Essay Prep

Question 1

Write a description of the medical problem your therapy will address

Answer 1

• Wet Age-Related Macular Degeneration (AMD) is a leading cause of vision loss among people aged 50 and older.
• It primarily affects the central vision, which is critical for essential day to day function
• Wet AMD is a complex multifactorial disease with the exact cause of the disease being unknown.
• However, genetic factors have been identified and smoking has been shown to increase risk.
• Furthermore, high levels of vascular endothelial growth factor (VEGF) have been associated with wet-AMD

Question 2

This disease is characterised by

Answer 2

• This disease is characterized by the abnormal growth of new blood vessels under the retina (choroidal neovascularization).
• These vessels are fragile, leaking blood or fluid into the macula, which damages the photoreceptors and leads to vision loss.

Question 3

Stat

Answer 3

Around 1 in 3 people over the age of 75 are affected by AMD, with the “wet” form being more aggressive compared to “dry” and is responsible for 90% of severe vision loss cases.

Question 4

Describe the interventions currently in use to address that medical problem

Answer 4

• The conventional treatment for wet AMD involves the use of anti-VEGF drugs (e.g., ranibizumab, Aflibercept) that are injected directly into the eye.
• These drugs bind VEGF or compete for its receptor, blocking its activity, thus blocking angiogenesis

Question 5

Limitations of these interventions

Answer 5

Several significant limitations:
- Expense: Anti-VEGF drugs are costly, with Aflibercept being the costliest drug to the Australian government 2019-2020, costing almost $400 million
- Frequent Administration: Due to the degradation of the drug, patients require intravitreal injections every 4-8 weeks, leading to a high treatment burden and discomfort.
- Infection Risk: Repeated injections carry an increased risk of infection or other complications.
- Limited Efficacy: Not all patients respond adequately to treatment, and vision may still deteriorate.

Question 6

How your proposed therapy might overcome those
limitations

Answer 6

• A cutting-edge development in the treatment of wet AMD is gene therapy using the rAAV-sFLT-1 vector.
• This therapy involves the transfection of retinal cells to produce sFLT-1, a soluble form of the VEGFR1 receptor, and a highly potent inhibitor of VEGF.
• Unlike conventional therapies, this gene therapy could provide a continuous, long-term inhibition of VEGF, reducing the need for frequent injections and potentially offering a more cost-effective and sustained treatment approach

Question 7

Hypothesis

Answer 7

Transfection of retinal cells with sFLT-1, using the rAAV vector will significantly reduce retinal VEGF activity, inhibiting choroidal neovascularization and preserving photoreceptor integrity in the Kimba mouse model. This approach will provide a significant, longer lasting improvement in vision compared to current anti-VEGF injections, in those without either treatment

Question 8

Experimental design

Answer 8

• Subjects: Kimba mouse model, which naturally exhibits features similar to human wet AMD, including neovascularization, without the need for artificial induction of AMD.
• Mice will be divided into three groups, each containing 15 mice (statistically significant results, accounting for potential variability): rAAV-sFLT-1 treated, standard anti-VEGF drug treated, and a control group with no treatment
• Gene Therapy Administration: Deliver the rAAV-sFLT-1 vector via a single intravitreal injection. This vector will enable retinal cells to produce sFLT-1 continuously, inhibiting VEGF activity.
• Standard Treatment Group: Administer standard anti-VEGF injections (Aflibercept) every 4 weeks to mirror the conventional treatment regime

Question 9

Outcome measures

Answer 9

• Fluorescein Angiography: Perform at baseline and at several time points (1, 4, and 8 weeks) to monitor changes in blood vessel leakage and neovascularization.
• Electroretinography (ERG): Conduct ERG to assess retinal function by measuring the electrical responses of various cell types in the retina
• Biodistribution Studies: Perform biodistribution analysis to assess the dissemination of the rAAV-sFLT-1 vector in various tissues. This will involve sampling tissues beyond the retina to confirm that the vector remains localized or to evaluate any off-target spread.
• Quality Control (QC): Include repeated measurements and independent verification of sFLT-1 expression using Western blot analysis to ensure consistent gene expression.

Question 10

Ethics

Answer 10

• Experiments will be conducted following Australian ethical guidelines for the humane treatment of animal models.
• Efforts will be made to minimize discomfort, and use of animals will be justified by the potential to develop a more effective treatment for wet AMD.
• Three Rs (Replacement, Reduction and Refinement) will be adhered to)

Question 11

Stats

Answer 11

Use ANOVA to analyze differences across groups, with significance determined at p<0.05. Results will be further validated by performing redundant tests, including RNA analysis for sFLT-1 expression.

Question 12

Potential difficulties / limitations of your experimental approach

Answer 12

• Variable Gene Expression: There might be variability in how much sFLT-1 each cell produces, which could lead to inconsistent treatment outcomes.
• Long-Term Effects Unknown: The long-term safety and efficacy of continuous sFLT-1 expression remain uncertain.
• Animal Model Limitations: Results from Kimba mice might not translate directly to humans due to species differences. Further studies would be needed before clinical trials.

Question 13

Future studies

Answer 13

• Explore Controlled Expression: methods to regulate sFLT-1 expression levels, potentially using inducible gene expression systems to manage treatment effects and minimize risks.
• Larger Animal Models: Before clinical trials, testing in larger, more human-like animal models (such as non-human primates) would help to better assess the translatability of the gene therapy’s effects and safety.
• Long-Term Safety Trials: Conduct extended studies to monitor for delayed adverse effects, to establish the long-term safety profile of continuous VEGF inhibition.

Question 14

General ethical limitations of these kinds of treatments

Answer 14

• Informed Consent
• Justice and Accessibility
• Animal Welfare in Preclinical Testing
• Managing Patient Expectations (Avoiding Overpromising)