Nucleic Acid Aptamer Selection Against FasR for Inhibition of Nerve Cell Apoptosis

Owais Jamil

Fall 2012
September 4, 2012

N50 RNA Pool, FasR/CD95

Nucleic Acid Aptamer Selection Against FasR for Inhibition of Nerve Cell Apoptosis

Abstract: 

Permanent damage to the nervous system can be caused by severe physical trauma to the peripheral nervous system. Following this trauma, one of the ways loss of function occurs is secondary damage to the central nervous system, which occurs in the form of neuron death. If this can be prevented, victims will be able to retain motor and cognitive ability (2).

The Fas Receptor (FasR, also known as CD95) is a member of the tumor necrosis factor protein family that found on the surface of somatic cells and functions in cell death. When the Fas ligand (FasL) binds to FasR, a death inducing signaling complex (DISC) is formed (1). This triggers a downstream signaling cascade ending with apoptosis. In nervous system peripherals that have experienced trauma, FasR and FasL have been shown to be present in high quantities (4).

Apoptosis is the process of programmed cell death triggered by various signals or inducing factors. In the event of a spinal cord injury, damage occurs at the cellular level as a result of apoptosis. In healthy nervous systems, apoptosis pathways are used to eliminate unused neuronal connections and eliminate damaged cells (3). However, in the case of severe trauma, cell death can become uncontrolled, eliminating not only damaged cells but also healthy cells (2). These damages are irreversible and can lead to severe loss of nervous system function, and even death.

Specific Aim:
In the nervous system, when FasL binds to FasR, the DISC begins the pathway within the cell to lead to its death. To prevent this, perform the SELEX method to select an RNA aptamer with a high binding affinity for FasR. This aptamer would serve as a means to inhibit the function of FasR in healthy neurons that surround damaged nervous tissue, in hopes of preventing increased damage to the irreparable nerve tissue.

Figure 1. By inhibiting the function of FasR in healthy neurons surrounding trauma, loss of function can be minimized.

FasR/CD95 can be purchased from BD Pharmingen (Catalog#554256) at the price of $255 for 500 micrograms. (http://www.bdbiosciences.com/ptProduct.jsp?prodId=10379&catyId=745875)

References:

1. Kim, J.W., Choi, E., O Joe, C. 2000 “Activation of death-inducing signaling complex (DISC) by pro-apoptotic C-terminal fragment of RIP” Oncogene (19). 4491-4499

2. Beattie, M.S. 2004 “Inflammation and apoptosis: linked therapeutic targets in spinal cord injury” Trends in Molecular Medicine (12). 580-583

3. Groene, H-J., Herr, I., Krammer, P.H., Martin-Villalba, A. 2006 “Control of neuronal brancing by the death receptor CD95 (Fas/Apo-1)” Cell Death and Differentiation (13). 31-40

4. Shohami, E., Trentz, O., Kossman, T., Morganti-Kossman, M.C. 2007 “Immunohistochemical characterization of Fas (CD95) and Fas Ligand (FasL/CD95L) expression in injured brain: Relationship with neuronal cell death and inflammatory mediators” Histol Histopathol (22) 235-250

Nucleic Acid Aptamer Selection against Endoglin (CD105) for the Inhibition of Tumor Cells

Vicki Oladoyin

September 18, 2012

FRI: Aptamer

N40B Pool, RNA, CD105

Nucleic Acid Aptamer Selection against Endoglin (CD105) for the Inhibition of Tumors

Abstract:

           Endoglin (CD105) is a cell membrane glycoprotein expressed on cellular lineages within the vascular system and is involved in blood vessel development (Fonsatti 2003). It has been discovered that endoglin may be involved with tumors associated with the vascular endothelium, because endoglin has been found to be over-expressed on proliferating endothelial cells of both peri-and intra-tumoral blood vessels (Fonsatti 2003).  For research purposes, an aptamer against mouse endoglin will be selected via in vitro SELEX (systematic evolution of ligands by exponential enrichment).  Furthermore, the practical implications of using an aptamer selected against endoglin to inhibit the formation of tumors will be discussed.

Specific Aim 1: Identifying an Aptamer Against Endoglin

Indentifying a high affinity and specific RNA aptamer against endoglin would be beneficial, because endoglin is expressed on over-proliferating endothelial cells as represented in Figure 1.  An aptamer selected against endoglin would therefore be useful for diagnosis and possibly the development of a therapeutic drug to inhibit the over-proliferation of tumor cells.

Specific Aim 2: Identifying an Anti-Endoglin Aptamer

            Identifying an anti-endoglin aptamer would be a useful tool for the studying of the functions and processes of the endoglin glycoprotein in the human body and its role in the formation of blood vessels.  This could then lead to a better understanding of angiogenesis, the physiological process involving the growth of new blood vessels from pre-existing vessels (Wahl 2004).

Endoglin (CD105) protein can be purchased from Sino Biological Inc. at a price of $290 per 100ug; its call number is 50407-M08H.  It has a calculated molecular mass of 61.2kDa.  The cost per round will be about $35.50. This amount will allow eight rounds of selection to be performed using 200 pmols of the target per round.  The company's phone number is 86-400-890-9989.

• for a link to the proposal click here
• for a link to the first progress report click here
• for a link to the second progress report click here
• for a link to the final manuscript click here

References

1.  Abdalla S., and M Letarte. "Hereditary Haemorrhagic Telangiectasia: Current Views on Genetics and Mechanisms of Disease."J Med Genet (2006): 97-110.

2.  Dallas, Nikolaos, Shaija Samuel, Ling Xia, Fan Fan, Michael Gray, Sherry Lim, and Lee Ellis. "Endoglin (CD105): a marker of tumor vasculature and potential target for therapy." Clinical cancer research : an official journal of the American Association for Cancer Research 14.7 (2008): 1931-1937.

3.  Ellis, L. (2008) “Endoglin (CD105): A Marker of Tumor Vasculature and Potential Target for Therapy” Clinical Cancer Research. 14:1931.

4.  Ester, F. (2003) “Endoglin (CD105): a powerful therapeutic target on tumor-associated angiogenetic blood vessels.” Oncogene. 22:6557-6563.

5.  Fonsatti E, Sigalotti L, Arslan P, Altomonte M, Maio M. Emerging role of endoglin (CD105) as a marker of angiogenesis with clinical potential in human malignancies. Curr Cancer Drug Targets. 2003 Dec;3(6):427-32.

6.  "HHT Foundation International: About HHT." HHT Foundation International: Hereditary Hemorrhagic Telangiectasia – Osler-Weber-Rendu. N.p., n.d. Web. 2 Sept. 2012.

7.  P. Shannon Pendergast, H. Nicholas March, Dilara Grate, Judith M. Healy, Martin Stanton. "Nucleic Acid Aptamers for Target Validation and Therapeutic Application." J Biomol Tech. 2005 September;16(3): 224-234. Arbl.cvmbs.colostate.edu.

8.  Siemann DW. "Vascular Targeting Agents". Horizons in Cancer Therapeutics: From Bench to Bedside. 2002;3(2):4-15.

9. Stoltenburg, R., C. Reinemann, and B. Strehlitz. "SELEX—A (r)evolutionary Method to   Generate High-affinity Nucleic Acid Ligands." Biomolecular Engineering 24.4 (2007): 381-403.

10. Wikström, P., Lissbrant, I. F., Stattin, P., Egevad, L. and Bergh, A. (2002), Endoglin (CD105) is expressed on immature blood vessels and is a marker for survival in prostate cancer. Prostate, 51: 268–275. doi: 10.1002/pros.10083.

Nucleic Acid Aptamer Selection Against Basic Fibroblast Growth Factor (FGF-2)

Nucleic Acid Aptamer Selection against Basic Fibroblast Growth Factor (FGF-2)

Jessica Nguyen, 18 September, Fall 2012

R50 RNA Pool against Basic Fibroblast Growth Factor

Abstract:

Although science and medicine have developed rapidly over the last decade, some diseases and conditions still evade present diagnostic and therapeutic techniques. For instance, cardiovascular disease, a class of disease that involves vessels of the heart, is the primary cause of death in the industrialized world with approximately 73 million Americans having some form of the disease ⁽¹⁾. Cancer, the uncontrolled growth of abnormal cells, is also considered to be one of the deadliest diseases of the modern world. Although these diseases seem unrelated, scientists have discovered that one protein, basic fibroblast growth factor (FGF-2), plays a vital role in both these conditions and various other conditions of the body. This research proposes that by finding an aptamer (an oligonucleotide with binding properties) against FGF-2 (also known as FGF-b), these diseases and others can be resolved with more efficiency and specificity.

FGF-2 has many effects on the body and is naturally present in basement membranes and in the extracellular matrix of blood vessels ⁽²⁾. It is a single-chain polypeptide growth factor that plays a significant role in the processes of wound healing, inducing angiogenesis or the growth of new blood vessels from existing ones, and supplementing undifferentiated growth in human embryonic stem cells ^{(1), (3), (4),(5)}. Because this protein has such a prominent role in many functions of the human body, an aptamer against FGF-2 could have multiple therapeutic and diagnostic effects in the body and could prove to be very promising. However, despite the many benefits of an aptamer against this protein, the specific aims of this research focus on the role of FGF-2 on cancer progression. 

Specific Aim 1: The first specific aim of this research will be to discover an aptamer that has a high binding affinity for FGF-2.  Seven rounds of selection have already been completed, along with one binding assay of Rounds 2, 4, and 6 (results showed that although positive binding was constant over rounds, negative binders did decrease) and sequencing of the Round 6 pool. More rounds of selection hope to be completed, and a binding assay done with clones found during sequencing will be conducted to test for high affinity binding between the clone and the target.    

Specific Aim 2: The next aim of this research is to modify the aptamer so that it can be used to diagnose and track the progression of cancer in the human body. Modifications that can be used include fluorescence.

Specific Aim 3: The third specific aim of this research will be to modify this aptamer so it can be used for drug delivery. This can be accomplished by collaboration with other labs that have discovered a functioning therapeutic against cancer but have no way of selectively targeting the abnormal cells (Figure 1). 

Figure 1: Specific Aims of Aptamer Research. FGF-2 plays an important role in the angiogenesis and metastasis of cancer. By targeting this protein target for aptamer selection, diagnostic and drug delivery systems can be created that inhibit the progression of cancer.

FGF-2 (17.3kDa) can be purchased from ScienCell Research Laboratories (877.602.8549) for $175 for 50 micrograms (CAT: 104-02). Each round would cost roughly $11.67. Currently, there is some available in the Aptamer -20°C fridge. 

Click here for full proposal. 

Click here for first progress report. 
Click here for second progress report.
Click here for final progress report.

References: 
1. House, Stacey L., et al. "Cardiac-Specific Overexpression of Fibroblast Growth Factor-2 Protects Against Myocardial Dysfunction and Infarction in a Murine Model of Low-Flow Ischemia." 108 (2003).

2. Soulet, Fabienne, et al. "Fibroblast Growth Factor-2 Interactes with Free Ribosomal Protein S19." 289 (2001): 591-596. 6 April 2012.

3. Zhang, Shuang-Xia, et al. "Gekko Sulfated Glycopeptide Inhibits Tumor Angiogenesis by Targeting Basic Fibroblast Growth Factor." (2012).

4. Pereira, Renata C., Aris N. Economides and Ernesto Canalis. "Bone Morphogenetic Proteins Induce Gremlin, a Protein That Limits Their Activity in Osteoblasts." 141.12 (2000).

5. Liu, Yanxia, et al. "A novel chemical-defined medium with bFGF and N2B27 supplements undifferentiated growth in human embryonic stem cells." 346 (2006).

6. Stoltenburg, Regina, Christine Reinemann and Beate Strehiltz. "SELEX - A revolutionary method to generate high-affinity nucliec acid ligands." 24 (2007).

7. Stovall, Gwen. "Protocol - Selection - Filter Based RNA." 2012.

8. Golden, Mace C., et al. "Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers." 81 (2000).

9. McCauley, Thomas G., Nobuko Hamaguchi and Martin Stanton. "Aptamer-based biosensor arrays for detection and quantification of biological macromolecules." 319 (2003).