YB - Viral Vectors II

Question 1

What are the steps involved in gene therapy? (6)

Answer 1

• Identify the affected gene (via sequencing and mutation analysis)
• Clone the healthy version of the gene
• Load the gene into a suitable vector
• Deliver the vector to target cells
• Transport therapeutic gene into the nucleus
• Integrate the gene into host DNA and ensure it functions properly

Question 2

What are key characteristics of HSV-1? (7)

Answer 2

• Causes cold sores; infects epithelial cells (lytic phase)
• DNA is enclosed in a capsid, surrounded by a tegument, and enclosed in a lipid envelope.
• Does not integrate into host genome
• Can remain latent in neurons for years
• Reactivated by stress
• Affects ~67% globally; neurotropic enveloped virus
• Large dsDNA genome (~150 kb), encodes ~80 genes for structure & replication

Question 3

What are the steps of HSV-1 propagation? (6)

Answer 3

Attachment & Entry

• Virus binds to host membrane receptors and fuses its envelope with the membrane, releasing the nucleocapsid into the cytoplasm.

Uncoating & DNA Entry

• The capsid is uncoated, and viral DNA enters the nucleus.

Genome Replication

• Inside the nucleus, viral DNA undergoes replication, producing multiple DNA copies.

Transcription & Translation

• Viral DNA is transcribed into mRNA, which is translated in the cytoplasm into capsid and spike proteins.

Nucleocapsid Assembly

• Capsid proteins return to the nucleus, combine with replicated DNA to form new nucleocapsids.

Budding & Release

• Nucleocapsids bud through the inner nuclear membrane, acquire envelopes in the Golgi, and exit the cell via exocytosis.

Question 4

Why is HSV-1 useful in gene therapy? (4)

Answer 4

• High capacity for large transgenes (150 kb)
• Can infect neurons and persist lifelong
• Latency-active promoters (LAP1/LAP2) drive long-term expression
• Can be engineered to become non-toxic and oncolytic

Question 5

Q5: What are some challenges of using HSV vectors? (4)

Answer 5

• Complex production process and difficulty in scaling.
• Requires retention of essential viral genes for functionality.
• Highly immunogenic, raising safety and tolerance concerns.
• Natural neurotropism may limit targeting to other tissues.

Question 6

Q6: What are common modifications to HSV for therapy? (3)

Answer 6

• Make virus replication-defective or restricted
• Re-targeting the virus to new cells - Altering surface proteins
• Insert therapeutic transgenes

Question 7

Q7: What were the findings of the NP2 Phase 1 trial for HSV-1 gene therapy? (5)

Answer 7

• Used modified HSV-1 (nrHSV-1) carrying PENK
• Delivered PENK gene to patients with chronic pain
• Virus reached dorsal root ganglia and caused PENK expression
• No serious adverse events
• Pain relief in middle/high dose groups

Question 8

Q8: What are HSV amplicon vectors? (5)

Answer 8

• Engineered plasmids with a transgene, an origin of replication (ori), and a packaging signal (pac).
• Introduced into packaging cells with a helper virus providing replication machinery.
• Undergo rolling circle replication, creating concatemeric DNA.
• The pac signal guides DNA cutting and packaging into viral particles.
• Final amplicon contains multiple copies of the transgene.
• Lacks viral coding genes → low toxicity.

Question 9

Q9: What are the pros and cons of HSV amplicon vectors? (5/2)

Answer 9

Pros

• Large transgene capacity (\~150 kb)
• High transgene copy number
• Broad cell tropism
• Simple vector construction
• Limited toxicity

Cons:

• Poor DNA stability
• Inadequate for long-term or continuous expression

Question 10

Q10: What are replication-defective HSV vectors? (2)

Answer 10

• Contain deletions in essential replication genes.
• Can only replicate in engineered cell lines providing the missing genes.
• Risk of recombination with wild-type HSV in the patient.

Question 11

Q11: What are attenuated HSV vectors? (2)

Answer 11

• Lack non-essential genes (deletions)
• Can replicate in vitro, but are restricted in vivo.
• Used as vaccines, oncolytic viruses, or gene delivery tools

Question 12

Q12: What makes HSV useful for vaccination? (4)

Answer 12

• Induce strong, long-lasting immune responses.
• HSV DNA remains episomal, avoiding genome integration.
• Retain tk gene, allowing selective killing with aciclovir.
• Used in development of multiple vaccine types.

Question 13

Q13: How does aciclovir act as a kill switch in HSV-based therapies? (4)

Answer 13

• Aciclovir is phosphorylated by HSV-TK enzyme, not by human kinases.
• Phosphorylated aciclovir accumulates in HSV-infected cells.
• Inhibits DNA replication, leading to cell death.
• Acts as a safety mechanism for unwanted viral activity.

Question 14

Q14: Why is HSV-1 effective for nervous system gene therapy? (2)

Answer 14

• Has neuroinvasiveness and neurovirulence genes
• Can spread retrograde and anterograde, crossing synapses
• Can spread trans-synaptically, enabling widespread neuronal gene delivery.

Question 15

Q15: What are mechanisms of HSV oncolytic virus activity? (3)

Answer 15

• Direct lysis or apoptosis of infected cancer cells through viral replication.
• Indirect killing of uninfected neighboring cells via immune signaling and apoptosis. Prevents spread
• Stimulation of anti-tumor immunity, activating APCs and T cells.

Question 16

Q16: What are the stages of immune activation by oncolytic HSV in cancer therapy? (6)

Answer 16

1. Oncolytic virus infects and replicates in cancer cells.
2. Infected cells undergo lysis, releasing new virions and tumor antigens.
3. APCs (antigen-presenting cells) capture tumor antigens.
4. APCs activate T cells specific to tumor antigens.
5. T cells infiltrate tumors and kill more cancer cells.
6. The cycle continues, enhancing systemic immune response.

Question 17

Q17: How does T-Vec treat melanoma? (4)

Answer 17

• Selective replication in tumor
• Lysis of tumor cells, releasing antigens.
• Immune activation against tumor antigens
• Systemic immune response kills distant cancer cells

Question 18

Q18: What was ONYX-015 and how did it perform in trials? (4)

Answer 18

• A modified adenovirus designed to replicate in p53-deficient tumors.
• Used in a Phase III trial with chemotherapy for head and neck cancer.
• Administered via intratumoral injection.
• Combination therapy was well tolerated but did not improve survival.

Question 19

Q19: What therapeutic transgenes are used in HSV-based cancer therapies? (3)

Answer 19

• IL-12 → stimulate T cell & NK response
• Angiostatin → blocks tumor blood vessels (angiogenesis)
• TRAIL → induces cancer cell apoptosis

Question 20

Q20: What are key features and benefits of AAV as a gene therapy vector? (5)

Answer 20

• Single-stranded DNA virus, \~4.7 kb genome.
• Non-pathogenic and well tolerated in humans.
• Can infect dividing and non-dividing cells.
• Provides long-term gene expression.
• Integrates into a specific site on chromosome 19 (AAVS1).
• Easy to engineer

Question 21

Q21: What are limitations of AAV vectors? (2)

Answer 21

• Small genome capacity (\~4.7 kb) limits capacity for large or multiple genes.
• Many people have pre-existing antibodies which can neutralize the vector.

Question 22

Q22: How does AAV deliver a transgene into host cells? (6)

Answer 22

• AAV binds to cell surface receptors.
• Enters via endocytosis, forming an endosome.
• Escapes from the endosome into the cytoplasm.
• Uncoating releases viral ssDNA.
• ssDNA is converted into dsDNA in the nucleus.
• The transgene is transcribed and translated into protein.

Question 23

Q23: What immune barriers hinder AAV gene therapy? (4)

Answer 23

• Pre-existing neutralizing antibodies (NAbs) block infection.
• Innate immunity activation via TLR2 and TLR9 causes inflammation.
• CD8+ T cells target AAV capsid fragments on MHC I → kill transduced cells.
• Antibodies to transgene product may block therapeutic protein function.

Question 24

Q24: What are 3 genetic targets in AAV-based cardiovascular therapy? (3)

Answer 24

• PCSK9 – inhibits LDLR recycling; targeting it reduces LDL cholesterol.
• ANGPTL3 – inhibits lipoprotein lipase; suppression lowers lipids and CVD risk.
• ApoC3 – negatively regulates triglyceride metabolism; loss reduces CVD risk.

Question 25

**Q25: What are key design factors for AAV-based gene therapy in tumors? (4)**

Answer 25

* **Terminal repeats** must remain intact. * Requires specific **capsid proteins** for tissue targeting. * Must include an appropriate **packaging signal**. * **Promoters** must be either **ubiquitous** or **tissue-specific**, and genes often require mini formats due to size limits.

Question 26

**Q26: What are the main obstacles in large-scale AAV production? (3)**

Answer 26

* **Upstream (cell culture)** – suspension cells are scalable but hard to transfect. * **Downstream (purification)** – difficult to separate full from empty capsids. * **Chromatography** – may damage vectors or reduce yield; extreme pH can be harmful.

Question 27

**Q27: What are 3 advanced methods for improving AAV production? (3)**

Answer 27

* **TESSA system** – doxycycline-controlled helper adenovirus; 30x yield, no contamination. * **Synthetic biology in yeast** – being explored, but suffers low yield/potency. * **dbDNA system** – uses rolling circle replication to produce large quantities of closed circular DNA.

Question 28

**Q28: What are other viruses explored for gene therapy? (3)** **A:**

Answer 28

* **JX-594 (vaccinia virus)** – showed 75% 1-year survival in liver cancer vs 18% control. * **PVSRIPO** – hybrid poliovirus/rhinovirus for glioma; non-neurotoxic. * **Seneca Valley Virus (SVV-001)** – natural RNA virus, promising for glioblastoma & medulloblastoma.