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BioSafety Considerations

BioSafety Considerations. For Viral Vectors. Biosafety Considerations of Viral Vectors. Lifestyles of the Small & Infectious Terminology of Gene Expression Viruses to Vectors Vectors and More Vectors. Lifestyles of the Small and Infectious. Very Basic Virology. What Are Viruses?.

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BioSafety Considerations

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  1. BioSafety Considerations For Viral Vectors

  2. Biosafety Considerations of Viral Vectors • Lifestyles of the Small & Infectious • Terminology of Gene Expression • Viruses to Vectors • Vectors and More Vectors

  3. Lifestyles of the Small and Infectious Very Basic Virology

  4. What Are Viruses? • Small, infectious obgligate intracellular parasites whose genome is either DNA or RNA. • The viral genome is replicated within a host cell and uses cellular systems to direct the synthesis of other virion components. • These newly synthesized components are used to assemble progeny virions which are responsible for transmission of the viral genome to the next host cell.

  5. Common Viral Strategies • All viruses package their genome inside a particle that mediates transmission of the viral genome from host to host. • The viral genome contains the information for initiating and completing an infectious cycle. • All viruses are able to establish themselves in a host population.

  6. Terminology of Gene Expression What is…???

  7. cDNA : a DNA copy of an mRNA • Only contains the protein coding domain (not introns) • When transcribed the RNA requires no processing • Can be translated directly • Transgene • A gene that is put into some sort of expression vector to be delivered into a cell

  8. Expression Cassette • Piece of DNA that contains all the elements necessary for a gene to be expressed in a cell.

  9. Elements of an Expression Vector Termination signals are tailored to prokaryotic or eukaryotic systems: eukaryotic cells require polyA signal. Promoters are tailored to type of cell system: bacteria, mammalian, insect, etc.

  10. Cis&Trans Acting Elements • Cis – present on the same piece of DNA/RNA being acted on; cannot function separately. • Promoters, signaling sites • Trans – elements that act on a different piece of DNA/RNA than the one they are present on. • Viral replication proteins, RNA polymerase

  11. Complementing Cell Lines • Complementation: Providing trans-acting functions to rescue a nucleic acid that is missing, or mutant in, those functions. • By stably transfecting cells to express a gene product necessary for viral replication, can then grow viruses deleted for that gene. • HEK293 cells express Ad E1; will complement E1 deleted adenoviruses.

  12. Viruses to Vectors Biosafety Considerations

  13. Why Use Viruses? • Viruses have evolved to efficiently condense, package, & deliver nucleic acids to cells. • Relatively easy to generate & renew. • Infect a wide variety of cell types. • Production of proteins w/ authentic post translational modifications. • Potential for regulated production. • Potential for in vivo gene delivery.

  14. Concerns About Recombinant Viruses • Pathogenicity of parental virus. • Can engineer to be repl. incompetant • Cytopathogenicity of vector. • eg. Spike proteins on Adenovirus • Requirements for specialized facilities. • Scale-up considerations. • Training requirements.

  15. Replication Incompetent Viruses • To avoid potential pathogenicity of viral vectors – disable them so they cannot replicate in target cells. • Usually accomplished by deleting genes that provide necessary trans-acting functions from the vector genome. • Introduction of genes & defective vector into cells results in synthesis of vector genomes & packaging of the defective genomes into virus particles.

  16. Reconstitution of Replication Competent Virus (RCV) • During amplification & packaging of defective viral vector genomes they can, at low frequency, re-acquire the genes necessary for autonomous growth, making the virus replication competent again. • Occurs by recombination.

  17. Strategies to Avoid RCV Reconstitution • Split genomes: putting replication genes on different DNA constructs • Remove viral regulatory regions • Produce as a transient single batch rather than continuous culture • Use non-host cell lines • reduces “rescue”

  18. Why Do Split Genomes Decrease RCV Risk?

  19. Pseudotyping of Viruses • The use of different viral surface proteins to affect the host range of the virus and/or to change physical properties of the viral particles. • Some viruses are surrounded by cell-derived membranes & foreign surface proteins can be included in these envelopes. • G glycoprotein of vesticular stomatitis virus • Eliminates ability to regenerate original host range

  20. Vectors …and more Vectors

  21. Typical Viruses Used • Retrovirus/Lentivirus • Adenovirus • Baculovirus (insect cells) • Poxvirus (vaccinia, fowlpox) • Herpesvirus • Alpha virus (SFV, sindbis, VEE) • Adeno-Associated Virus

  22. Common Viral Vectors

  23. Retroviruses • Based on murine leukemia virus • Risk Group 1 and 2 • Simple genomic structure: all the retroviral genes can be removed & supplied extraneously • Three genes: gag, pol, env

  24. Biosafety Concerns • Amphotropic viruses are capable of infecting human cells, therefore biosafety concern is on effects of the expressed gene • Replication competent retroviral breakthroughs

  25. Lentiviruses • Complex retrovirus, based on HIV genome

  26. Lentivirus • Capable of infecting non-dividing cells • Provirus DNA must enter nucleus for integration of DNA to occur • Simple retroviruses rely on dissolution of nuclear membrane during mitosis • Lentiviruses encode nuclear localization signals which transport complex into nucleus

  27. Biosafety Concerns for Lentivirus • Generation of replication competent virus • Infection of non-target cells • Inappropriate expression of gene product in non-intended cell type • Insertional mutagenesis • “Rescue” by other human pathogenic viruses

  28. Insertional Mutagenegis • Lentivirus integrates into the host chromosome at random. • Possible to integrate in area such that downstream LTR could function as a promoter for growth regulation.

  29. “Rescue” • Host genome may contain endogenous retrovirus • Recombination event could lead to reactivation or “rescue” of replication competent virus

  30. How Is Safety Engineered Into Lentiviral Vectors? • Lentivirus- 3rd generation Lentivirus System. Significantly modified for biosafety. • Packaging vector lacks both LTRs and expresses only gag and pol. • Rev is supplied in trans on a separate vector. • The vector expressing the packaged viral genome has a self-inactivating LTR and expresses no viral gene products • The envelope protein is VSV-G and is also expressed on a separate vector • Packaged virus expresses no viral gene products

  31. Adenoviruses • Risk Group 2 • Deletions of the E1 region render the virus replication incompetent; can be propagated in a complementing cell line (293 cells) • Airborne Transmitted; infects broad range of cells

  32. Advantages High titers Infects wide ranges of cells Infects dividing and quiescent cells Virion stability Disadvantages Have had some adverse events Transient expression Problems w/ subsequent administration in gene therapy Based on human pathogen RCA breakthroughs Adenovirus Vector

  33. Baculovirus • Risk group 1 agent • Enveloped virus w. double stranded DNA genome • Insect pathogen that does not propagate in mammalian cells • Used for many years to express proteins; will also deliver genes and mediate expression in mammalian cells

  34. Baculoviruses as Gene Delivery Vehicles for Mammalian Cells • Virus will deliver its DNA into mammalian cells • Viral promoters are not activated in mammalian cells but if expression cassette is included with a mammalian promoter, it will be expressed • No overt deleterious effects on mammalian cells have been shown

  35. Advantages Ease of Use Broad host range Efficiency & lack of toxicity Non-replicative in mammalian cells Inactivated by human complement Limitations Relatively uncharacterized Potential for maintenance of viral DNA No evidence of in vivo delivery Potential expression of viral gene products Baculovirus Vectors

  36. Vaccinia Virus • Risk Group 2 • Enveloped virus w/ double stranded DNA genome • Extensive use as recombinant protein expression vector • Wide host range • Clone large DNA fragments (> 20 Kb) • Used as a live vaccine against smallpox

  37. Advantages Broad host range Easy to generate viruses Accepts large inserts High expression level Molecular virology well understood Limitations Lytic infections Readily transmisible agent Vaccination requirement Scale-up considerations Vaccina Virus Vector

  38. Avipox Vectors • Poxviral vectors are replication competent • Poxviruses that infect avian species (fowlpox and canarypox) are replication defective in mammalian cells • Vectors based on these viruses might provide a safer alternative to vaccinia based vectors for in vivo use

  39. Alpha Viruses • Risk Group 2 and 3 agents • Enveloped virus w/ single stranded RNA genome • Arthropod-borne viruses; replicate in cells from vertebrates & invertebrates • Consequence of human alphavirus infection can be mild or significant disease. • Main in vivo application is to expression of antigen to elicit an immune response

  40. Recombinant Alphaviruses • Vector systems have been developed from three alpha viruses: • Sindbis virus (SIN, risk group 2); • Semliki Forest Virus (SFV, risk group 2/3); • Venezuelan Equine Encephalitis (VEE, risk group 3) • SIN and SFV systems are commercially available

  41. Advantages High expression level Broad host range High titers Ease of use Limitations Cytopathic Replication competent virus Based on human pathogen Large Scale production Alphaviral Vectors

  42. Risk Group 2 HSV1 & HSV2: Enveloped virus w/ double stranded DNA genome Replicates in the nucleus Dual life cycle: lytic growth in epithelial cells; latent infection in neuronal cells Herpes Virus

  43. Advantages Accepts large inserts Broad host range High titer Latency in neuronal cells persistence of expression Disadvantages Virus spread by direct contact Complex, extensive engineering of vectors Latency not well-understood Herpesviral Vectors

  44. Adeno-Associated Virus (AAV) • Risk group 1 agent • Human parvovirus-not associated with any disease • Majority of population is seropositive • Requires a helper virus (adeno or herpes coinfection for replication) • AAV integrates into the host cell chromosome (19) • Lack of initiation of immune response

  45. Advantages Infects multiple cell types No viral genes in vector Long term gene expression (persistence of genome) No immune response Limitations Limited insert size Helper virus contamination Large Scale production Genome persistence not understood AVV Vectors

  46. Simian Virus 40 Rhabdovirus Influenza Virus Poliovirus Hepatis B Virus Epstein Barr Virus Parvovirus Chimeric viruses Adeno-retroviral Adeno-AAV Alpha-retroviral Alpha-rhabdoviral Emerging Vectors

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