基因治療

　�。ǘ�)基因的轉運

　　目前已有多種基因轉運的方式，其基本原則是將外源基因運到細胞內。已使用的有病毒載體和非病毒載體兩大類。

　　病毒載體：病毒具有一些獨特的性質如多數(shù)病毒可感染特異的細胞，在細胞內不易降解；RNA病毒能整合到染色體以及基因水平較高等。因此病毒載體是良好的基因轉運載體。目前已被用作載體的病毒有逆轉錄病毒、腺病毒、腺相關的病毒。皰疹病毒和肝炎病毒等。逆轉錄病毒用作載體時需進行幾步改造：（1)將天然的野生型RNA前病毒轉變成DNA載體，并插入欲轉移的相關外源基因。其基本原則是用標記基因和外源基因替代病毒的編碼基因，如圖23-2所示。（2)制備輔助細胞為載體DNA提供其喪失的功能，如圖23-3所示。（3)將載體DNA導入輔助以產(chǎn)生病毒載體。如圖23-4。（4)病毒載體感染靶細胞，外源基因在細胞內得以表達，如圖23-5所示。

STEP 1

INSERTING FOREIGN DNA INTO THE RETROVIRAL PROVIRUS

Wild-Type Provirus

圖23-2 Scientists use recombinant DNA techniques to replace the gag,pol,and env genes with one or more foreign genes.The foreign genes can be inserted in several different patterns.in this example,a selectable marker gene(neo)replaces the viral gag and pol genes and　a human gene replaces the env gene.

STEP 2

MAKING THE HELPER CELL

Desiging the Helper Vius

圖 23-3 The helper cell should meet two basic requirements:(1)it should provide functions missing from the the vector virus,and(2)it should not be capable of producing viable virus particles. The crucial element in the development of a successful helper cell is the design of the helper virus.Researchers use recombinant DNA techniques to disable the helper virus in the test tube-one of the most common measures is removal of the Psi fragment.The Psi-deficient helper virus produces all of the normal viral proteins, but cannot package its own RNA because it lacks the appropriate packaging signal. Helper virus DNA is inserted into the genome of the helper cell using chemical techniques.

STEP 3

PRODUCING THE VECTOR

圖23-4 Scientists use chemical measures or an infection technique to insert recombinant vector DNA (including a human gene) into helper cells.Because the vector provirus contains the Psi sequence, the vector RNA genome is automatically encapsulated by viral proteins produced by helper virus DNA in the helper cell. The resulting viral particles are released by budding from the helper cell membrane. The vector virus is capable of only one infection because it lacks the information needed to make viral proteins.

STEP 4

INFECTING THE TARGET ELL

圖23-5 Researchers infect target cells (such as human bone marrow cells)with the vector virus in two different ways: they mix them with helper cells producing the virus, or they bathe them in fluid harvested from the helper cell culture.When the vector provirus is integrated into the target cell DNA ,enzymes from the target cell treat it as an integral part of the genome. Cellular enzymes do the work necessary to make proteins from the foreign genes located between the two viral L TRs.

　　非病毒載體：這類載體的發(fā)展較快，目前主要是脂質體，一些具有受體功能的載體也呈現(xiàn)出誘人的前景，關于常見載體的優(yōu)缺點列于表2。

表23－2　常見基因轉運載體的優(yōu)缺點

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