Gene Therapy for Breast Cancer, Research Proposal Example

Breast cancer affects the lives of many women. One of the more aggressive forms of breast cancer occurs when the human epidermal growth factor receptor (HER2/neu) is positive. The prognosis for survival is poor and HER2/neu positive breast cancers are usually not responsive to chemotherapy and radiation. For this reason, it is imperative that further options, such as gene therapy, are explored. One form of gene therapy uses antisense oligonucleotides to down-regulate the HER2/neu oncogene, inhibiting the development of malignant cells. Though laboratory studies have found this type of gene therapy effective, further research is required before administering this experimental therapy on human subjects. The purpose of this proposal is to increase understanding of antisense therapy in treating HER2/neu positive breast cancer in vitro so this treatment will be safe for testing in human subjects.

The HER2/neu is a proto-oncogene which regulates the function of cell growth, division, and repair. Healthy cells have two HER2/neu gene copies and when in balance, breast cells grow normally. But when HER2/neu is overexpressed, cancer often results. Overexpression of this proto-oncogene occurs in approximately 20-30% of breast cancer cases. Transmembrane tyrosine kinase is the oncogene for the HER2/neu and encodes a human epidermal growth factor receptor type 2, which is located on chromosome 17q1. The high number of HER2/neu proto-oncogenes causes an overexpression of the receptors on the cell membrane. HER2/neu positive breast cancer is often treated with methods which are specific to membrane receptors. Trastuzumab (Herceptin) is a common drug which is given to patients with HER2/neu positive breast cancer. Trastuzumab targets HER2 proteins on cancer cells and prevents them from proliferating and encourages apoptosis. It is often administered in conjunction with chemotherapy; however it does have side effects such as allergic reactions and congestive heart failure. Due to some of the side effects, trastuzumab may not be appropriate for all patients with HER2/neu positive cancer. When this occurs, other methods, such as gene therapy, should be considered.

Gene therapy is an experimental treatment method which induces a purified preparation of genetic material in the form of DNA or RNA into cells for the treatment and prevention of diseases. The primary goal of gene therapy is to “introduce therapeutic material into the target cells, where it becomes active and exerts the intended therapeutic effect” (Mammen, Ramakrishnan, Sudkaker, and Vijayalakshimi 196-200). One method of gene therapy attacks the malignant cells to stop the development of cancer. Malfunctioning genes are first identified and then replaced, corrected, or eliminated. The patient may then be supplied with a copy of a gene which functions normally. Another approach is to introduce new genetic material into the body, which would to enhance its disease-fighting abilities. Gene therapy targets either somatic or germ cells. In somatic gene therapy, the recipient’s genome is altered, but the change is not inherited. When targeting germ cells, the new genetic code has a high chance of being passed on the recipient’s offspring.

Gene therapy may be beneficial for preventing, treating, or curing HER2/neu positive breast cancer because of the genetic component of this type of cancer. HER2/neu positive breast cancer specifically involves the overexpression of HER2/neu receptor. HER2/neu encodes a tyrosine kinase receptor protein, p185^HER/neu embedded in the cell membrane. When this receptor is activated, extracellular signals are sent to the nucleus to increase the receptor’s expression. In 2002, a study by Wilson, Roberts, Leek, Harris, and Geradts surmised that a significant number of genes are either up- or down-regulated in HER2/neu-positive breast cancer cells. The sensitivity for regulation makes gene therapy a good candidate for fighting HER2/neu positive breast cancers.

Genes must be introduced into target cells with a vector, or carrier. Vectors may be administered intravenously or injected directly into specific tissues in the body to be received by target cells. A benefit of using a vector is that increases the stability of the genetic material. The added stability is beneficial when working with the unstable phosphorothioate backbones of oligonucleotides. Another benefit of a vector is that less foreign genetic material is required to achieve the desired result (Dias, and Stein). Vectors may be administered intravenously or injected directly into specific tissues in the body where it is taken up by target cells. Vectors are available in the form of viruses and liposomes. Viruses are the most common type of vectors used in gene therapy due to their ability to identify specific cells, insert their own genetic material, and alter the target cell’s DNA or RNA. Though precautions are taken and viruses are genetically altered to make them safe, there is a risk because viruses usually infect more than one type of cell; they may infect healthy cells or germ cells and affect offspring. However, once this process is better understood, it may be possible to use genetically altered viruses to prevent the development of cancer cells in future generations. Liposomes are nonviral vectors which transport therapeutic DNA into a target cell’s membrane. Though this method is simpler than viral vectors; its challenge lies in that it requires a large amount of DNA to achieve the desired effect (Mammen, Ramakrishnan, Sudhaker, and Vijayalakshmi 196-200). Even though it may be somewhat of a safer process to use a liposome over a virus, the aggressive nature of HER2/neu positive breast cancer warrants the consideration of using a virus as a vector.

Antisense therapy is a form of genetic therapy that uses oligonucleotides to treat illnesses caused by malfunctioning genes. Oligonucleotides are short fragments of nucleic acids in the form of DNA or RNA. Many times their use is limited because the degradation process which inhibits cellular proliferation can be toxic to other healthy cells. The 2002 study by Dias and Stein cautions against using oligonucleotides for this very reason. However, if this anti-proliferative effect can be harnessed to destroy malfunctioning DNA strands in tumors, oligonucleotides can be used to treat patients with HER2/neu positive breast cancer. This may be possible by using a virus as a vector, as viruses can be programed to target specific cells.

When the genetic sequence of a specific disease is known, it is possible to synthesize a strand of DNA, RNA, or a chemical analogue that will bind to the messenger RNA (mRNA) of a targeted gene and inactivate it. Antisense oligonucleotides, specifically phosphothorothioates, can be used to selectively inhibit gene expression. Dias and Stein (2002) report that phosphorothioates are “highly soluble and have excellent antisense activity” and are “capable of activating RNAase H activity.” RNAase H is an enzyme that hydrolyzes RNA strands as well as the RNA/DNA duplex. RNAase H also allows for the completion of new DNA synthesis by removing the RNA primer. Oligonucleotide-assisted RNAase H-dependent reduction of targeted RNA expression can be quite efficient, reaching 80–95% down-regulation of protein and mRNA expression” (Dias, and Stein). It is proposed that RNAase H-dependent oligonucleotides will be used in this study of antisense therapy due to their ability to effectively degrade mRNA.

In 2000, a study by Roh, Pippin, Green, Boswell, Hirose, Mokadam, and Drebin found that antisense oligonucleotides exert a dose-dependent and sequence-specific mechanism for targeting HER2/neu in overexpressing breast cancer cells. HER2/neu-specific antisense oligonucleotides were successful in inhibiting only the growth of cancer cells that overexpress HER2/neu, while leaving healthy cells intact.

Antisense oligonucleotides will be injected into the tumor cells using a genetically altered virus as a vector. The intended biochemical targets are the malignant cells which encourage the overexpression of HER2/neu cells. The elimination of the HER2/neu cells will cause the tumors to self-destruct. Antisense oligonucleotides can down-regulate the expression of HER2/neu mRNA and the oncogene p185^HER2/neu tyrosine kinase. This process stops the cell cycle in G₀/G₁ phases and results in the death of the cancer cells and a decrease in cells in the S and G2/M phases of the cell cycle. “These results suggest that HER2/neu overexpression in breast cancer cells alters cell cycle regulatory mechanisms controlling G1 progression or the G1/S transition” (Roh, Haeri, Pippin, Green, Boswell, Hirose, and Drebin 6138-43). The proliferation of breast cancer cells, such as BT474 are increased in proportion to a rise in levels of p185^HER2/neu. The antisense oligonucleotides bind to complementary mRNA base-pairing sequences and cause the destruction of the complementary mRNA strands with the nuclease RNAase H. Antisense oligonucleotides specifically inhibit the expression of HER2/neu and BT474 breast cancer cell growth (Roh, Haeri, Pippin, Green, Boswell, Hirose, and Drebin 6138-43). The study also found that antisense therapy to enhance the effectiveness of other cancer treatments, such as radiation and chemotherapy. The sensitivity of the receptors to chemotherapy and radiation is increased and makes the malignant cells more likely to self-destruct. The increased sensitivity is important because it is estimated that one of the reasons for the resistance to chemotherapy may be due to the ability of the HER2/neu to inhibit cell death.

The mechanism of this method is complex and further testing on human subjects is required. By affecting the coding mRNA, antisense oligonucleotides can change the genetic sequence of the cancer cells. Even though the virus vector has its risks, it is more effective in delivering genetic material to the cancer cells. Because HER2/neu positive breast cancer is so aggressive, it is necessary that stronger methods are taken to help fight this disease. The study by Roh et al in 2000 points out that there are many flaws in previous studies. There is concern that high concentrations of oligonucleotides may be toxic to cells due to their phosphorothioate backbones. When combined with the risks of using a virus as a vector, apprehension is understandable. But by genetically altering the viruses to target only highly proliferating tumor cells, there may be a greater chance of fighting HER2/neu positive breast cancers. If these flaws can be addressed, the prognosis for survival increases for the women struggling with HER2/neu positive will increase.

The purpose of this proposal is to research the outcome of using antisense therapy for the treatment of HER2/neu positive breast cancer in vitro. The overexpression of the HER2/neu receptor will be inhibited using antisense oligonucleotides. RNAase H will be administered to attack the mRNA sequences of the HER2/neu positive breast cancer cells. The tyrosine kinase receptor protein, p185^HER/neuwill be the target cell to stop the overexpression of HER2/neu. Even though Roh et al (2000) found antisense oligonucleotides to be specific in inhibiting the growth of cancer cells that overexpress HER2/neu while leaving healthy cells intact, the vector to be used is a genetically altered virus which will further cement the safety of this aggressive procedure. After this is proven to be both a safe and effective option for HER2/neu positive breast cancer, it may be administered into human subjects.

Work Cited

Dias, Nathalie, and C.A Stein. “Antisense Oligonucleotides: Basic Concepts and Mechanisms.”Molecular Cancer Therapeutics. 1.347 (2002): n. page. Web. 27 Jul. 2013 <http://mct.aacrjournals.org/content/1/5/347.full>.

Hillig, Thore, Jorgen Thode, Marie Breinholt, Maria-Benedicte Franzmann, Carsten Pedersen, Flemming Lund, Henrik Mygind, and Gyorgy Soletormos. “Assessing HER2 Amplification by IHC, FISH, and Real-Time Polymerase Chain Reaction Analysis (Real-Time PCR) Following LCM in Formalin-Fixed Paraffin Embedded Tissue from 40 Women with Ovarian Cancer.” APMIS. 120.12 (2012): 1000-07. Web. 24 Jul. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3533780/>.

Mammen, Biju, T Ramakrishnan, Uma Sudhaker, and Vijayalakshmi. “Principles of Gene Therapy .” Indian Journal of Dental Research. 18.4 (2007): 196-200. Web. 24 Jul. 2013. <http://www.ijdr.in/article.asp?issn=09709290;year=2007;volume=18;issue=4;spage=196;epage=200;aulast=Mammen>.

Roh, Haeri, James Pippin, Douglas Green, Craig Boswell, Christopher Hirose, Nahush Mokadam, and Jeffrey Drebin. “HER2/neu Antisense Targeting of Human Breast Carcinoma.” Oncogene. 19.53 (2000): 6138-43. Web. 24 Jul. 2013. <http://www.nature.com/onc/journal/v19/n53/full/1204001a.html>.

Wilson, Katherine, Helen Roberts, Russell Leek, Adrian Harris, and Joseph Geradts. “Differential Gene Expression Patterns in HER2/neu-Positive and -Negative Breast Cancer Cell Lines and Tissues.” American Journal of Pathology. 161.4 (2002): 1171-85. Web. 24 Jul. 2013 <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1867314/>.