YB - Viral Vectors I Flashcards
(23 cards)
What is gene therapy? (1)
Treatment of a genetic disease by introducing function-altering genetic material into a patient using delivery vectors.
What are the requirements for successful gene delivery? (4)
- Safe
- Efficient uptake of DNA into host cells
- Long-term expression
- Sufficient levels of gene expression
Why are viruses preferred for gene delivery? (1)
Because they are highly efficient at introducing genetic material into cells.
What are the main types of viral vectors used in gene therapy? (4)
- Retroviruses
- Adenoviruses
- Herpesviruses
- Adeno-associated viruses
What are some general features of viruses? (6)
- Ancient intracellular parasites
- Cannot replicate on their own
- Efficient at infecting and evading the immune system
- Constantly evolving
- Contain ss/ds RNA or DNA
- Surrounded by a capsid; often with an envelope containing targeting proteins
What are key characteristics of retroviral vectors? (3)
- Most studied viral vector system
- Have a small ssRNA genome (~7–9 kb)
- Genomic size limits large gene inserts
What are important retroviral genome elements? (5)
- LTR (long terminal repeat)
- Ψ (packaging signal)
- GAG (encodes capsid)
- POL (encodes reverse transcriptase)
- ENV (encodes envelope protein)
What are the steps of retroviral infection? (5)
- Entry & uncoating – Virus enters cell and sheds envelope
- Reverse transcription – RNA → DNA via reverse transcriptase
- Integration – Viral DNA integrates into host genome
- Transcription & translation – Host transcribes and translates viral genes
- Assembly & release – New virus particles form and bud from the cell
How are retroviral vectors safely packaged? (5)
- Use a packaging cell line
- Transfect with vector containing Ψ+ signal and therapeutic gene
- Host provirus (Ψ–) provides GAG, POL, ENV proteins
- Proteins assemble around Ψ+ RNA
- Budding releases viral particles with envelope
What disease did Ashanti DeSilva have? (1)
ADA-SCID – Severe Combined Immunodeficiency caused by adenosine deaminase deficiency
What are the steps of the ADA gene therapy process? (8)
- Collect T cells from the patient
- Expand the cells in culture
- Construct retroviral vector with functional ADA gene
- Infect T cells with the vector
- Retroviral DNA integrates into host T cell genome
- Select ADA+ T cells
- Transfuse ADA+ T cells back into the patient
- ADA gene expression restores immune function
What were the results of the ADA-SCID gene therapy? (3)
- ADA gene expression was detected in blood cells
- Reduced need for external ADA protein
- Patient was also treated with purified ADA protein
What causes X-SCID and what are its effects? (3)
- Caused by mutation in IL2RG gene
- Results in absence of T and NK cells
- B cells are nonfunctional
What serious side effect was observed in some X-SCID gene therapy trials? (1)
Development of acute lymphoblastic leukemia
What are advantages of retroviral vectors? (2)
- High gene transfer efficiency
- High gene expression
What are limitations of retroviral vectors? (3)
- Small insert size (~7–7.5 kb)
- Can only infect dividing cells
- Potential toxicity
What are features of adenoviral vectors? (3)
- Large dsDNA genome (~36 kb)
- Efficient infection of epithelial cells
- Used in gene delivery trials
What was the Jesse Gelsinger trial and its outcome? (5)
- Trial for ornithine transcarbamylase deficiency (OTC)
- Jesse had mild OTC and was treated with adenoviral vector
- Suffered a fatal immune reaction
- Pre-existing antibodies worsened the response
- Revealed conflict of interest by lead researcher
What are features of first-generation adenoviral vectors? (2)
- E1 gene deleted to prevent replication
- Allows transgene insertion
What are features of second-generation vectors? (2)
- E1, E2, and E4 genes deleted
- Reduced immune response and longer gene expression
What defines third-generation (gutless) adenoviral vectors? (3)
All viral genes removed except ITRs and packaging signal
Carry up to 36 kb of genetic cargo
Require helper virus for production
What are disadvantages of adenoviral vectors? (3)
- Do not integrate into host genome → temporary expression
- Many people have pre-existing immunity
- Poor for hematological cancers (limited to epithelial targets)
What are advantages of adenoviral vectors? (4)
- High transduction efficiency in both dividing and quiescent cells
- Epichromosomal expression (no genome alteration)
- Broad tissue tropism (e.g., liver, lung, heart, brain)
- Scalable production
