Selected Conference Abstracts

 

1st Australian Neurodegeneration and Dementia Conference 2015

Crossing the blood brain barrier: a novel strategy targeting neurodegenerative disorders

J Macdonald, P Houghton, W Duan & S Shigdar

School of Medicine, Deakin University, Waurn Ponds, Geelong, Victoria, Australia

 

Neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease are incurable and debilitating conditions. Affecting millions of people of worldwide, these conditions are becoming more prevalent due to increases in life expectancy. While symptoms can be lessened through pharmacological intervention, there remains no treatment available capable of slowing down or stopping the degeneration and death of neurons in the brain. Limited treatment options can largely be attributed to the restrictive nature of the blood brain barrier (BBB), which through its highly selective nature limits drug bioavailability. An emerging strategy to overcome this barrier is to target active transport mechanisms present on the BBB. Given its high expression levels, the transferrin receptor has been studied extensively for this purpose. While promising results have been produced using monoclonal antibodies, nucleic acid based aptamers have emerged as a superior alternative due to their increased stability, safety profile and minimal batch-to-batch variation. From this, we have engineered DNA aptamers specific for the transferrin receptor which can act as a transport system for the delivery of therapeutics across the BBB. Aptamer specificity and sensitivity against both transferrin positive and negative cells has been confirmed via flow cytometry. The ability of the aptamers to be efficiently internalised was established via confocal microscopy. Transcytosis across the BBB was initially confirmed in an in vitro model subsequent to testing in an in vivo model which confirmed specific internalization within 10 minutes of injection.  The results from the study highlight the great potential these aptamers have to be developed for the delivery of therapeutic agents across the BBB for the treatment of neurodegenerative disorders.

 

The 2015 Alzheimer's Disease Congress

Novel therapeutic strategies to target Alzheimer’s disease

J Macdonald, P Houghton, W Duan & S Shigdar

School of Medicine, Deakin University, Waurn Ponds, Geelong, Victoria, Australia

 

Neurodegenerative disorders such as Alzheimer’s disease are becoming more prevalent due to longer life expectancies. While pharmacological treatments can temporarily improve symptoms, there are no treatments available that can slow or stop the malfunction and death of neurons in the brain. One of the major problems associated with the treatment of neurodegenerative disorders is bioavailability as the blood brain barrier (BBB) is exceptional at preventing drugs from crossing into the brain. Several strategies have been proposed, such as disrupting the integrity, or inhibiting the drug efflux pumps on the BBB. However, these techniques will also allow other toxic substances access to the brain leading to potentially devastating side effects. A novel strategy is to use active transport mechanisms present on the BBB to transcytose, or slingshot, drugs into the brain. The transferrin receptor has been studied extensively for its potential to serve as a high affinity target for the delivery of a variety of drug vehicles. Aptamers have emerged as an alternative to conventional antibodies due to their safety profile, increased stability and minimal batch-to-batch variation. Here we describe the generation of nucleic acid aptamers to the transferrin receptor suitable for in vivo delivery of therapeutics across the BBB. The transferrin receptor aptamers were truncated and their specificity and sensitivity was confirmed against both transferrin receptor positive and negative cell lines. Their ability to be internalised was confirmed using confocal microscopy and colocalisation studies confirmed a similar uptake to the transferrin antibody. Transcytosis across the BBB was tested in vitro, prior to in vivo testing in a mouse model. The results from this study confirm the potential for these aptamers to be utilised for therapeutic delivery of drugs across the BBB for the treatment of neurodegenerative disorders.

 

Aptamers 2015

Getting the brain to take its medicine

Joanna Macdonald, Wei Duan & Sarah Shigdar          

School of Medicine, Deakin University, Waurn Ponds, Geelong, Victoria, Australia

Presenter(s): Dr Sarah Shigdar

Email: Sarah.Shigdar@deakin.edu.au

 

Survival rates for patients with brain cancer or brain metastases have increased only slightly in the last twenty years. This is partly due to the effectiveness of the blood brain barrier at keeping toxic substances out of the brain. Novel approaches to breach this barrier are now being investigated, including the use of actively targeting a transporter on the blood brain barrier. Transferrin is one such receptor that can be used to effectively ‘slingshot’ a cargo into the brain. However, once drugs enter the brain, in order to minimise cytotoxicity to the healthy brain cells, a second active targeting mechanism is required to ensure the drugs reach their target cells – the tumour cells. Aptamers are easy to combine, due to their nucleic acid nature and thus, we have generated a bi-functional aptamer that binds to two different targets, the transferrin receptor on the blood brain barrier and EpCAM, a marker of metastatic cancer cells. We confirmed the specificity and sensitivity of this aptamer using flow cytometry with a number of cell lines positive for either transferrin receptor or EpCAM. Additionally, we assessed the ability of this aptamer to transcytose into the brain using an in vitro model of the blood brain barrier. Successful transcytosis was confirmed via endocytosis into EpCAM positive cancer cells in the base of a transwell. The ability of the aptamer to transcytose in a living system was confirmed by tail vein injection of 2 nmoles of bifunctional aptamer. The results of this study confirm initial proof of concept that aptamers targeting the transferrin receptor can breech the blood brain barrier and be used as effective agents to deliver therapeutics into the brain.

 

 

Lorne Cancer Conference 2015

Targeting Triple Negative Breast Cancer Brain Metastases

Joanna Macdonald, Wei Duan, Sarah Shigdar

Schoool of Medicine, Deakin University, Waurn Ponds, Australia, 3216

 

Triple negative breast cancer (TNBC) relapses more frequently then hormone receptor-positive subtypes and is often associated with poor outcomes. This is due to the fact that TNBC is more likely to spread to the brain, where current management strategies do not drastically alter outcomes. Indeed the overall survival for patients with a diagnosis of brain metastases is 4.3 months. Therefore, there is an urgent requirement for better therapeutic strategies. Having generated chemical antibodies against the cell surface marker, EpCAM, we sought to functionalise this aptamer to target brain metastases. A recent development has shown that targeting the transferrin receptor on the blood brain barrier can transport molecules into the brain via receptor-mediated transcytosis. We have generated a chemical antibody to the transferrin receptor and demonstrated that it can indeed enter the brain in an in vivo mouse model. Indeed, this aptamer specifically entered the brain within 10 minutes of tail vein injection. We have attached a second chemical antibody, targeting EpCAM, to this transcytosing aptamer and confirmed its specificity and sensitivity using flow cytometry against transferrin receptor positive or negative cell lines, as well as EpCAM positive or negative cells lines. Furthermore, we have shown that it is specifically internalized via receptor-mediated endocytosis into MDA-MB-231 cells. As well, we have attached the common chemotherapeutic, doxorubicin, to this bi-functional aptamer, and demonstrated that it is specifically internalized within the targeted cells. These results demonstrate that this bi-functional aptamer-doxorubicin conjugate has potential for the specific targeting and treatment of brain metastases in TNBC patients. Moreover, by specifically targeting the cancer cells in the brain, this novel modality is likely to mitigate the neurotoxic effects of chemotherapeutic agents on the healthy brain tissue.

 

 

IABCR 2014

Robust detection of cell surface markers in paraffin embedded tissue: Use of chemical antibodies in the diagnosis of breast cancer

Sarah Shigdar & Wei Duan

 

Antibodies have proven to be effective in immunohistochemistry for the diagnosis of tumours and for the detection of cell surface markers prior to commencement of immunotherapy. However, batch-to-batch variation and cross-reactivity can limit their effectiveness. Chemical antibodies, also known as aptamers, are small pieces of DNA or RNA that are generated in a similar manner to antibodies – incubation with the target protein – and bind in exactly the same manner as antibodies via the ‘lock and key’ mechanism. Aptamers show very high specificity to their target and, as they are chemically synthesised, they can be easily derivatised with detection moieties at one or many pre-specified locations along their sequence, depending on the application. Having generated an aptamer targeting EpCAM, we tested its sensitivity in number of invasive ductal carcinoma cases using a chromogenic staining system, and showed that aptamers directed against EpCAM demonstrated superior sensitivity in paraffin embedded tissues. In a number of cases, conventional monoclonal antibodies failed to detect low levels of EpCAM in the breast tumour or the lymph node. This aptamer showed no non-specific staining or cross-reactivity and displayed a much more robust detection of EpCAM. Notably, sensitive staining was achieved in less than 15 minutes. From a clinic-pathological point of view, this has both prognostic and therapeutic significance, the latter due to the potential for immunotherapy trials. Results from EpCAM immunotherapy trials has been mixed, as there is currently no EpCAM antibody that is robust enough to be able to detect EpCAM expression in all pathological tissues. Therefore, the results from this study have important clinical significance for breast cancer diagnosis and suggest that aptamers have a promising future in the diagnostic laboratory.

 

 

Aptamers 2014

Aptamers as effective cancer stem cell targeting modalities

Sarah Shigdar, Dongxi Xiang, Wei Duan

Schoool of Medicine, Deakin University, Waurn Ponds, Australia, 3216

 

Aptamers are becoming known for their ability to replace other agents, such as antibodies, in diagnostic and therapeutic applications. Within cancer research, aptamers can be very versatile molecules, capable of being used for in vitro diagnostic applications, therapeutics and in vivo molecular imaging. Having generated an aptamer against the cancer cell marker, EpCAM, we sought to develop this aptamer into a smart drug delivery vehicle. Using a common chemotherapeutic, doxorubicin, we intercalated this drug into the stem of the aptamer and tested its effectiveness to prevent the formation of tumour spheres, an indication of metastatic ability. The number of spheres formed was much reduced and the spheres were significantly smaller. When cancer cells were treated ex vivo and then injected subcutaneously into mice, no mouse formed tumours, in contrast to those treated with free drug. Using an in vivo model of colon cancer, we have shown a significant reduction in tumour size following a 3 day treatment schedule. When the cells from these tumours were serially transplanted in additional mice, 80% of mice failed to form a tumour even when 1,000,000 cells were transplanted, proving that we have ‘knocked out’ the self-renewal and proliferative ability of these tumour cells. These results show the potential of our aptamer-doxorubicin conjugate to potentially eradicate all of the cells within a tumourous mass and prevent remission. Importantly, the results in this study were achieved using a 4-fold lower concentration of doxorubicin to that used in the clinical setting, suggesting that this targeted delivery system can reduce the detrimental side effects experienced by cancer patients. The combination of aptamers with conventional drugs will likely represent the next step in the arsenal against cancer.

 

 

Lorne Cancer Conference 2014

The new kid on the block – are aptamers more effective targeted therapeutics?

Sarah Shigdar, Dongxi Xiang, Tao Wang, Hadi Al Shamaileh, Wei Duan

Schoool of Medicine, Deakin University, Waurn Ponds, Australia, 3216

 

Aptamers are short single-stranded DNA or RNA sequences that fold into complex three-dimensional structures capable of binding to specific targets in the same manner as antibodies. Due to their small size, aptamers can penetrate tumourous masses at a much more effective rate than therapeutic antibodies. Having generated aptamers against two well recognised cancer cell surface markers, EpCAM and CD133, and shown that these aptamers are efficiently internalised via receptor-mediated endocytosis, we sought to functionalise these aptamers into targeted therapeutic agents. We intercalated a common chemotherapeutic, doxorubicin, into the stem of the aptamer and tested its effectiveness to prevent the formation of colorectal tumour spheres, an indication of metastatic ability. Both the sphere size and number were significantly smaller/reduced following treatment with our novel therapeutic. When HT29 colorectal cancer cells were treated ex vivo and injected subcutaneously into mice, no mouse formed tumours, in contrast to those treated with free drug. Additionally, following an in vivo three-day treatment schedule of HT29 xenograft tumour, we showed a significant reduction in tumour size. When the cells from these tumours were serially transplanted into additional mice, 80% of mice failed to form a tumour, even when 1,000,000 cells were transplanted, indicating that we have ‘knocked out’ the tumourigenic potential of these cancer cells. These results show the potential of our aptamer-doxorubicin conjugate to potentially eradicate the tumourgenic/cancer initiating  cells within a tumourous mass, and prevent relapse. Importantly, the results in this study were achieved using a 4-fold lower concentration of doxorubicin to that used in the clinical setting, suggesting that this targeted delivery system could improve the therapeutic index of doxorubicin, and reduce the detrimental side effects experienced by cancer patients. Therefore, the combination of aptamers with conventional drugs will likely represent one of the new  arsenals against cancer.

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