AAV Based Combination Therapies to Improve Glioblastoma Treatment Efficacy

Research Question: Do AAV-based combination therapies improve glioblastoma treatment efficacy?

Background: Historically, brain cancers have been treated with either chemotherapy, surgery, radiation therapy, or a combination of these. While these methods can be effective for some cases of brain cancer, for glioblastoma, a highly aggressive form with a 5-year survival rate of less than 10%, these standard treatments grant patients a median survival time of only 14.6 months. This highlights the need for more targeted and effective treatments. Adeno-associated virus (AAV) gene therapy enables targeted modification of tumour cells. It allows for the delivery of therapeutic genes directly into cancer cells, offering a more precise approach compared to standard treatments. Recent studies suggest that AAV-based therapies may improve outcomes, particularly when combined with complementary strategies such as immunotherapy and chemotherapy. There have been very few studies conducted on this topic. In fact, over the last five years, there have been only 24 studies aimed at exploring the use of AAV in combination with chemotherapy to treat brain cancers, as shown by the Venn diagram above. This study argues that AAV-mediated gene delivery, when used in combination with immunotherapy or chemotherapy, represents a mechanistically versatile platform capable of overcoming key limitations of glioblastoma treatment, including immune evasion, therapeutic resistance, and tumour heterogeneity.

Hypothesis: Through this study, I predict that my findings will demonstrate that AAV-based gene therapy, when combined with chemotherapy or immunotherapy is more effective in treating glioblastoma than existing approaches, such as chemotherapy alone, radiation therapy, or surgery.

Methods:

A PRISMA flowchart was used to keep track of studies evaluated and selected.

An initial pool of 40 studies was identified, of which 5 met the inclusion criteria following abstract and full-text screening.

Studies were included if they were primary research articles involving AAV-based gene delivery in glioblastoma models (in vitro or in vivo) and reported outcomes such as survival, tumour regression, or immune response.

Review articles and studies not involving brain tumours were excluded. Following title and abstract screening, the most relevant studies were selected for evaluation.

Webplotdigitizer was used to extract numerical data from the figures analyzed. The data was then converted into multiple graphs and charts through Microsoft Excel. The created graphs were then analyzed and compared to determine the relative findings of the study.

A classification approach was used to group therapies and identify patterns across studies.

Two studies primarily focused on increasing immune response.

Two studies focused on driving suicide gene expression and inducing tumour cell apoptosis.

Lastly, one study discussed the novel approach of differentiating tumour cells into neuron cells.

This was done to showcase three separate approaches to combatting glioblastoma using AAV gene therapy.

The data collected across studies was then compared based on survival percentages, tumour area reduction, and reductions in tumour cell viability.

The first image shows the PRISMA flowchart that outlines the selection process of the papers chosen for this study. The following figures were adapted from primary studies to illustrate key therapeutic strategies and to compare outcomes.

Results:

Across the studies analyzed, AAV-based therapies demonstrated enhanced effectiveness when combined with monotherapies such as chemotherapy or immunotherapy.

In von Roemeling et al., the combination of AAV6-CXCL9 with anti-PD-1 immune checkpoint blockade resulted in approximately 50% survival in the GL261 model and 25% survival in the KR158 model over a period of 90 days.

In GuhaSarkar et al., through using AAV-IFN-β, the most effective treatment condition maintained approximately 60% survival at around day 50.

Hingtgen et al. demonstrated that the combination of AAV-S-TRAIL with temozolomide (TMZ) reduced tumour cell viability by approximately 65–70%, based on bar graph comparisons of treated versus monotherapy conditions.

King et al. reported a very strong survival outcome, with approximately 83% survival at day 60 in the group treated with AdTK and AdFlt3L.

Jiang et al. further showed that AAV-mediated delivery of NeuroD1 (ND1) resulted in approximately a 65% reduction in tumour size compared to control conditions, based on relative bar heights in tumour area measurements.

Combination therapies showed consistently high survival outcomes (up to ~80%). Combination therapies also displayed significant reduction of both tumour size, and cell viability (up to ~65-70%).

The graphs created above demonstrate the increased effectiveness of treatment options when AAV mediated gene therapy was used, in comparison to monotherapies.

These findings support a shift from single-strategy treatments toward combinatorial therapies.

This analysis demonstrates that AAV-mediated therapies show strong potential for improving glioblastoma treatment outcomes, particularly when used in combination with complementary therapies. It shows that using combination therapies outperform monotherapies and results in increased treatment efficacy. The specific treatment options discussed in this study vary while still showing similarly promising results. These recent pre-clinical studies demonstrate how AAV can be strategically used to sensitize cancer cells to chemotherapy, drive suicide gene expression, deliver cytotoxic chemicals, and drive differentiation of cancer cells, all of which were shown to consistently outperform monotherapies. Current treatments that mainly consist of monotherapies, unfortunately, fall short of the threshold needed to provide humans with survival benefits that outperform brain cancer when untreated, consequently, the future of brain cancer treatment should be more heavily focused on exploring the potential of combination therapies using AAV.

Data extracted using WebPlotDigitizer and analyzed in Microsoft Excel further supported these findings, revealing that combination therapies consistently produced higher survival rates (up to ~80%) and greater reductions in tumour size and cell viability (up to ~65–70%) compared to monotherapies.

Overall, the results demonstrate that AAV based therapies, particularly when combined with monotherapies, significantly improve therapeutic outcomes in pre-clinical glioblastoma models.

Conclusions: The possibility of using AAV as a vessel for gene delivery will hopefully result in new discoveries and developments in the area of glioblastoma and brain cancer research in general as it is an extremely under researched topic. The addition of combining the gene therapy treatment with chemotherapy adds another factor of novelty as there are only 24 papers, consisting mostly of review articles, on this topic as previously mentioned. Throughout history, treatments have only consisted of monotherapies. Radiotherapy, chemotherapy, and neurosurgical removal, are all examples of these monotherapies that are the usual form of treatment for patients with brain cancer. While these treatments are not ineffective, they have mixed survival rates; for example, the median survival of glioblastoma patients after radiotherapy is 12.1 months, and has a two year survival rate of 10.4%. Therefore, using monotherapies in combination with AAV gene therapy is a newly developed idea that increases the efficacy of these treatments, especially when combined with chemotherapy, and should be further researched.

Future Research: There are shared shortcomings across each of the papers discussed, although they hold promise for future research. While short-term results were promising, long-term in vivo models will need to be conducted to ensure effectiveness and safety in humans. Most treatment regimens were tested in vitro and in animal models, raising questions about whether these treatments are truly translational and will achieve the same response in humans.

Future Areas of Study:

Investigating the feasibility of clinical trials for this treatment in particular, as well as researching other clinical trials of AAV based treatments in general.

Incorporating a larger number of studies that focus on AAV based therapies for glioblastoma to compare the effects to the studies used in this project.

Further research to explore long-term treatment outcomes. Most studies analyzed focused on short-term survival or tumour reduction, but long-term effectiveness and safety remain uncertain.

Researching these factors in extended in vivo models would be an important next step.

I would like to thank my parents who supported me throughout this entire journey. I am so grateful for their patience and reassurance.

I would also like to thank my chemistry teacher, Dr. Jeffery Murphy, for his support throughout this project. He provided valuable feedback that helped refine my project and improve the quality of my work. He guided me in understanding how to analyze data more effectively.

Thank you to the Fraser Valley Regional Science Fair for giving me this amazing opportunity to present my work at the Canada Wide Science Fair!

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