18 - Viruses as Therapeutics Flashcards
What are beneficial uses of viruses?
- Gene replacement therapy
- Facilitate genetic modification of cells
- Create recombinant vaccines to promot immunity to infectious agents and tumors
- Act as targeted killers of cancer
Define gene therapy?
The transfer of new genetic material to the cells of an individual with resulting benefit to the individual.
What is germline therapy? What is somatic gene therapy?
Germline: modifies gene that may be passed on to subsequent generatons
Somatic: genetic modifications restricted to somatic cells, with no effect on the germline
What are two approahed for gene therapy?
Ex vivo: cells removed from patient and exposed to the vector in cell culture
In vivo: vector introduced directly into the patient
What can serve as gene delivery agents?
Viruses, chemicals, DNA loaded vesicles (liposomes), DNA-protein complexes, or physical: microinjection, direct injection of DNA, “DNA gun”
What are the three most common vectors used in gene therapy clinical trials?
- Adenovirus
- Retrovirus
- Naked/Plasmid DNA
What is X-linked severe combined immunodecifiency (X-SCID)? What occurs if left untreated?
Pts present with no T or NK lymphocytes and non-functional B cells; caused by defects in the gene for the common gamma chain (Yc)
In untreted, X-SCID is fatal in the first years of life due to infection susceptibility.
What is one way that you can use immunotherapy to target a tumor?
- Surgical resection or tumor
- Isolation of tumor-infiltrating leukocytes
- Selection of tumor-reactive T cell clones
- Propagation of tumor reactive clones
- Precondition pt with chemo/radiation and then transfer T cells with IL-2
What is a second way that you can use immunotherapy to target a tumor?
- Collect peripheral blood lymphocytes
- Modify them with viral/nonviral gene transfer (TCR/CAR)
- Propagate the tumor-reactive T cells (TCR+/CAR+)
- Precondition pt with chemo/radiation and then transfer T cells with IL-2
Decsribe how retroviruses can be used as vectors for gene delivery?
- Replicate viral genes with exogenous genes
- Retain LTR (integration) and Psi sequence (packaging)
- Supply deleted gene product in trans: packaging cell lines, heper virus, helper plasmids.
- Infect cells to introduce exogenous genes
What are the steps in the production of a retroviral vector?
- Start with cultured cells and transfect plasmid DNAs that will express gag, pol, and env proteins in the cell
- Gag, pol, and env proteins will be constituitively expressed in the packaging cells.
- Transfect, transfer, or shuttle plasmid with gene of interest into the packaging cell line to produce vector particles.
What are the advantages to using retroviruses as vectors?
Efficient delivery of foreign genes
Control over host range and cell and tissue tropism (pseudotyping, tissue-specific promoters)
What are the disadvantages to using retroviruses as vectors?
“random” integration into chormosomes, insertional mutagenesis
Maintaining high levels of gene expression
Dependence on target cell proliferation with MMLV-based vectors
MLV-LTR contains a strong, T cell specific ______.
Enhancer
What will lentivirus vectors do in nondividing cells?
The preintegration complex with Vpr, integrase, and matrix protein will cross the nuclear membrane in nondividing cells.
What are safety concerns associated with lentivirus vectors?
Multi-plasmid systems for creating packaging cells
Self-inactivating vectors: deletion in U3 region of right LTR
Control of vector tropism
What are the characteristics of adenovirus? Where does it replicate?
Linear dsDNA genome
Replicates in the nucleus, no integration
Gene transcription regulated with immediate early, early, and late classes of mRNAs.
How us adenovirus used as a vector?
Delete certain viral genes to make room for therapeutic gene of interest.
Some viral genes (E1) Are essential for replication of adenoviurs so they must be provided in trans by the complementing cell line.
Vector is transfected into the complementing cell line (293 cells) to produce vector particles.
What do adenovirus vectors do when they get into the cell?
Vector particles are used to deliver the therapeutic gene of interest into the target cells of the patient.
Vector DNA enters the nucleus of the cell but does NOT integrate
Therapeutic product make in the cell that have been infected by adenoviral vectors
What are problems associated with adenovirus vectors?
Short term gene expression - no integration of recomb DNA into host genome
Immune response - limits success of repeated use vectors; E3 region deletions increases immune response
Size restrictions on inserted genes
What are solutions to the adenovirus vector problems?
Better packaging cell lines
Better understanding of the immune resposne to adenovirus
What is the structure of Vaccinia virus expression vectors?
Large dsDNA genome that does cytoplasmic replication.
Virus encoded enzymes: RNA pol, capping, methylating, polyadenylating
Viral promoters: viral RNA pol, not cellular pol II, cellular promotos not recognized by vaccina pol
What are the three basic strategies for foreign gene expression with vaccinia vectors?
- Plasmid contruction with foreign gene + vaccinia promoter
- Chimeric gene into the vaccinia genome via homologous recombination
- Foregin gene into non-essential site in vaccinia genome: Thymidine kinase (TK) gene is the most popular site
What are two other poxviruses? Do they multiply in human cells?
Canarypox
Modified Vaccinia Ankara (MVA): attenuated vaccine of poxvirus
Do not multiply in human cells
What are properties of retrovirus vectors? (high yield)
Stable integration, long-term gene expression
Potential for insertional mutagenesis
Replication incompetent
What are properties of adenovirus vectors? (high yeild)
No integration, short-term gene expression
Vigorous immune response to vector
Replication incompetent
What are properties of poxvirus (vaccinia) vectors? (high yield)
No integration
Replication competent
Live, attenuated vaccines
What does it mean if a virus is an oncolytic wild virus? What are examples?
Some wild-type vises have natural oncolytic activity in human tumors - ie that destroy tumors
Exp: myxovirus, bovine herpesvirus 4, reovirus, coxsackie virus, parvovirus
Besides oncolytic vild viruses, how else can viruses be used to treat cancer? What are examples?
Genetically manipulated viruses specifically targeted to infect and kill cancer cells.
Examples: adenovirus, herpers simplex virus, vaccinia virus, and poliovirus.
What modifications to adenovirus allow tumor-specific tropism?
- Deletion of E1A or E1B regions so viruses will only replicate in cells with specific dysfunctions in cell cycle checkpoint pathways
- Incorporation of tumor-specific promoters
- Improvement of transduction efficiency in tumor cells, often done by altering virus cell attachment protein
Called conditionally replicating adenovirus (CRAd)
What does WT adenovirus need to replicate in a normal cell? What does the mutant have that allows it to grow in cancer cells?
WT adenovirus needs E1B to block p53 in order to replicate in normal cells.
ONYX-05 (with mutant E1B) will not grow in normal cells but will in cancer cells with mutant p53
What is modified on adenovirus to alter cell attachment and allow the virus to be specific for tumor cells?
Fiber protein.
What are characteristics of Reovirus wilf type strain? What is its replication normally restricted by?
No midifications by recombinant DNA
Inherent tumor selectivity for cells with an activated Ras activity
In normal cells, reovirus replication is restricted by the activation of RNA-activated protien kinase (PKR)
How can reovirus be used as a viral vector?
Upregulation of ras pathway blocks PKR action, which is ideal for replication of reovirus.
The virus can then grow in very high quantities and kill tumor ells.
What are characteristics of oncolytic wild-type viruses? (high yield)
Replicate better in some cancer cells than they do in normal cells.
Human infections normally result in either mild or subclinical disease.
What are characteristics of viruses that are genetically menipulated to target cancer cells? (high yield)
Conditionally replicative - grow better in cancer cells than in normal cells
Can use tropism of wild-type virus
Tropism can be altered by changing attachment protein
