Viral vectors. Part I. 27th October 2014
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1 Viral vectors Part I 27th October 2014 Prof. Józef Dulak, PhD, DSc Department of Medical Biotechnology Faculty of Biochemistry, Biophysics and Biotechnology Room 3.025/3.07 Phone jozef.dulak@uj.edu.pl
2 Genetic syringes - vectors AAV vectors (adeno-associated) Retroviral/lentiral vectors Adenoviral vectors Plasmid DNA
3 Vectors Non-viral/plasmids Viral naked DNA Lipoplexes RNA Retroviruses (including lentiviruses) DNA Adenoviral AAV Herpes complexes with Viroplexes other chemicals (lipoplexes enriched In specific viral proteins) 3
4 Viral vectors Integrating Lentiviral -retroviral -AAV (limited) Non-integrating Adenoviral HSV Baculoviral Integration depends on: -LTR sequences and integrase (retroviruses) - ITR seqeuences and rep proteins (AAV) 4
5 Episomal transgene Advantage - no risk of insertional mutagenesis Disadvantage - transient expression - repeated treatments may be necessary 5
6 Viral vectors used in clinical trials of gene therapy 1. Adenoviruses 2. Retroviruses a) oncoretroviruses b) lentiviruses 3. Adeno-associated viruses 4. Herpes simplex virus 6
7 Integrated transgene Advantage: - perpetual - may provide a stable expression and a cure Disadvantage: - random insertion may lead to silencing of a transgene or inactivation or dysregulated activation of host genes - unknown, long-term effects of the transgene 7
8 Viral vectors follow the rule of their parent viruses Nature, 8
9 The fours barriers of successful gene therapy Kay M, Nature Rev Genetics, 2011
10 Principle of generating viral vector Cis- sequences trans- sequences Verma & Weitzman, Ann Rev Biochem
11 Principles of generating viral vectors 1. Removal of most genes coding for viral proteins from the viral genome, in particular those potentially pathogenic 2. Maintenance of the cis-acting sequences of the viral genomes not required for viral replication; in particular those determining inclusion of the genomes within the viral particles (packaging signal - y) 3.Expression of the viral proteins required for viral replication within the virus producing cells (packaging cells) these proteins can be expressed from genes encoded by transiently transfected plasmids or a previously egineered cellular genome or by a helper virus simultaneously infecting the packaging cells. M. Giacca, J Control Release, 2012
12 Genetic strategy for engineering a virus into a vector Sequences in cis Sequences in trans (the vector lacks the Y sequence, hence it is not packed into the capsid) Packaging cell Kay et al., Nature Medicine 7, (2001) 12
13 Strategies for viral vectors constructions Helper-independent (not useful for gene therapy) required genes are delivered in trans RM Twyman, Gene transfer to animal cells
14 Retroviral vectors Family Retroviridae 1. Oncoretroviruses: a) Alpharetrovirus Avian leucosis virus (RSV) b) Betaretrovirus Mouse mammary tumor virus (MMTV) c) Gammaretrovirus Murine leukemia virus (Mo-MLV) d) Deltaretrovirus Bovine leukemia virus e) Epsilonretrovirus Walleye dermal sarcoma virus 2. Lentivirus HIV, HTLV, BLV 3. Spumavirus human spumavirus (human foamy virus HMV) 14
15 Retroviruses Gag core proteins, matrix, nucleocapsid Pol reverse transcriptase and integrase Env envelope glycoproteins 15
16 Genomic organization of retrovirus The retroviral genome is flanked by a long terminal repeat sequence (LTR). LTR region contains all signals necessary for gene expression, including the enhancer, promoter, transcription initiation, transcription terminator and polyadenylation signal. The size of the LTR varies considerably among the different retroviruses. The integrated provirus has two LTRs; the 5' LTR normally acts as an RNA polymerase II promoter whereas the 3' LTR functions as the terminator sequence. An LTR is composed of three elements U3, R and U5 regions. - U3 includes most of the transcriptional control elements and carries the promoter/enhancer sequence. It is the unique, non-coding region of nucleotides which is in the first part of the genome to be reversely transcribed, forming the 3' end of the provirus genome - R region is usually a short ( nucleotide) sequence that provides the sequence homology for strand transfer during reverse transcription of the RNA genome. U5 region carries the poly A-site and, together with the R region, determines the poly A addition. It is a unique non-coding region of 200 1,200 nucleotides that forms the 5-' end of the provirus after reverse transcription. Both U3 and U5 regions contain the Att sites required for integration 16
17 Retroviral infectious cycle All retrovirus genomes consist of two molecules of RNA, which are s/s, (+)sense and have 5' cap and 3' poly-(a) (equivalent to mrna). These vary in size from ~8-11kb RM Twyman, Gene transfer to animal cells
18 Essential and non-essential elements in retroviruses Verma & Weitzman, Ann Rev Biochem 2005
19 How to make a vector from a virus? 1. Separation of genes and sequences acting in-trans into different plasmid vectors in order to avoid viral reconstruction through recombinantion - coding sequences, required for formation of infectious particle act in-trans - in-trans sequences are delivered in separate plasmids and are present in genome of packaging cells 2. Construction of a plasmid containing in cis required sequences plus a therapeutic gene 3. the construct is introduced into the packaging cell line, providing the structural viral proteins in trans 19
20 Construction of retrovirus vector 1. Construction of retrovirus vector as a recombinant plasmid in E.coli 2. Introduction of a plasmid into a packaging cell line 3. Incorporation of vector transcripts into transmissible virus particles 20
21 Structure of a plasmid used for production of retroviral vectors therapeutic gene reporter gene 21
22 Production of retroviral vectors Separate transfection of three vectors 22
23 Stages of retroviral construction (1) Transfection of recombinant plasmid into the packaging cell previously stably transfected with helper plasmids 23
24 Stages of retroviral construction (2) 24
25 Features of retroviral vectors Due to destruction of a natural, well-functioning gene composition, required for the formation of infectious virus, the resulting system of retroviral vector synthesis suffers from: - low efficiency of packaging of vectors in comparison to wild type viruses - formation of a large number of defective particles, which inhibit the transduction efficiency 25
26 Binding of retroviral vector to a cell surface Pseudotyping -one species of retrovirus is capable of incorporating the envelope protein of another species or a different virus -the endogenous or heterologous envelope protein can be provided in trans to a replication-defective vector that lack the envelope coding sequence - can be used to alter the tropism or increase the titer of the vectors 26
27 G protein of vesicular stomatitis virus (VSVG) An envolope virus belonging to the family Rhabdoviridae - Binds the phospholipids present on virtually all mammalian cell membranes & triggers endocytosis of the viral particles - Lowering the ph in endosomes activates the fusiogenic properties of VSVG, which determine fusion of the viral envelope with the endosomal membrane and release of the virion content into the cytosol
28 Retroviral vectors can be used for transduction in many species 28
29 Types of retroviral vectors 1. Ecotropic - infect only rodent cells (Eco-R receptor) 2. Xenotropic - infect most mammalian except rodent 3. Amphotropic - infect all mammalian (receptor Ram- 1 and Glvr-1) 4. Pantropic - infect various species - VSV glicoprotein recptor is controversial; but VSV binds ubiquitous phosphatidylserine 29
30 Targeting retroviral vectors to specific cell types 1. Neuronal RVG/VSVG chimeric proteins a fusion product between the external domain of Rabies virus glycoprotein and the cytoplasmic domain of VSVG 2. LCMV-GP lymphotropic choriomeningitis virus specific infection of glioma cells while sparing neurons for brain cancer therapy 3. Targting to dendritic cells by binding to C-type lectins, using the proteins from - glycoprotein from Sindbis virus - gp64 a major envelope protein of baculovirus - Aura virus glycoproteins
31 Significance of retroviral vectors Retroviral gene delivery is the method of choice for gene expression in higher organisms because it is generally faster, more reliable, and has broader utility than alternative gene transfer methods. Infection efficiencies of >90% Efficient transduction of "difficult" cell types including primary, explant, embryonic stem (ES) and embryonic carcinoma (EC) cells The host range of retroviruses has been expanded by pseudotyping the vectors with heterologous viral glycoproteins and receptor-specific ligands. This is possible because one species of retrovirus is capable of incorporating the envelope from another species or type of retrovirus. Therefore, the envelope protein can be provided in trans so that the virus produced can infect cells based on the tropism of that envelope protein Transduction without variability or loss of expression 31
32 Retroviral Vectors as Gene Delivery Tools The ability to transduce a variety of cell types The ability to integrate efficiently into the genomic DNA of the dividing or mitotically active recipient cells The ability to express the transduced gene at high levels Capacity for long-term persistence and stable transmission of the gene to all future progeny of the transduced cell Up to 6.5 kb of foreign gene sequence can be packaged in a retroviral vector. This is adequate for most applications Ability to be manufactured in large quantities to meet very stringent safety specifications 32
33 Retroviral vectors for ex vivo gene therapy Particularly used for ex vivo transduction of hematopoietic stem cells Kaufman et al., EMBO Mol Med. 2013
34 Potential risk of application of retroviral vectors gag structural proteins pol reverse transcriptase env envelope proteins long-term expression & integration into cellular genome LTR gag pol env LTR retrovirus random integration risk of insertional mutagenesis LTR transgen LTR Retroviral vector This has happened in several cases of otherwise succesful gene therapy trials (to be discussed during next lectures)
35 Side effects of MMLV-based retroviral vectors prompted investigations of the mechanisms of integration and search for the new, safer vectors 35
36 HIV genome Sakuma et al., Biochem J
37 HIV life cycle HIV receptors primary CD4 receptor & coreceptor CXCR4 on T lymphocytes Or CCR5 on macrophages, DC, activated T cells Sakuma et al., Biochem J
38 Lentiviral vectors Lentiviral vectors are based on: HIV-1, HIV-2 SIV FIV Naturally infect cells expressing CD4 change to a VSVG results in a broad range of transfectable cell types Transfect non-dividing cells: thanks to the capacity of pre-integration complex (PIC) which forms in the cytosol, to actively cross the nuclear membrane thanks to the interaction of the PIC proteins (integrase, matrix, Vpr) with the proteins of nuclear pore 38
39 Essential and non-essential elements in lentiviruses Verma & Weitzman, Ann Rev Biochem 2005
40 Consecutive generations of lentiviral vectors Only 3 out of 9 HIV genes are left 40 Sakuma et al., Biochem J 2012
41 Production of lentiviral vectors A transient production system: 1. HEK 293T cells immortalized cells from human kidney 2. Four plasmids i) plasmid containing the gene of interest ii) packaging plasmid with gag and pol sequences iii) plasmid containing the rev sequence (necessary for the efficient expression of gag and env iv) plasmid containing the envelope sequence
42 Self-inactivating lentiviral vectors
43 Self-inactivation of lentiviral vectors Deletion of 3 -LTR elements, including its TAT-boc, Sp-1, NF-kB and NFAT binding site This reduces the likehood of: 1. Propagation of spontaneously replication-competent recombinant HIV-like viruses; 1. insertional activation of cellular oncogenes by residual promtoer activities of integrated LTRs; 3. Mobilisation of integrated vectors by wild-type virus; 4. Transcriptional interference and suppresion by LTRs
44 Safety of lentiviral vectors 1. Replication deficient 1. Six out of nine genes involved in the lentiviral pathogenecity are removed; env vif viral infectivity factor Vpr viral protein r Vpu viral protein u Nef negative regulatory factor Tat transactivator 3. Self inactivated Self-inactivating lentiviruses: deletion of 299 bp in 3 LTR causes after transduction inactivation of 5 LTR, decreasing the risk of recombination and vector mobilisation 4. Integration properties different than oncoretroviruses
45 Sites of integration of retroviral vectors GETTING INTEGRATED:Different host factors may affect a retroviral vector's pre-integration complex (PIC). The factors influencing the integrations are unknown D. Trono, Science 13 June 2003
46 Better safety profile of lentiviral vectors in relation to integration properties TSS transcription start sites A. Nawrouzi et al., Viruses
47 Comparison of oncoretroviruses and lentiviruses Self-inactivating vectors have deletion in 3 LTR Nuclear transport possible: proteins involved: integrase, one of Gag protein, Vpr 47
48 Advantages of lentiviruses over oncoretroviruses 1. Safety profile (as above) 2. Ability to infect non-dividing cells 3. Larger capacity 4. Higher transduction efficacy
49 Benefits and drawbacks of lentiviral vectors Pros 1. Relatively high coding capacity (~ 9 kbp) 2. Relatively low immunogenecity 3. Host immunologically naiive to vector 4. Highly efficient in dividing and non-dividing tissues 5. Made into efficient non-integrating vectors Cons 1. Relatively low titres 2. Difficult to scale production 49
50 Titer (miano) of viral vectors Concentration of viral particles and/or virions which are able to transduce the cells The titer represents only a small fraction of a total number of viral particles 50
51 Retroviral vectors properties Capacity Ability to integrate Tissue specificity kb oncoretroviruses 8-9 kb lentiviruses yes yes Ability to transfect non-dividing cells Duration of expression no oncoretroviruses yes lentiviruses long-term Level of expression Safety moderate risk of insertional mutagenesis 51
52 Parent viruses and derived vectors M. Giacca, J Control Release, 2012
53 Application of retroviral/lentiviral vectors in gene therapy 1. Inherited diseases: a) metabolic disorders eg. hypercholesterolemia b) haemophilia c) Hemoglobinopathies (thalasaemia) c) immunodeficiency diseases 2. Transfer of a suicide gene thymidine kinase prevention of graft versus host reaction GvH (przeszczep przeciw gospodarzowi) in patients after allogeneic bone marrow transplantation 3. Transfer of genes to stem cells 53
54 Transduction of bone marrow stromal cells Plasmid-GFP AAV serotype 6 (48h after tramsduction Efficacy~ 5-10 % Efficacy ~ 20% Adenoviral-GFP Retroviral-GFP Efficacy ~ 60% Efficacy 40-60%
55 Transduction of bone marrow stromal cells with lentiviral vectors Efficacy : 97,3%
56 Texts to read! 56
57 Gene Transfer, Clinical Research and Ethics wygłosi Associate Professor Jonathan Kimmelman, PhD z McGill University w Kanadzie. Wykład będzie miał miejsce w dniu 28 października 2014 r., o godz w Auli Nowodworskiego UJ CM, ul. św. Anny 12, Kraków. ZBMho-1
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