Viral vectors in virology

definition,converting a virus into a vector,main types og viral vectors,principle of retroviral gene transfer,

1. Viral vectors By Abisha

2. V ectors : 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

3. A viral vector is a virus which has been modified in a laboratory environment for purpose of introducing genetic material into a cell. To form a viral vector, remove the genes in the virus that cause disease . 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

4. 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 vector genome The helper DNA can be delivered as a plasmid or helper virus , or it can be stably integrated into the chromatin of the packaging cell .

5. Pathogenicity functions and the sequences that are required for encapsidation are eliminated from the helper construct so that it cannot be packaged into a viral particle. The vector genome contains the transgenic expression cassette and is flanked by inverted terminal repeats and cis acting sequences that are required for genome encapsidation . 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 virus particles

6. 6 Origin of replication Promoter Cloning site What are main features of a vector? Protein purification tags Reporter genes: Targeting sequence Antibiotic resistance Epitope Genetic markers Features

7. 7 Origin of replication Promoter Cloning site for replication and maintenance of vector in host cell. to drive transcription of vector's transgene Also to drive transcription of other genes in vector such as the antibiotic resistance gene allow for the insertion of foreign DNA into the vector through ligation

8. 8 Protein purification tags Some expression vectors include proteins or peptide sequences that allows for easier purification of expressed protein. Reporter genes: Targeting sequence that allow for identification of plasmid that contains inserted DNA sequence . that directs the expressed protein to a specific organelle in the cell or specific location

9. 9 Antibiotic resistance Epitope allow for survival of cells that have taken up the vector in growth media containing antibiotics through antibiotic selection . allows for antibody identification of cells expressing the target protein . Genetic markers allow for confirmation that the vector has integrated with the host genomic DNA .

10. Vectors perform their functions in two ways mostly: Transcription: Expression: Prokaryotes expression vector: Promoter Ribosome Binding Site (RBS) Translation initiation site there are two types of expression vector: Prokaryotes expression vector Eukaryotes expression vector

11. Viruses are highly evolved natural vectors for the transfer of foreign genetic information into cells. But to improve safety, they need to be replication defective. So the viruses can be used as vehicles to carry 'good' genes into a human cell. Eukaryotes expression vectors: These require sequences that encode for: Polyadenylation tail Minimal UTR length Kozak sequenc e

12. 1/4/2017 13 Key properties for a viral vector Safety Low toxicity Stability Cell type specificity Identification

13. 1/4/2017 14 Safety Viral vector should be modified in such a way as to minimize the risk of handling them. Usually involves the deletion of a part of the viral genome critical for viral replication Low toxicity viral vector should have a minimal effect on the physiology of the cell it infects.   Stability Some viruses are genetically unstable & can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of work conducted using a viral vector and is avoided in their design.

14. 1/4/2017 15 Cell type specificity Most viral vectors are engineered to infect as wide a range of cell types as possible. viral receptor can be modified to target the virus to a specific kind of cell. Viruses modified in this manner are said to be pseudo typed. Identification Viral vectors are often given certain genes that help identify which cells took up the viral genes. These genes are called Marker s

15. 1/4/2017 16 What are main types of viral vectors? DNA viral vectors Adenovirus Adeno -Associated virus (AAV) Herpes virus RNA viral vectors Lentivirus Retrovirus

16. Retroviral Vectors Retroviral vectors are commonly used and known to integrate into the genome of the infected cell in a stable and permanent fashion . Reverse transcriptase in the virus allows integration into the host genome. There are two types of retroviral vectors: replication-competent and replication defective . Usually replication-defective vectors are preferred in practice as they allow for several rounds of replication due to their coding regions.

17. 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 to function. Instead, the genome enters the cell DNA via reverse transcription and is incorporated in a random position of the cell genome.

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

19. Adeno -associated viral vectors Similarly to adenoviral vectors, adeno -associated viral (AAV) vectors can deliver genetic material to dividing and non-dividing cells. It is a small virus that is known to affect humans with a very mild immune response. 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 of DNA.

20. Herpes Simplex Virus Vectors This type of viral vector has the ability to deliver large-scale quantities of exogenous DNA. The primary concerns with the use of herpes simplex virus to deliver genetic material are cytotoxicity and the maintenance of transgene expression.

21. 1/4/2017 22 In Gene therapy In vaccines production: Main applications of viral vectors Applications; Cancer- transfer suppressor gene Immunology- Transduce haemotopoietic stemcell (HSC) Stem cell biology -Transfer Human stemcell

22. 1/4/2017 23 In Gene therapy: Gene therapy is a technique for correcting defective genes responsible for disease development . There are following delivery system for gene therapy: Physical methods Non-viral vectors Viral vectors Virus is usually “crippled” to disable its ability to cause disease and viral methods have proved to be the most efficient to date

23. 25 In Gene therapy: 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 gene therapy.

24. In vaccination

25. 1/4/2017 28 In vaccines production: Viruses expressing pathogen proteins are currently being developed as vaccines against these pathogens, based on the same rationale as DNA vaccines . T-lymphocytes recognize cells infected with intracellular parasites based on the foreign proteins produced within the cell. 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 as vaccines .

26. 1/4/2017 29 This vaccine approach involves: inserting influenza virus genes into a different carrier virus, or vector, that is used as a vaccine. DNA or RNA-encoding influenza proteins, such as hemagglutinin , are engineered into a vector that infects humans but does not cause disease.

27. With a microbial vector vaccine , the vector itself, including the influenza genetic material, is injected directly into a person. The harmless vector virus can then express the proteins necessary to prompt an immune response.

28. 1/4/2017 32 Retrovirus Structure: Outer coat: Inner core:

29. 1/4/2017 33 Retroviral mediated gene transfer: Transfection of packaging cell line   Virus Collection Transduction in target cells

30. 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 infectious particles, as well as the nucleic acid sequence of interest that will be packaged within them for delivery.  retroviral vectors produce replication defective, or self-inactivating, particles. This allows for delivery of the desired sequence, without continued viral replication in the target cells.   1.Transfection of packaging cell line

31. 1/4/2017 35 The packaging cells produce infectious particles , whose genome only encodes sequences from the transfer plasmid, which can be used to transduce the target cells. The highest concentration of virus is typically produced between 48-72 hours. 2- Virus Collection   To collect the virus, remove the supernatant from the packaging cell line. The purified virus containing the desired gene can be stored at -80 °C until needed.

32. 1/4/2017 36 3-Transduction in target cells Viral Envelope glycoprotein interacts with the cellular receptor and enters the actively dividing cells. 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 nucleic acid .  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 replication defective. Some transfer vectors also contain a marker such as a fluorescent reporter or drug selection marker for the selection of transduced cells.

33. Insertion of gene of interest Transfection Harvest the supernatant Transduction Integration of GI into the cell DNA Production of protein of interest

34. 1/4/2017 38 Principle of retrovirus vector gene transfer . The structural genes gag, pol and env are deleted from the virus vector which instead carries the therapeutic gene and the psi packaging signal.

35. Therefore, the vector requires complementation of the deleted structural genes for formation of recombinant infectious virus particles , which is mediated by the helper cell . 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 are produced. 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 therapeutic gene.

36. Adenovirus vectors are based on serotypes 2 and 5. Therapeutic genes are placed into the deleted E1 region of the viral genome, driven by internal promoters. The function of E1 for production of viral particles is provided by the complementing cell line expressing E1. Structure of adenoviral vectors and principle of adenovirus production.

37. This cell line produces viralstocks at high titres for infection of desired cells and tissues. 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 viral DNA.

38. 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 the promoter, the transgene and the pA -site. Structure of adeno -associated virus (AAV) vectors

39. Comparison between bacterial & viral mediated gene transfer: Viral mediated DNA insert length is upto 30kbp More transfection effeciency Long term persistence and stable gene transfer Bacterial mediated DNA insert length is upto 10 kbp comparitively less transfection effeciency 1/4/2017 44

40. Thankyou