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Table of Contents
1. Cancer and CLL
2. miRNA & PTEN
3. DNA Origami
4. References


Cancer is a group of diseases broadly characterized by the uncontrolled growth, immortalization, and spread of transformed cells throughout the body ultimately resulting in mortality via multi-organ failure. In 2013, there were approximately 1.7 million new cases of cancer, and about 585 thousand people were expected to die of the disease[1]. However, the five year survival rate of all cancers diagnosed between 2002 and 2008 is 68%, up from 49% in 1977[1].

Figure 1: Mortality rates of different classes of cancer in the United States 1930-2009[1].

Leukemia is a type of cancer that develops when the bone marrow produces abnormal stem cells, which fail to undergo normal differentiation, programmed cell death, yet continue to reproduce. The unhealthy cells crowd out healthy cells in the blood, resulting in multi-organ system failure which can be fatal. Chronic lymphocytic leukemia (CLL), one specific type of the disease, is characterized by the accumulation of abnormal, mature non-functional B-lymphocytes that either evade programed cell death or undergo uncontrolled proliferation[1]. It is the most common form of leukemia in the western hemisphere, accounting for nearly 38% of all leukemia cases[2]. In 2013, around 16 thousand new cases were diagnosed, with approximately 4,500 estimated deaths[3]. Over 60% of those diagnosed were in adults over 65 years of age. The outlook for CLL has improved dramatically over the last three decades due to the continued improvement of previously established treatments. The 5-year survival rate is 82%, and most patients live for 10 years or more if the cancer is detected in stage A[2].

The OSU-CLL cell line is an immortalized cell line created by Dr. John Byrd and his colleagues at The Ohio State University to promote mechanistic in vitro and in vivo studies of CLL[4]. OSU-CLL cells displace aberrant co-expression of CD5 protein, and trisomy in chromosomes 12 and 19, all of which are common indicators of CLL[4]. Studies have also shown that OSU-CLL cells show similar motility and proliferation behaviors when compared to primary CLL cells. OSU-CLL is also readily transferable to mice for in vivo studies[4].

Currently, one of the most well-known treatment for CLL is chemotherapy, which refers to the use of chemical therapeutics that targets biological growth and survival pathways to ultimately kill rapidly dividing cells. However, this also causes undesired side effects such as severe immunosuppression, hair loss, drug resistance[5]. Monoclonal antibody immunotherapies are also a major treatment regimen for CLL patients which engage specific receptors on the B-CLL cell surface to induce apoptosis[6]. With the rare exception of allogeneic bone marrow transplantation[7], the disease is non-curative. Thus, novel therapeutic strategies are required.

One recently developed specific type of chemotherapy, antisense therapy, utilizes complementary base pairing to bind RNA molecules in cancerous cells. Antisense therapy involves targeting a specific messenger RNA (mRNA) strand that is overexpressed in cancerous cells (the sense strand) and introducing a complementary strand (the antisense strand) to inhibit translation. A previous study revealed that targeting the growth inducing protein Bcl-2 with antisense therapy in patients showed a reduction in tumor size implicating antisense therapy as a viable therapeutic option[8]. However, a potential drawback to antisense therapy is susceptibility to nuclease-dependent degradation as well as significant limitations in cellular uptake efficiency[9] . Thus, novel approaches to antisense therapy are necessary. One such approach is DNA nanotechnology as a novel antisense delivery system. This approach offers several advantages over current liposomal nanodelivery systems including increased biocompatibility, low cytotoxicity, and failure to induce immunogenicity[10]. In addition, the programmability and precise control over the number and arrangement of antisense molecular therapeutics on the surface of DNA nanostructures offers unprecedented design modalities. Furthermore, previous studies have shown an ability of cells to uptake DNA nanostructures in a highly efficient manner[10]. Taken together, these findings suggest that DNA nanostructures functionalized with antisense molecular modifications may serve as a powerful novel antisense therapeutic approach.

2. miRNA & PTEN

Messenger RNA (mRNA) is transcribed from DNA and translated in the ribosomes within the cytoplasm to produce proteins, which regulate and catalyze all processes in the cell. However, in cancerous cells, proteins responsible for growth and division are overexpressed, while those responsible for inducing apoptosis and regulating growth are underexpressed. MicroRNAs (miRNAs) are small, single stranded RNA molecules which have a vital role in regulating almost all cellular processes, especially with respect to gene expression levels. They are imperfect complements to mRNA strands, allowing them to bind to mRNA at the 3’-UTR and prevent protein translation. The first miRNA strand, Lin-4 was discovered in 1993 where it was observed that the lin-4 gene could regulate the expression of another gene, lin-14 [11]. It was also discovered that lin-4 didn’t code for a protein, and that a 22 nucleotide sequence of transcript was complementary to part of the lin-14 sequence. Therefore, it was hypothesized that the lin-4 transcript was blocking the expression of the lin-14 gene. This breakthrough led others to start studying how genes might regulate other genes, and thousands of subsequent miRNAs have been identified and characterized

[Figure 2]: Life Cycle of miRNA

As reviewed in Jinju Han et al.’s report[12], microRNAs are coded by portions of the genome previously regarded as “junk DNA”. Certain miRNAs are encoded for by portions of the genome that do not code from proteins, while others are translated from introns which have been spliced from mature RNA. miRNAs which have their own genes are transcribed as long strands of primary miRNA (Pri-miRNA). Pri-miRNA can be several hundred nucleotides in length and can include several miRNA strands. Each individual miRNA has a hairpin-loop structure on the pri-miRNA. Two enzymes are responsible for recognizing, cutting and removing the hair-pin structures, which are known as pre-miRNA. The pre-miRNA are exported from the nucleus to the cytoplasm, where the RNase enzyme DICER cuts the loop from the pre-miRNA. Cutting the pre-miRNA results in two mostly complementary RNA strands, which are then processed by the RNA-induced silencing complex (RISC).

The RISC selects an individual miRNA strand and the other strand is typically degraded. The strand that is less thermodynamically stable has been found to generally be selected for further processing[13]. The RISC then uses an argonaute protein to bind with the single stranded miRNA and facilitate proper orientation to bind with the target mRNA. If the miRNA is a perfect complement to the target mRNA, the duplex will be destroyed by the argonaute protein. In the case of imperfect binding (more common), the miRNA simply blocks expression of the mRNA. MiRNA’s need not bind perfect complementary to block mRNA, so an individual miRNA can block the expression of several genes. This imperfect binding also suggests that miRNA might preferentially bind to a perfectly complementary nucleic acid strand over the mRNA target.

MicroRNAs have been implicated in most cellular processes, including growth, development, and apoptosis. Because of their ubiquitous nature, miRNA dysregulation is a factor in most human disease. In cancer, some miRNA are overexpressed, while other are underexpressed. Predictably, miRNAs which should limit growth and proliferation are underexpressed in cancer, while those which limit apoptosis and cell cycle control are overexpressed. In Chronic Lymphocytic Leukemia (CLL), miR - 21 and miR - 155 are chronically overexpressed[14]. miR - 21 targets genes such as PTEN and Bcl2, both of which are tumor suppressors[15][16]. With elevated levels of miR - 21, PTEN and Bcl2 are suppressed, allowing cells to grow uncontrollably. Thus, miR-21 serves as an attractive target for antisense therapy in CLL cells.

[Figure 3]: PTEN and its effects on the Akt/PKB pathway.

Phosphatase and Tensin Homolog (PTEN) is a classic tumor suppressor protein and plays an integral part in the Akt/PKB signalling pathway, which controls cell death[17]. PTEN catalyzes the dephosphorylation of PIP3, a phospholipid in the cell membrane, resulting in the product PIP2. This reaction inhibits the Akt/PKB pathway, leading to cell death. In many types of cancer, including CLL, PTEN activity is low resulting in apoptotic evasion and cellular proliferation, resulting in a cancerous phenotype. PTEN has also been shown to induce apoptosis when it is overexpressed, as it activates a specific apoptotic pathway which detaches the cell from the extracellular matrix[17]. When PTEN levels were elevated in breast cancer cells, their cell cycle was arrested and soon became apoptotic[18]. Therefore, if PTEN levels could be elevated and restored in CLL cells due to antisense delivery via DNA nanostructures, it would be possible to induce apoptosis. Thus, we hypothesize that DNA nanostructures functionalized with miR-21 complementary oligomers will be effectively uptaken by CLL cells to induce apoptosis through elevation of PTEN expression levels. .

3. DNA Origami

DNA origami is a technique which utilizes the Watson-Crick base-pairing of DNA strands to construct nanostructures with precise control of geometry. It was first developed in 2D by Paul Rothemund in 2006[19] and later expanded to 3D in 2009. DNA origami works by combining a multi-kilobase loop of phage DNA, known as the scaffold, with smaller, synthesized staple DNA strands (~40 nt). The staples bind to the scaffold in a piecewise manner, folding it into the desired shape (Figure 4). To ensure structural integrity, the staples must transition between helices, forming Holliday junctions.

Figure 4: Scaffolded DNA Origami [24]

As a nanoscale platform, DNA origami has several attractive features, including consistency and spatial control. DNA origami structures can be functionalized with a variety of molecules by incorporating single stranded DNA overhangs into the structural staples used to fold DNA origami. This can help to attach proteins, nanoparticles, or to control complex mechanisms[20]. DNA origami also shows potential for therapeutic applications. The structures are inherently biocompatible and the size is appropriate for cellular endocytosis[21]. Furthermore, the ability to functionalize molecules opens doors for biosensing. Theoretically, a targeting molecule such as folate could be attached to an origami structure, along with a fluorescent molecule and a chemotherapeutic drug. A promising example for therapeutic applications was shown recently with a DNA origami cage capable of releasing it’s cargo when exposed to a certain signal[22]. Other recent studies have focused on delivering intercalating drugs, which naturally bind between DNA base-pairs. Anti-cancer intercalators are an ideal cargo for a DNA origami vehicle, and this approach has been used to successfully induce apoptosis in breast cancer cells[23].

4. References

[Figure 2]: JWF Catto, Et al. MircroRNA in Prostate, Bladder, and Kidney Cancer: A Systematic Review. European Eurology, 2011.
[Figure 3]: S Phin, Et al. Genomic Rearrangements of PTEN in Prostrate Cancer. Frontiers in Oncology, 2013.
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