VIROLOGY. Engineering Viral Genomes: Retrovirus Vectors
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1 VIROLOGY Engineering Viral Genomes: Retrovirus Vectors
2 Viral vectors
3
4 Retrovirus replicative cycle
5 Most mammalian retroviruses use trna PRO, trna Lys3, trna Lys1,2 The partially unfolded trna is annealed via at least 18 nt at its 3 end to a site on RNA genome called the primer-binding site (pbs) The diploid retroviral genome
6 The reverse transcription process -1
7 The reverse transcription process -2
8 Characteristics of retroviral integration
9 Assembly of a retrovirus from polyprotein precursors
10 Sequences important in packaging of retroviral genomes
11 Favorable Features of Retroviruses as Vectors Well characterized Easily to manipulate (genomes 7-9 kb) They require 3 trans (gag, pol, env) and 7 major cis-active control elements (U3, R, U5, PBS, SD, Ψ, SA ) in order to replicate Stability of recombinants vectors (plasmids) High efficiency of gene transfer Most are replication-defective Stable and precise integration of the transgene Low immunogenicity Can be pseudotyped to infect a broad range of cells
12 Retrovirus as vectors Replication-incompetent vectors -They bear deletions of some or all of the viral genes - They retain cis-acting viral sequences necessary for transmission (U3, R, U5, PBS, SD, Ψ, SA) - They need to be propagated in packaging cell lines that provide in trans gag, pol, env Replication-competent vectors (RCR) -Avian vectors (up to 2 kb inserts)
13 Replication-incompetent vectors
14
15 Retroviral vectors
16 Retroviral vectors
17 Production of Recombinant Retrovirus in the Packaging Cell
18 Infection of a Target cell and Expression by a Replication-Incompetent Retrovirus Vector
19 Infection of target cells and expression by a replication-incompetent retrovirus vector Production of infectious retroviral particles containing the gene of interest
20 Tropism of Retrovirus Vectors Retrovirus tropism is determined at three levels: 1) Viral envelope proteins (gpsu); 2) Nuclear translocation and integration -defined by structural features of p30 CA 3) Transcriptional activity of the LTR in the transfected cell - MLV LTR is active in most mammalian cell types
21 Retroviridae host-cell receptors and co-receptors A A E The envelope determines which cells the retrovirus enter
22 Host-range of MoMuLV-derived Vectors Ecotropic glycoprotein, gp70, allows infection of rat and mouse cells Amphotropic glycoprotein gp70 endows a murine virus with a very broad host range (mouse, human, chicken, dog, cat, mink cells) Polytropic receptors can be utilized for retrovirus entry. Pseudotyping the retroviral envelope with the VSV G protein confers a host range capable to infect mammalian, fish, frogs and insect cells
23 Host-range of Retroviral Vectors G
24 Development of retroviral vector design
25 Replication-defective retroviral vectors LTR Internal promoter IRES
26 Retroviral expression vectors
27 Retroviral expression vectors
28
29 Picornaviruses IRES and their use in molecular biology
30 Packaging cell: produces viral proteins from stably integrated genes
31 Production of recombinant retroviral stocks by stable and transient producer cell lines
32 RetroPack PT67 cell line is a NIH/3T3-based packaging line expressing the 10A1 viral envelop
33 Disadvantages of Retrovirus Transduction System Post-mitotic cells cannot be transduced Unable to transduce large (>11kb) DNA fragments Random integration and genome rearrangement (risk of insertional activation of cellular genes)
34 Retrovirus vectors - a research lab application: generation of a GFP-expressing retroviral vector by using the Retromax system
35 Retromax system: choice of vectors
36 Retromax system: choice of packaging vectors Ecotropic (usually (MoMuLV) Amphotropic (from 4070 MulV) Gibbon Ape leukemia virus (GALV) 10A1 (MuLV) mouse and rat cells only (not human) most mammalian cells (no hamster) many mammalian cells (including hamster) most mammalian cells (including hamster)
37 Retromax system: choice of packaging vectors The pcl-eco packaging vector. The gene coding for env was replaced with env gene from different MULV strains (4070A and 10A1) to create pcl-ampho and pcl-10a1 packaging vectors
38 Retromax system: pcl packaging vectors
39 Retromax system: outline of the procedure Prepare 293 cells Co-transfect with retroviral vector containing gene of interest and an appropriate packaging vector After 48 h, harvest virus containing supernatant Infect target cells, either for titer determination or for gene expression Split infected target cell and grow for selecting stable expressing cell lines (G418 selection)
40 Retromax system: construction of pclxsn- GFP vectors 1/2 IRES GFP
41 GFP exp in infected HUVEC after 1 wk of G418 selection
42 VIROLOGY Engineering Viral Genomes: Lentivirus Vectors
43 Viral vectors
44 Retrovirus and Lentivirus vectors
45 Lentiviral vectors
46 Favorable Features of Lentivirus Vectors HIV-1 integrates its DNA and completes a replication cycle in fully differentiated, non dividing cells (macrophages). So, this virus must have a mechanims for the active transport of preintegration complexes into the nucleus. Vpr and a minor, phosphorylated, form of the matrix (MA/ p17) protein direct nuclear import of the HIV-1 preintegration complex. Nuclear localization signals have been found in the IN protein of HIV-1
47 Structure of a non-rcr (SIN) HIV-1 based vector
48 Development of self-inactivating vectors or SIN vectors Packaging plasmids Transfer vectors
49 Lentiviral SIN vectors
50 Production of a 2 nd generation Lentiviral SIN vector
51 Flow-chart production of a recombinant lentiviral vector
52 A 3 rd generation Lentiviral vector system
53 A 3 rd generation Lentiviral vector system gene of interest gag, pol rev VSV G
54 Biosafety Considerations for Research with Lentiviral Vectors
55 The first clinical trial of a lentiviral vector highlights the promise of this new class of gene-therapy vector
56
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