1、 Chapter 13 Gene Therapy 13.2 Gene transfer Two Ways of Gene Transfer vector objective gene in vivo ex vivo target cell Fed back into the body with recombinant target cells Direct entry of the target gene into the body The target gene was introduced into the target cells and cultured in vitro The wa
2、y of gene transfers 1. in vivo therapy （ in vivo） It refers to the direct introduction of the target gene into the relevant tissues and organs of the body, so that it can enter the corresponding cells and express it. 2. ex vivo therapy （ ex vivo） The target gene was introduced into the target cells
3、in vitro, and the cells were returned to the patients after screening and proliferation, so that the gene could effectively express the corresponding products. 1. Virus-mediated gene transfer system Viral vector-mediated gene transfer is highly efficient and viral vector is the most commonly used ge
4、ne therapy vector. One of the most used is the retroviral vector. （ 1） Retroviral vector +ssRNA Reverse transcriptase Core protein Membranin Human immunodeficiency virus (HIV) Retroviral Life Cycle I Retroviral Life Cycle Retroviral Life Cycle （ 1） Each end has a long terminal repeat LTR. 1. Structu
5、ral characteristics of retroviral provirus （ 2） LTR consists of three parts: U3、 R and U5. A. U3 with enhancers and promoters ； B. Viral integration sequences at both ends of U3 and U5, respectively ； C. There are also poly (A) tailed signals in the R region. 1. Structural characteristics of retrovi
6、ral pre-virus （ 3） Viruses have three structural genes: gag、 pol and env. （ 4） There are sequences () and splice donor sites (SD) and shear acceptance sites (SA) necessary for viral packaging downstream of the 5 LTR 2. Retroviral-mediated gene transfer system (1) Retroviral vector Retains the packag
7、ing signal of the virus particle while miss the virus particle packaging protein gene; it can accommodate foreign genes, but cannot package ifself into a proliferating virus particle. LTR therapeutic gene LTR 2. Retroviral-mediated gene transfer system (2) Auxiliary cell line It is constructed from
8、a different defective retroviral infection. Cells are capable of synthesizing package proteins, however, due to the lack of packing model, it alone cannot generate reproducing virus particles. gag pol env Retroviral packaging system The deletion of retroviral structural gene gag、 env and pol did not
9、 affect the activity of other parts. Packaged pseudovirus particles are easy to isolate and prepare. Retrovirus transfers genetic material efficiently into target cells. Provirus is efficiently integrated into the target cell genome by LTR, which is beneficial to the permanent expression of exogenou
10、s genes in target cells. 3. Characteristics of retroviral vectors 4. Main Disadvantages of Retroviral Vector Random integration with potential risk of insertion mutation and activation of oncogenes; Retroviral vectors have a relatively small insertion capacity, only accommodating foreign genes below
11、 8 kb. （ 2） Adenovirus Vector (AV) （ 2） Adenovirus Vector (AV) Adenovirus is a macromolecular (36 kb) double-stranded non-encapsulated DNA virus. It enters the cell through receptor-mediated endocytosis, and then the adenovirus genome is transferred into the nucleus, maintained in vitro, i.e., not i
12、ntegrated into the host cell genome. Advantages of adenovirus 1. High efficiency of gene introduction; 2. Wide host range; 3. Gene transduction is independent of cell division. 4. Recombinant adenoviruses can be absorbed orally through the gut, or inhaled by spray or administered intratracheal. 5. A
13、denovirus vector has a large capacity and can insert 7.5 kb fragment of foreign genes. Disadvantages of adenovirus 2.Host immune response leads to transient adenovirus vector expression. 3. There are two ways that may produce replicative adenoviruses: the vector recombines with helper cells or the w
14、ild-type virus replicates in the patient. 4. Poor targeting. 1.It cannot be integrated into the genomic DNA of target cells. Gene expression is relatively short-lasting. （ 3） adenovirus associated virus vector AAV Virus Particle Adenovirus-associated virus (AAV) is a class of single-stranded linear
15、DNA defective viruses, spatially a bare icosahedron-particle without envelope . Its genome DNA is less than 5 kb. AAV cannot be replicated independently: only in the presence of auxiliary viruses, replication and lysocytic infection occur, otherwise the establishment of lysogenic latent infection. S
16、tructure of AAV Virus Genomic DNA REP: Virus Replication Gene CAP: Genes encoding capsid proteins ITR： Reverse end repeats Characteristics of AAV Mainly latent infection; AAV can be efficiently while site-specifically integrated into human chromosomes, thus exogenous genes can express steadily and c
17、onsistently, without potential risk of inactivating tumor suppressors and activating proto-oncogenes due to random integration. Defects of AAV carriers: Small capacity, currently only accommodate exogenous DNA fragment of up to 5 kb; Infection efficiency is lower than retroviral vectors. Infection i
18、n 40-80% of adults in nature, thus easily stimulate immune rejection. viral gene therapy vector Integration characteristics Safety Infection cell Clone capacity Retroviral vector Random integration, high efficiency May cause disease Segmentation cells 7kb Adenovirus vector Disintegrate, may be lost
19、High security Segmentation cells Unseparated Cell adenovirus associated virus vector Site-directed integration High security Segmentation cells Unseparated Cell 5kb 7.5kb 2. Non-viral vector-mediated gene transfer system （ 1） Liposome mediated gene transfer Fundamental : Cationic liposome (monomers)
20、 automatically embeds target DNA molecules upon mixing with each other, and then the post-embedding mixture transfer DNA to target cells by endocytosis upon incubation with the cells. liposome mediated gene transfer (2)Receptor-mediated transfer technology The binding of membrane receptor and its hi
21、gh-affinity ligand can be used to mediate cell- or tissue-targeted DNA transfer. Receptor-mediated DNA transfer （ 3 ） Direct gene injection technology For example: intramuscular injection of coagulation factor IX gene, which can lead to the production of hemophilia required coagulation factor IX. Review ： 1. What are the kinds of ways of gene transfer for gene therapy? 2. Illustrate the application of gene transfer technology in gene therapy.
