Viruses as Therapeutics Flashcards
Three uses of viruses in therapeutics
- Provide Gene Replacement Therapy
- Create recombinant vaccines to promote immunity to infectious agents and tumors
- Act as targeted killers of cancer cells
Gene therapy definition
The transfer of new genetic material to the cells of an individual with resulting benefit to the individual
Germline gene therapy vs. Somatic gene therapy
Germline: modifies gene that may be passed on to subsequent generation
Somatic: genetic modifications restricted to somatic cells, with no effect on the germline
Two general approaches to delivery
- ex vivo: cells removed from patient and exposed to the vector in cell culture
- in vivo: vector introduced directly into patient
Top 3 indications addressed by gene therapy clinical trials
- Cancer diseases
- Monogenic disease
- Infectious diseases
Top 3 gene types transferred in gene therapy clinical trials
- Antigen
- Cytokine
- Tumor supressor
Gene delivery agents
- Viruses
- Chemical - transfection
- DNA loaded vesicles (liposomes)
- DNA-protein complexes
- Physical (microinjection - direct injection of DNA)
X-SCID is due to molecular defects in the gene for the _______ ______ ______
common gamma chain (γc)
Retrovirus vectors for gene delivery
- Replace viral genes with exogenous genes
- Retain LTRs (integration), Ψ sequence (packaging)
- Supply deleted gene products in trans
- Packaging cell lines
- Helper virus, helper plasmids
- Infect cells to introduce exogenous genes
Creation of packaging line
- Start with normal cultured cells
- Transfect plasmid DNAs that will express gag, pol, and env proteins in cells
- Genes encoding gag, pol, and env will integrate into the genome of the packaging cells
- gag, pol and env proteins will be constitutively expressed in the packaging cells
Transfecting vector plasmid with gene of interest into packaging cell line to produce vector particles
- Vector plasmid will integrate into the genome of the packaging cells
- Vector RNA will be transcribed by host-cell RNA polymerase
- Vector RNA will be packaged into vector particles by the viral proteins that are being synthesized in the producer cells
Retrovirus vectors
Advantages:
Disadvantages:
Advantages:
- Efficient delivery of foreign genes
- Control over host range, cell and tissue tropism
Disadvantages:
- Random integration into chromosomes, insertional mutagenesis
- Maintaining high levels of gene expression
- Dependence on target cell proliferation with MMLV (leukemia)-based vectors
Retrovirus LTR contains a stron T-cell specific _______
enhancer
Lentivirus Vectors
Preintegration complex with Vpr, integrase, and matrix protein will cross the nuclear membrane in nondividing cells
Lentivirus Vectors - Safety Concerns
- Multi-plasmid systems for creating packaging cells
- Self-inactivating vectors
- Deletion in U3 region of right LTR
- Control of vector tropism
Adenovirus - Basic Biology
- Linear dsDNA genome
- Replicates in the nucleus - no integration
- Gene transcription regulated with immediate early, early and late classes of mRNAs
Creating adenovirus construct
- Delete certain viral genes to make room for therapeutic gene of interest
- Some deleted viral genes are essential for the replication of adenovirus so they must be provided in trans by the complementing cell line
Deliver of adenovirus vector construct
- Transfect vector DNA construct into the complementing cell line (293 cells) to produce vector particles
- 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 target cell but DOES NOT integrate
- Therapeutic product synthesized in the cells that have been infected
Adenovirus Vectors - Problems
- Short term gene expression - no integration of recombinant DNA into host genome
- Immune response - limits success of repeated use of vectors
- Size restrictions on inserted genes
- **Solutions: **Better packaging cell lines - “gutless vectors”
Vaccinia Virus Expression Vectors - Biology
- Large, dsDNA genome
- Flexibility in the size of DNA that can be packaged
- Cytoplasmic replication
- Virus encoded enzyme
- RNA polymerase
- capping, methylating, polyadenylating
- Viral promoters
- Viral RNA polymerase, not cellular pol II
Basic strategy for foreign gene expression with vaccinia vectors
- Plasmid construction
- Foreign gene + vaccinia promoter
- Chimeric gene → vaccinia genome (homologous recombination)
- Foreign gene → non-essential site in vaccinia genome
- TK (Thymidine kinase) gene is the most popular site
- Selection: TK(-) virus, TK(+) virus-BUdR sensitive
Viruses as Oncolytics
Oncolytic Wild Viruses - some wild-type viruses have natural oncolytic activity in human tumors
Genetically manipulated viruses specifically targeted to infect and kill cancer cells
Conditionally replicative Adenovirus (CRAd)
Modifications in adenovirus that allow tumor-specific tropism:
- Deletion of E1A or E1B genomic regions - viruses will only replicated in cells with specific dysfunctions in cell cycle checkpoint pathways, like some cancer cells
- Incorporation of tumor-specific promoters
- Improvement of transduction efficiency in tumor cells, often accomplished by altering the virus cell attachment protein
Reovirus (wild-type strain)
No modifications by recombinant DNA
- Inherent tumor selectivity for cells with an activated Ras activity
- In normal cells reovirus replication is restricted by the activation of the RNA-activated protein kinase (PKR)
