基因治疗

,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2014/12/6,#,Gene Therapy,Background,Cancer,CCVD:,Cardio-CerebrovascularDiseases,Viral Disease,GeneticDisease:,more than 2000 diseases,CNS: CentralNervousSystemDisease,ImmuneDiseases,Diabetes,Human beings fight against all kind of diseases,Background,Chemical drugs,Surgery,Physical Therapy,Transplantationtherapy,Immunotherapy,Regenerativemedicine,Different treatments to the diseases,Genetherapy,Background,Definition:,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 to attenuate abnormal gene,expression in mRNA level,Genome editing to correct mutant gene sequence: ZFN,TALEN, CRISPR-Cas9,Background,Applications:,Gene therapy can be applied in many diseases, such as cancer, viral disease, genetic disease, et al,. And mostly appliable to the,single-genedisorders.,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.,Background,Technicalproblem: Barriers to the target,DNA/RNA is unstable in the bloodstream, can be immunogenic and does not readily cross membranes to enter cells.,Nuclease, renal filtration,Poor selectivity and inefficiency of enrichment in the target cell or tissue.,Background,Different delivery tools,Virus Vector:,Retrovirus, Lentivirus, Adenovirus, Adeno-associated virus (AAV),Herpes Simplex Virus,(HSV-1),et al.,Non-virus method:,Cyclodextrin Polymers, Lipids, Peptide, Antibodies, Aptamers, and small molecules,Background,Virus based transduction,Widely studied RNA virus,Wide host range,Infect dividing cell and just infect once,Integrate into host genome,Low Immunogenic,Long expression period,Insertion mutation by random integration and oncogenicity,Cant infect non-dividing cell and low virus titer,Retrovirus:,Background,Virus based transduction,RNA virus derived from HIV,Can infect non-dividing cell,Low Immunogenic,Long expression period,Insertion mutation by random integration and oncogenicity,Low virus titer,Lentivirus:,Background,Virus based transduction,DNA virus,High virus titer (up to,10,14,VP/ml,) and high infection efficiency,Wide host range and large transgene capacity ( 37kb ),Infect dividing and non-dividing cell,Low integration level, exist as episome in host cell,No insertion mutation by random integration and oncogenicity,Short expression period (5-20 days),Complex procedure and manipulation,Potential immunogenic and inflammatory response,(,Jesse Gelsinger, 18 years old, died from severe immune response caused by adeno-,virus based gene therapy in 1999),Adenovirus:,Background,Virus based transduction,DNA virus without pathogenicity,Specific host range,Infect dividing and non-dividing cell,Low integration level, exist as episome in host cell,No insertion mutation by random integration and oncogenicity (site-specific integrate into 19 chromosome),Long expression period,No immunogenic and inflammatory response,Small transgene capacity ( 3kb ),Low virus titer (,10,12,VP,/ml,),Complex procedure and manipulation,Host range limitation,Adeno-associated virus (AAV),Background,Virus based transduction,DNA virus,High infection efficiency,Infect dividing and non-dividing cell,Neurotropic virus,Large transgene capacity ( up to 150kb ),Long expression period,High immunogenic and inflammatory response and necrosis,Herpes Simplex Virus-1,Background,Virus based transduction,HSV/AAV,Ad/EBV,HSV/EBV,Ad/AAV,Ad/retrovirus,Hybrid virus vector,Background,siRNA-based gene therapy,Background,Non-virus method,Chemical modification,C,hemical modification can,make the RNA be resistant to,the nuclease cleavage.,Background,Non-virus method,Cyclodextrin polymer,nanoparticles,Can deliver both siRNA and plasmid DNA,First applied in 1999,Targeted: ligand,Low toxicity,Steric stabilization,No measured innate immune responses when administered intravenously,Background,Non-virus method,Liposome,Can deliver both siRNA and plasmid DNA,Protect entrapped oligonucleotides from nuclease degradation and renal clearance,Promote cellular uptake and endosomal escape,They include the use of cationic or ioniz-,able lipids, shielding lipids, cholesterol,and targeting ligands,Background,Non-virus method,Conjugate delivery,F,irst reported in 2007,P,EG: shielding effect,G,alNAc ligand was essential for both uptake by hepatocytes and in vivo silencing activity.,O,ther 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 nearly 7 weeks,Background,Non-virus method,Conjugate delivery,ASGPR, on hepatocytes,Triantennary GalNAcsiRNA,Both subcutaneous and intravenous administration of this conjugate revealed great accumulation of siRNA in the liver and improved knockdown of the target gene.,Background,Self-assembly of oligonucleotide nanoparticles,Conjugate delivery,3D-DNA tetrahedra,Background,CentralNervousSystemDisease,BBB,：,BloodBrainBarrier,Retrovirus:,cant infect neuron,ex vivo,in vivo,Non-virus:,Virus,Injection by Neurosurgical steretactic operation,Receptor on the brain microvascular endotheliocyte,Process with mannitol to improve the permeability,Lentivirus:,infect neuron, long period expression,HSV-1:,neurotropic virus, but with high immunogenic,inflammatory response and necrosis,can specifically infect spinal marrow and astroglia,cell in brain through tail intravenous injection,AAV9,：,Background,Hepatic Cell,Retrovirus:,in vivo,Non-virus:,Virus,Lentivirus:,AAV,：,Local injection: low expression level,Intravenous injection: nuclease cleavage,Hydrodynamic injection: work well on mice model,Low selectivity and genome integration,Low selectivity and genome integration,Hepatotropic AAV serotype,Background,Hepatic Cell,hydrodynamic injection,Plasmid DNA (60,g) and ssDNA oligo (60,g) suspended in 2ml saline were injected via the tail vein in 5-7 seconds into 8-10 weeks old Fah mut/mutmice.,Hereditary tyrosinemia type I (HTI),Fumarylacetoacetate hydrolase (FAH),homozygous GA point mutation of the last nucleotide of exon 8 of,Fah,Fah5981SB mouse model,Treatment effect,Treatment effect,33.5% 3.3%,0.40 0.12%,Side-effect analysis,Indel and off-target effect,Side-effect analysis,Indel and off-target effect,Indel and off-target effect,Side-effect analysis,mismatch-specific Surveyor nuclease assay,Discussion,T,ransient expression of Cas9, sgRNA and a co-injected ssDNA by nonviral,hydrodynamic injection is sufficient correct the genome mutation of the mouse,model of HTI.,I,mprovements to CRISPR delivery methods and repair efficiency will be,required for its broad clinical applications.,F,urther studies will be,required,to evaluate the extent of off-target effects,particularly in vivo, and strategies to reduce off-target.,Thank you!,