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Schematic diagram of a generic viral vector

A vehicle (e.g. a plasmid) used to transfer the genetic material such as DNA sequences from the donor organism to the target cell of the recipient organism. Schematic diagram of a generic viral vector.

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Schematic diagram of a generic viral vector

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  1. A vehicle (e.g. a plasmid) used to transfer the genetic material such as DNA sequences from the donor organism to the target cell of the recipientorganism Schematic diagram ofa generic viralvector

  2. A viral vector is a virus which has been modified in a laboratoryenvironmentfor purposeofintroducing genetic material into acell. • To form a viral vector, remove the genes in the virus that causedisease. • Then replace those genes with genes encoding the desired effect (for instance, insulin production in the case of diabetics).. This procedure must be done in such a way that the genes which allow the virus to insert its genome into its host's genome are left intact

  3. Converting a virus into a vector A packaging (helper) construct, containing viral genes derived from the parental virus that encode structural proteins and proteins that are required for vector genome replication, is introduced into a packaging cell line along with a construct that contains the vectorgenome The helper DNA can be delivered as a plasmid or helper virus, or it can be stably integrated into the chromatin of the packagingcell.

  4. Pathogenicity functions and the sequences that are required for encapsidation are eliminated from the helper construct so that it cannot be packaged into a viralparticle. The vector genome contains the transgenic expression cassette and is flanked by inverted terminal repeats and cis acting sequences that are required for genomeencapsidation. Some vector genomes retain viral genes that are relatively inactive, as a result of the elimination of viral early genes that are required for their transcription. Viral structural proteins and proteins that are required for replication of the vector DNA are expressed from the packaging construct and the replicated vector genomes are packaged into virusparticles

  5. Reporter genes: Origin of replication Antibiotic resistance Targeting sequence Promoter Cloning site Genetic markers Protein purification tags Epitope 6

  6. Origin of replication for replication and maintenanceof vector in hostcell. • to drivetranscription of vector'stransgene • Also to drive transcription of other genes in vector such as the antibiotic resistancegene Promoter • allow for the insertion offoreign DNA into the vector through ligation Cloning site 7

  7. Reporter genes: that allow for identification ofplasmid that contains inserted DNAsequence. Targeting sequence that directs the expressed protein to a specific organelle in the cell or specificlocation Some expression vectors include proteins or peptide sequences that allows for easierpurificationof expressedprotein. Protein purification tags 8

  8. allow for survival of cells that have taken up the vector in growth media containing antibiotics through antibioticselection. Antibiotic resistance allows for antibody identification of cells expressing the targetprotein. Epitope allow for confirmation that the vector has integrated with the host genomicDNA. Genetic markers 9

  9. Vectors perform their functions in two waysmostly: • Transcription: • Expression: • there are two types of expressionvector: • Prokaryotes expressionvector • Eukaryotes expressionvector • Prokaryotes expressionvector: • Promoter • Ribosome Binding Site(RBS) • Translation initiation site

  10. Eukaryotes expressionvectors: • These require sequences that encodefor: • Polyadenylationtail • Minimal UTRlength • Kozak sequence • Viruses are highly evolved natural vectors for the transfer of foreign genetic information intocells. • But to improve safety, they need to be replicationdefective. • So the viruses can be used as vehicles to carry 'good' genes into a humancell.

  11. Key properties for a viralvector Safety Cell type specificity Lowtoxicity Stability Identification 1/7/2017 13

  12. Viral vector should be modified in such a way as to minimize the risk of handlingthem. • Usually involves the deletion of a part of the viral genome critical for viralreplication Safety viral vector should have a minimal effect on the physiology of the cell itinfects. Lowtoxicity • Some viruses are genetically unstable & can rapidly rearrange theirgenomes. • This is detrimental to predictability and reproducibility of work conducted using a viral vector and is avoided in theirdesign. Stability 1/7/2017 14

  13. Most viral vectors are engineered to infect as wide a range of cell types aspossible. • viral receptor can be modified to target the virus to a specific kind of cell. Viruses modified in this manner are said to be pseudotyped. Cell type specificity Viral vectors are often given certain genes that help identify which cells took up the viral genes. These genes are calledMarkers Identification 1/7/2017 15

  14. What are main types of viralvectors? • DNA viralvectors • Adenovirus • Adeno-Associated virus(AAV) • Herpesvirus • RNA viralvectors • Lentivirus • Retrovirus 1/7/2017 16

  15. RetroviralVectors • Retroviral vectors are commonly used and known to integrate into the genome of the infected cell in a stable and permanentfashion. • Reverse transcriptase in the virus allows integration into the host genome. • There are two types of retroviralvectors: • replication-competentand • replicationdefective. • Usually replication-defective vectors are preferred in practice as they allow for several rounds of replication due to their coding regions.

  16. Lentiviral Vectors • Lentiviruses are a type of retrovirus that are able to integrate into non-dividing cells and do not require mitotic cell division in order tofunction. • Instead, the genome enters the cell DNA via reverse transcription and is incorporated in a random position of the cellgenome.

  17. AdenoviralVectors • Adenoviral vectors have a wide range of action and are able to deliver nucleic acids to both dividing and non-dividingcells. • Adenoviruses are often responsible for respiratory, gastrointestinal and eye infection that affecthumans. • As a result, research is currently being conducted to investigate the use of adenoviral vectors in applications of gene therapy and vaccination.

  18. Adeno-associated viralvectors • Similarly to adenoviral vectors, adeno-associated viral (AAV) vectors can deliver genetic material to dividing and non- dividingcells. • It is a small virus that is known to affect humans with a very mild immuneresponse. • As a result AAV vectors have beneficial properties for gene therapy that are effective with limited negative effects. However, the utility of this type of vector is significantly limited by its restricted capacity ofDNA.

  19. Herpes Simplex VirusVectors • This type of viral vector has the ability todeliver • large-scale quantities of exogenousDNA. • The primary concerns with the use of herpes simplex virus to deliver genetic material are cytotoxicity and the maintenance of transgene expression.

  20. Applications; • Cancer- transfer suppressorgene • Immunology-Transduce haemotopoieticstemcell(HSC) • Stem cell biology -Transfer Humanstemcell • Main applications of viralvectors • In Genetherapy • In vaccines production: 1/7/2017 22

  21. In Genetherapy: • Gene therapy is a technique for correcting defective genes responsible for disease development. • There are following delivery system for genetherapy: • Physical methods • Non-viralvectors • Viralvectors • Virus is usually “crippled” to disable its ability to cause disease and viral methods have proved to be the most efficient todate 1/7/2017 23

  22. In Genetherapy: • If the pathogenicity of a specific virus, such as adenovirus, can be eliminated while the efficiency of gene transfer and expression is retained, the gene may be well suited for genetherapy. 5 2

  23. In vaccinesproduction: • Viruses expressing pathogen proteins are currently being developed as vaccines against these pathogens, based on the same rationale as DNAvaccines. • T-lymphocytesrecognizecellsinfectedwithintracellular parasites • based on the foreign proteins produced within thecell. • A viral vaccine induces expression of pathogen proteins within host cells. Since viral vaccines contain only a small fraction of pathogen genes, they are much safer and sporadic infection by the pathogen is impossible. • Adenoviruses are being actively developed asvaccines. 28

  24. This vaccine approachinvolves: • inserting influenza virus genes into a different carrier virus, or vector, that is used as avaccine. • DNA or RNA-encoding influenza proteins, such as hemagglutinin,are • engineered into a vector that infects humans but does not causedisease. 1 29 /7/2017

  25. With a microbial vector vaccine, the vector itself, including the influenza genetic material, is injected directly into aperson. • The harmless vector virus can then express the proteins necessaryto • prompt an immuneresponse.

  26. RetrovirusStructure: • Outercoat: • Innercore: 1/7/2017 32

  27. Retroviral mediated gene transfer: • Transfectionof • packaging cell line • VirusCollection • Transductionin • targetcells 33

  28. 1.Transfection of packaging cellline • The production of replication defective virus is accomplished when packaging cells are transfected with plasmids that express all of the viral proteins necessary to generate infectiousparticles, • as well as the nucleic acid sequence of interest that will be packaged within them fordelivery. • retroviral vectors producereplication • defective, or self-inactivating,particles. • This allows for delivery of the desired sequence, without continued viral replication in the targetcells.

  29. 2-VirusCollection • The packaging cells produce infectious particles, whose genome only encodes sequences from the transfer plasmid, which can be used to transduce the targetcells. • The highest concentration of virus is typically produced between 48-72 hours. • To collect the virus, remove the supernatant from the packaging cellline. • The purified virus containing the desired gene can be stored at -80 °C until needed. 1/7/2017 35

  30. 3-Transduction in targetcells • Viral Envelope glycoprotein interacts with the cellular receptor and enters the actively dividingcells. • The viral dsDNA of retroviruses is not capable of passing through the nuclear pore complex and requires breakdown of the nuclear membrane during mitosis for integration of nucleicacid. • The viral genome enters the nucleus and desired gene sequence is integrated into the host genome through the activity of restriction enzymes and Integrase while the other portion of viral genome is self inactivated and replicationdefective. • Some transfer vectors also contain a marker such as a fluorescent reporter or drug selection marker for the selection of transduced cells. 36

  31. Principle of retrovirus vector genetransfer . The structural genes gag, pol and env are deleted fromthe virus vector which instead carries the therapeutic gene and the psi packagingsignal. 38 1/7/2017

  32. Therefore, the vector requires complementation of the deleted structural genes for formation of recombinant infectious virus particles, which is mediated by the helpercell. These cells harbour the‘wild- type’retrovirus lacking the psi packaging signal. The retroviral vector is transduced into the helper cell where recombinant virus particles areproduced. These particles can be used to infect the target cells via specific receptors to start reverse transcription for random integration of the proviral DNA into the host genome where the vector starts expression of the therapeuticgene.

  33. Structure of adenoviral vectors and principle of adenovirus production. Adenovirus vectors are based on serotypes 2 and5. Therapeutic genes are placed into the deleted E1 region of the viral genome, driven by internalpromoters. The function of E1 for production of viral particles is provided by the complementing cell line expressing E1.

  34. This cell line produces viralstocks at high titres for infection of desired cells andtissues. After infection of target cells,viral particles enter the cytoplasmic endosome and deliver the viral DNA harbouring the therapeutic gene. Gene expression is performed from the epichromosomal viralDNA.

  35. Structure of adeno-associated virus (AAV)vectors The wild-type AAV consists of the viral genes rep and cap coding for the different rep (Rep78, Rep68, Rep52, Rep42) and cap (VP1, VP2, VP3) proteins, the AAV promoters (p5, p19, p40), the polyadenylation site (pA) and the inverted terminal repeats(ITR). In rAAV vectors, the viral rep and cap genes are replaced by a transgene cassette carrying thepromoter,the transgene andthe pA-site.

  36. Comparison between bacterial & viral mediated genetransfer: • Viralmediated • DNA insert length isupto 30kbp • Moretransfection effeciency • Long term persistenceand stable genetransfer • Bacterial mediated • DNA insert length is upto10 kbp • comparitively less transfectioneffeciency 1/7/2017 44

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