How We Started
The discovery of antibiotics was a significant advancement in medical sciences. Antibiotics are selective in that they eliminate bacterial infections without harming the patient. If cancer drugs could have the same degree of specificity for their targets, it would save and improve the quality of many lives. Research in this field has experienced variable levels of success, but significant barriers remain.
Unlike bacteria, cancer cells are mutated derivatives of cells, so they do not differ significantly from healthy cells in terms of structure and composition. However, cancer cells do have some distinctive characteristics. These features allow for targeted drug delivery, which is the use of a vehicle to deliver nonspecific drugs to only cancer cells. An example of targeted drug delivery that we are interested in are antibody-drug conjugates (ADCs). In recent years, ADCs have been a major topic of interest within the scientific community. Despite extensive research conducted, this therapeutic still has many problems.
We came up with a DNA based ADC alternative that solves these problems. We decided to design our system to deliver doxorubicin, a common chemotherapy drug, to acute myeloid leukemia cells.
Acute Myeloid Leukemia
Acute Myeloid Leukemia (AML) is a type of cancer originating from hematopoietic stem cells (HSCs) in the bone marrow. In AML, the common myeloid progenitor rapidly divides and develops into abnormal myeloblasts instead of differentiating and fully maturing. The consequent build up of undifferentiated myeloid blast cells crowds out healthy cells, and results in deficiencies of red blood cells, monocytes, granulocytes, and megakaryocytes.
Acute Myeloid Leukemia
Rapid onset and progression.
Caused by abnormal Myeloid stem cells.
Cancer of the blood.
Figure 1: A schematic of the differentiation of
Chemotherapy: A Conventional Treatment for AML
Currently, the most common treatment for AML is chemotherapy. This treatment involves injection of cytotoxic drugs into the patient’s bloodstream. The goal is to eliminate all of the cancerous cells while minimizing damage to the healthy cells. However, chemotherapy drugs are not specific to cancerous cells and consequently have side effects that may include fatigue, anemia, nausea, vomiting, and constipation.
Doxorubicin (DOX) is a chemotherapy drug that is often used to treat AML and many other types of cancer. By intercalating between a cell’s DNA, DOX inhibits the enzyme topoisomerase II, halting DNA replication. It is among the most effective anticancer drugs ever developed. However, when administered directly into the patient’s bloodstream, its off target-effects include congestive heart failure and treatment-related leukemia, as well as the side effects listed above.
Figure 2: Chemical structure of doxorubicin
Antibody Drug Conjugates
Antibody-drug conjugates (ADCs) are drug delivery therapeutics that can target chemotherapy drugs like doxorubicin to AML cells. They are composed of three components: a monoclonal antibody, a drug molecule, and a linker that holds the two together. The monoclonal antibody is specific to a receptor on the surface of cancer cells, and is therefore responsible for drug targeting. Binding of the antibody to its respective receptor results in the uptake of the ADC via endocytosis. Once in the cell the linker is degraded, either by enzyme hydrolysis or by pH degradation, to release the drug.
The therapeutic range is the difference between the minimum dose of a drug required to be effective and the minimum dose that is toxic. Since ADCs deliver drugs specifically to cancer cells, they have a wider therapeutic range than conventional chemotherapy drugs. This means that more aggressive doses of chemotherapy can be administered to patients, with fewer side effects.
Figure 3: Typical structure of antibody drug conjugates
In September 2017, the FDA approved Mylotarg or “
Choosing our Shape
Rigid, 3D DNA structures are more likely to be taken into the cell by endocytosis than non-rigid, 2D structures . Therefore, our structure had to be rigid and non-flat. Size was also an important consideration; we needed our structure to be small enough to easily pass through nuclear pores, and accumulate in the nucleus. Additionally, our structure had to have binding sites for at least two aptamers.
The structure we found that met all of these requirements was a 4 stranded tetrahedron . The tetrahedron is made from 4 unique strands, each folding into a triangular shape. The tetrahedron can be made as a completely double-stranded object, or with an overhang on any of the vertices, to which aptamers can be attached.
Figure 7: DNA tetrahedron created from 4 DNA strands, with overhang for aptamer attachment.
To direct our DNA-DOX structure to AML cells, we decided to use nucleotide aptamers to target the CD33 and CD71 receptors.
What are Nucleotide Aptamers?
Nucleotide aptamers are short strands of RNA or DNA that have a high affinity and specificity for a target molecule (in this case a protein). Aptamers can be made for any large molecule capable of hydrogen bonding.
For proteins, the process for determining an aptamer is called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). SELEX starts with solutions containing many different potential aptamers. Typically, the solution is run through an affinity column with the target molecule. The potential aptamers that have the highest affinity to the target are separated and amplified with PCR. This is repeated until a satisfactory aptamer has been obtained .
We used a RNA aptamer for protein CD71 , and tested 4 DNA aptamers for protein CD33 . The aptamers were modified by the addition of a sequence at the 5’ end complementary to the overhang, so they could be incorporated into the DNA tetrahedron.
Figure 8 is an image of the RNA aptamer for targeting the CD71 receptor.
Figure 8: CD71 RNA aptamer
Figure 9 is an image of the four aptamers that were tested for binding to the CD33 receptor. Aptamers 2 and 4 had the highest affinity to the target protein:
Figure 9: From left to right: CD33 Aptamer 1, CD33 Aptamer 2, CD33 Aptamer 3 and CD33 Aptamer 4
CD33 is a member of the sialic acid-binding receptor family (siglecs), which is myeloid specific. It is expressed by all myeloid progenitor cells, and at much lower levels compared to AML cells . Figure 10 is an image of the CD33 receptor.
CD71 is a transferrin receptor that is frequently expressed in less committed cells. It is expressed in AML cells in 84% of patients . An increase in CD71 expression causes increased cellular concentrations of free iron. This leads to oxidative stress and impairs cell differentiation. CD71 overexpression is linked to severe anemia (low red blood cell levels) and thrombocytopenia (low platelet levels) as a result of myeloid cell differentiation blockage. Figure 11 is an image of the CD71 receptor.
Figure 10: CD33 and CD33 receptor binding
Figure 11: CD71 and CD71 receptor binding
We chose DOX as the delivery drug because it can be loaded into DNA structures through simple incubation. DOX is fluorescent and is quenched by binding to DNA. The fluorescent properties of DOX allow for the use of a spectrophotometer to measure the amount of DOX that is loaded into the structure. When researching DOX loading into DNA, we found a lack of consensus on the amount of DOX that could be loaded into the tetrahedron. The numbers varied from 26 molecules of DOX , to 330 . To properly develop this system as a therapeutic, we needed to know the amount of drug delivered by the system.
Figure 12: DNA tetrahedron intercolated with DOX
Cellular Uptake of the Structure
Endocytosis is the process by which eukaryotic cells internalize molecules. This is done by the budding of the cellular membrane to form an endocytic vesicle on the inside of the cell . It plays a role in a variety of cellular functions, including nutrient uptake, regulation of plasma membrane proteins, cell motility, and mitotic facilitation. The CD33 receptor exhibits endocytotic capacities upon activation . This means once our DNA structure binds to the receptor, it will be be uptaken by the cell.
Project Execution: Labbook
Click on the diagram to be taken to the the relevant section of the lab book.
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Challenges with ADCs
- Lin J, Guo Y, Wang W. Challenges of antibody drug conjugates in cancer therapy: Current understanding of mechanisms and future strategies. Curr Pharmacol Rep. 2018;4(1):10-26.
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- Definition of milatuzumab doxorubicin
antibody drugconjugate NCI drug dictionary. National Cancer Institute.
Our Solution (problem fixing):
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CancerGeneTherapy. 2016;23(9):315-320.https://www.ncbi.nlm.nih.gov/pubmed/27514505. doi: 10.1038/cgt.2016.33.
- Sun P, Zhang N, Tang Y, Yang Y, Chu X, Zhao Y. SL2B aptamer and folic acid dual-targeting DNA nanostructures for synergic biological effect with chemotherapy to combat colorectal cancer. International journal of nanomedicine. 2017;12:2657-2672. https://www.ncbi.nlm.nih.gov/pubmed/28435250.
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KwonaIC, Ahn DR. Drug delivery by a self-assembled DNA tetrahedron for overcoming drug resistance in breast cancer cells. Chemical communications. 2011;49(1):2010-2012. https://www.ncbi.nlm.nih.gov/pubmed/22105188. doi: 10.1039/c1cc90182f.