共31頁。 BackgroundNon-virus method Cyclodextrin polymer nanoparticlesCan deliver both siRNA and plasmid DNAFirst ap

14、plied in 1999Targeted: ligandLow toxicitySteric stabilizationNo measured innate immune responses when administered intravenously第16頁，共31頁。 BackgroundNon-virus method LiposomeCan deliver both siRNA and plasmid DNAProtect entrapped oligonucleotides from nuclease degradation and renal clearancePromote

15、cellular uptake and endosomal escapeThey include the use of cationic or ioniz-able lipids, shielding lipids, cholesterol and targeting ligands第17頁，共31頁。 BackgroundNon-virus method Conjugate deliveryFirst reported in 2007PEG: shielding effectGalNAc ligand was essential for both uptake by hepatocytes

16、and in vivo silencing activity.Other targeting ligands has been explored, including peptides, antibodies, small molecules, glycans, lectins and nucleic acids.Dynamic PolyConjugates (DPC)99% knockdown of liver genes after a single 0.2 mg per kg dose in non-human primates, with the effect lasting near

17、ly 7 weeks第18頁，共31頁。 BackgroundNon-virus method Conjugate deliveryASGPR, on hepatocytesTriantennary GalNAcsiRNABoth subcutaneous and intravenous administration of this conjugate revealed great accumulation of siRNA in the liver and improved knockdown of the target gene.第19頁，共31頁。 BackgroundSelf-asse

18、mbly of oligonucleotide nanoparticlesConjugate delivery3D-DNA tetrahedra第20頁，共31頁。 BackgroundCentralNervousSystemDiseaseBBB：BloodBrainBarrierRetrovirus:cant infect neuronex vivoin vivoNon-virus:VirusInjection by Neurosurgical steretactic operation Receptor on the brain microvascular endotheliocyte P

19、rocess with mannitol to improve the permeabilityLentivirus:infect neuron, long period expressionHSV-1:neurotropic virus, but with high immunogenic,inflammatory response and necrosis can specifically infect spinal marrow and astroglia cell in brain through tail intravenous injectionAAV9：第21頁，共31頁。 Ba

20、ckgroundHepatic CellRetrovirus:in vivoNon-virus:VirusLentivirus:AAV：Local injection: low expression levelIntravenous injection: nuclease cleavageHydrodynamic injection: work well on mice modelLow selectivity and genome integration Low selectivity and genome integration Hepatotropic AAV serotype第22頁，

21、共31頁。 BackgroundHepatic Cellhydrodynamic injectionPlasmid DNA (60 g) and ssDNA oligo (60g) suspended in 2ml saline were injected via the tail vein in 5-7 seconds into 8-10 weeks old Fah mut/mutmice.第23頁，共31頁。Hereditary tyrosinemia type I (HTI) Fumarylacetoacetate hydrolase (FAH), homozygous GA point mutation of the last nucleotide of exon 8 of FahFah5981SB mouse model第24頁，共31頁。Treatment effect第25頁，共31頁。Treatmen