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Viruses and Gene Therapy The good news about viruses Quality of viral vector • • • • • • • • Size of insert Integration or not/and where Ability to obtain in high titer Transduction efficiency Target cell specificity - pseudotyping Expression efficiency/control Possible pathogenicity or lack No immune response developed Useful vectors • • • • • • Retroviruses including HIV Adenovirus Adeno-associated virus (AAV) Herpes simplex virus Vaccinia virus DNA vectors Transduction efficiency Integration Target cells Problems Retrovirus ~ 8 kb High Yes Dividing cells (pseudotyped) Insertional mutagenesis Adenovirus ~ 30 kb High No Cells w/ receptor Immunity AAV ~ 4kb High Random or Cells w/ site sp. receptor Small insert size HSV ~ 40 kb Low No Neurons Latency/ immunity Vaccinia ~ 25 kb High No Cells w/ receptor Immunity Virus Insert size Retroviruses • Require LTR and packaging signal – LTR modified not to be a promotor – Cloned gene inserted with strong promotor (pCMV) and may have IRES – HIV vectors may have some accessory genes if LTR promotor is used (TAT) Created using a packaging cell line – Genes for gag/pol/env on separate plasmid to get packaging – May have two or three plasmids for trans genes to reduce recombination • Advantages: – Stable integration – Can design to target HIV infected cells with suicide gene – Can add other promoters that respond only in specific cells • Disadvantages: – No replication and spread of vector – Integration may be random – Requires dividing cell to integrate and express • What would happen to a retroviral vector with pCMV and HSV-TK injected into brain tumor and treatment with ganciclovir? – No affect on neurons so can treat glioma – TK activates ganciclovir and kills cells • What would happen if used vector to add WT p53 gene to lung cancer tumor? Adenovirus vectors • Non-enveloped so no pseudotyping • Requires elimination of early gene (E1 or E3) and other nonessential genes and becomes defective • Packaging cell line has E gene integrated and expressed (less likely crossover) • “Gutless” vectors have only the inverted terminal repeats (ITR) and a packaging signal and get all other gene products in trans in packaging cell • Vector-infected cells are removed by immune system so transient response but that is reduced in gutless vector • May not work at all if host starts with some Ad immunity • Infects wide range of cells (common receptors) Tumor destruction by adenovirus • • • E1B region binds and inactivates p53 protein mutant adenovirus (dl1520) that cannot produce E1B won’t reproduce tumor cells lacking functional p53 can support replication of this virus • • • • C33A lacks p53 U2OS has p53 Circles = WT Squares = mutant Which side has the Wt? The mutant? Tumors in mice after treatment with Wt or mutant Adenovirus carries lots of DNA • Put human hepatitis B genome into ad cloning vector (removed E1 and E3 genes) • Hep genome also had GFP linked to pCMV as marker to recognize transduced cells • Used to create HepB cell line in mouse cells that produces infectious viruses from nonintegrated DNA • Infected mouse as well - small animal model • Crossed species barrier AAV - parvovirus • Requires adenovirus early genes to replicate - not related • ssDNA with promoter and two genes - capsid and replication protein • Terminal repeats allow for specific integration into genome if helper is not there • Vector has TR and Promoter - no rep and capsid; insert size is small • W/o rep integration is at random • Correcting hemophilia in dogs AAV vector with factor IX • Tumor reduction - AAV vector with angiostatin HSV vectors come in three varieties • Recombinant virus made replication deficient w/o IE gene • Replication conditional replicate in certain cell lines • Amplicon - bacterial plasmidbased • Neurotropic but problems with immunity • Large virus with many nonessential genes - can add 150KB HSV amplicons • Plasmid contains – HSV ORI – HSV packaging signal – IE promoter and gene of interest – Selection marker • Virus made in cell that provides all other proteins in trans