For





Lifestyles of the Small & Infectious
Terminology of Gene Expression
Viruses to Vectors
Vectors and More Vectors

Very Basic Virology




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.




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.

What is…???


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


Piece of DNA that contains all the elements necessary for a gene to be expressed in a cell.

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



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



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.

Biosafety Considerations







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.






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.

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



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.





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”



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

…and more Vectors








Retrovirus/Lentivirus
Adenovirus
Baculovirus (insect cells)
Poxvirus (vaccinia, fowlpox)
Herpesvirus
Alpha virus (SFV, sindbis, VEE)
Adeno-Associated Virus

BSL 1
Baculovirus
(insect cells)
Adeno-
Associated
Virus
BSL 2
Adenovirus
Poxvirus
(vaccinia, fowlpox)
BSL 2/3
Retrovirus/
Lentivirus
(HIV, SIV, HTLV)
Alphavirus
(semliki forest, sindbis, VEE)
Herpesvirus
(Epstein-Barr,
Herpes viruses)
Flavivirus



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



Amphotropic viruses are capable of infecting human cells, therefore biosafety concern is on effects of the expressed gene
Replication competent retroviral breakthroughs


Complex retrovirus, based on HIV genome


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






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



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.

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


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




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

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





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

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

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







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

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




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






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



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

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





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

Advantages Disadvantages




Accepts large inserts
Broad host range
High titer
Latency in neuronal cells persistence of expression



Virus spread by direct contact
Complex, extensive engineering of vectors
Latency not wellunderstood







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

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








Simian Virus 40
Rhabdovirus
Influenza Virus
Poliovirus
Hepatis B Virus
Epstein Barr Virus
Parvovirus

Chimeric viruses
– Adeno-retroviral
– Adeno-AAV
– Alpha-retroviral
– Alpha-rhabdoviral





A= viral vector system
B= expression construct
Quite often… A + B = A*
But sometimes… A + B = C

Need realistic risk assessment for
– Protection of personnel
– Guidance for containment & work practices




Patrick Condreay, PhD
– GloxoSmithKline Discovery Research
Flint, Enquist, Krug, Racaniello, Skalaka
– Authors: Principles of Virology
Bristol-Myers Squibb Central NJ IBC