Chapter 6 Flashcards
Toxicity
Difficult to separate viral metabolism from cellular metabolism
Toxicity example problem: NRTI
Nucleoside analogs for HIV treatment
NRTIs lack a 3’-hydroxyl group on the deoxyribose moiety.
As a result, following incorporation of an NRTI, the next incoming deoxynucleotide cannot form the next 5’-3’ phosophodiester bond needed to extend the DNA chain
Thus once an NRTI is incorporated, viral DNA synthesis is halted = chain termination
NOTE: nucleoside analogs are preferentially incorporated by viral reverse transcriptase (compared with host cell DNA polymerase)
PROBLEM: host mitochondria encode their own DNA polymerase, which is not as selective as to which nucleoside it incorporates = toxicity
Current antiviral drugs – approaches by life cycle
- Before cell entry
- During viral synthesis
- Assembly
- Release
Block Viral Synthesis
Reverse transcription
Integrase
Genome replication/protein production
Protein production
Nucleic acid based approaches
Oligonucleotides
Ribozymes
RNA interference
Aciclovir
- Effective against HSV and to a lesser extent VZV
- Prodrug
- HSV (dsDNA virus) produces a thymidine kinase
Oligonucleotides
Decoy oligonucleotides
Antigene inhibition
Antisense inhibition
Decoy Oligonucleotides
Copies of viral DNA or RNA regulatory sequences
Compete with viral genome or mRNA for binding -> regulatory protein will bind decoy sequence and be unavailable to bind actual genome or viral mRNA
Antigene Oligonucleotides
- Produce a ssDNA molecule complementary to genomic viral DNA
- Produces a triple helix
- Blocks ability for regulatory proteins to bind, and gene transcription
what is Antisense Inhibition
Design an RNA molecule that is complementary to the viral mRNA or RNA genome sequence
Binds viral mRNA or genomic RNA -> dsRNA
Innate Immune Stimulants
- Potential for broad-spectrum antiviral therapies
- Type I IFN therapy
what is Innate Immune Stimulants used to treat
Hepatitis B In combination with a nucleoside analog drug Hepatitis C In combination with ribavirin SARS during Toronto outbreak
4 classes of HIV antivirals (antiretrovirals)
- Reverse transcriptase inhibitors
- Integrase inhibitors
- Protease inhibitors
- Entry inhibitors
Limitations of Antiviral Drugs (3)
- No broad spectrum antiviral drugs
- Many of the drugs can’t cure the infection.
HIV, herpesviruses, papillomaviruses - Symptoms do not correlate with infection
Beneficial Uses of Viruses
Understanding biological systems
- Gene therapy
- Cancer prevention and control
- Control of harmful or damaging organisms
- Gene Therapy
- Use genes to treat or prevent disease
- DNA encoding the gene must be packaged within a vector, which is used to get the DNA inside the cell
what are the delivery systems
Ex vivo somatic
In vivo somatic
germ line
Ex vivo somatic
Ex vivo = ‘out of the living’, somatic = non-germ cells
Collect somatic cells from a patient, treat them and reintroduce
In vivo somatic
Introducing the DNA/vector to the patient, somatic cells altered within patient
Germ line
Introducing the gene into germ line cells
Allow therapy to be heritable and passed onto later generations
Not currently used for human therapy
Approaches
- Insertion of a functional gene
2. Gene targeting
what does Gene targeting
do?
- Can delete a gene, remove exons, add a gene, introduce point mutations
- Uses homologous recombination or engineered nucleases
what are Gene Therapy Vectors
non viral or viral therapy vectors
Advantages of AAV?
- Low immunogenicity (restricted to generating neutralizing antibodies)
- Lack of pathogenicity Needs a helper virus to produce infectious particles
- Integration of gene into a specific site of host genome (AAVS1)
- Specific to LPD, AAV targets muscle cells, the most important tissue contributing to a healthy LPL protein production
3 virus-based strategies to treat or prevent cancer:
- Vaccines
- Oncolytic viruses
- Viral vectors for therapeutics
Prophylactic (preventive) vaccine
Vaccine to prevent infection of a virus that causes cancer
Ex. hepatitis B vaccine (associated with hepatocellular carcinoma)
Ex. human papillomavirus (associated with cervical cancer)
Both subunit vaccines
Oncolytic Viruses
- Viruses can preferentially infect and kill cancer cells
- Are also immunogenic, recruit immune cells to site of infection/cancer cell killing = enhanced anti-tumor immune response
what are the two strategies
Oncolytic Viruses
Use recombinant DNA technology to engineer tumor selectivity into viruses that are pathogenic to normal cells
Use inherent tumor selectivity of naturally oncolytic viruses
Just beginning to understand how this selectivity works
Viral Vectors for Therapeutics
- Using a cancer cell tropic virus to deliver a therapeutic to cancer cells specifically
- In phase III clinical trials for chemotherapy- resistant metastatic sarcoma, pancreatic cancer, and breast cancer
Ex. Rexin-G
Retrovirus core: Surface proteins that are selective for cancer cells a dominant negative human G1 cyclin Arrests cancer cells in G1 Cells die by apoptosis.
Virus-mediated Pest Control
- Specificity
- Long-lasting effects
Problems with Virus-derived Pest Control
- High specificity
- Slow effects
- Environmental stability
- Viruses don’t like dry, heat
- pesticide companies
Baculoviruses
- Infect insects and other arthropods
- Can be used to treat butterfly and moth larvae and sawfly larvae infestations
- Use of the baculovirus-insect cell expression system to produce recombinant proteins
Baculoviruses vaccines advantages
post-translational modifications closer to humans (vs. yeasts) improved solubility higher protein yield could release VLPs reduced serum sickness development less chance of cross-species infections
Capsid/Subunit vaccines diaadvantages
Expensive (cell medium, purification
Time consuming (clone gene, transfect cells, wait for protein buildup)
Glycosylation sites are different from those made in human cells
Bacteriophage Therapy is
Killing a pathogen with its own pathogen
Viruses infecting bacteria
Very target-specific
Most numerous viruses on the earth
Bacteriophage Therapy life cycles?
Lytic cycle
Lysogenic cycle (latency): phage genome is integrated into the bacterial chromosome. Inserted DNA = prophage
Bacteriophage discovered by?
Frederick Twort by accident
named by Felix d’Herelle
Made filtrates from the patients’ fecal samples and found that the fecal filtrates inhibited growth of Shigella dysenteriae in vitro
“Phage Therapy”
Advantages of Phage Therapy
- Specificity means less side effects
- Using replicating phage means you can use low doses
- Phage replicate only at site of infection
- Phage are unaffected by antiobiotic resistance
- Free of gross toxicity
- Simple and fast to manufacture
- Can adapt to counter bacterial resistance
Disadvantages of Phage Therapy
- Not accessible for intracellular infections
- Need to identify pathogenic bacteria before treatment and ensure it is responsible for disease
- Difficult to administer
- Limited systemic use detected by the host immune system
- Facilitate horizontal gene transfer between bacteria
increased chance of resistance
Phage Therapy
Use bacteriophage to eliminate bacterial infections
Alternative to antibiotic treatments
