Fundamentals of Molecular Biology and Gene Therapy

CHAPTER 2 Fundamentals of Molecular Biology and Gene Therapy

Molecular biology is a relatively young scientific field, arising in the 1970s after the discovery of methods to study and manipulate DNA. The rapid development of these techniques enabled actual clinical application by the 1980s and 1990s. Many diagnostic laboratory tests routinely used in clinical medicine, as well as methods used to develop and mass-produce pharmaceuticals, make use of molecular biology techniques.

The growing field of molecular biology has also led to the development of novel therapeutic strategies for disease. A new understanding of genes and the products of their expression has allowed the emergence of a rising field heralded as “gene therapy.” This rapidly expanding field is founded on the ability to introduce genetic material into the body to modulate the expression of genes and to thereby treat disease or alter an ongoing pathologic process.

This chapter reviews basic molecular terminology and introduces the concept of gene and molecular therapy. Emphasis is placed on the rationale, methods, and progress to date for clinical application of these exciting therapies.

Fundamentals of Molecular Biology

The basic premise of molecular biology is to study cell function and regulation at the level of the genome. With this understanding comes insight into human disease because aberrances in cell function or regulation are the basis for most diseases. The following section reviews fundamental terminology and concepts on how information travels from the genetic code to the functional protein level. Included is a brief discussion on cell cycling, which is an important concept in tumor molecular biology.

Gene Expression

The information for conducting all aspects of cellular function is contained within molecules of DNA located in the nucleus. The actual length of a human DNA strand is 1.8 M; however, it is coiled around nuclear proteins called histones, which structure the folds and loops of DNA to allow compression into the microscopic size of the cell’s nucleus. Each strand of DNA contains thousands of genes, which are specific subunit sequences that are coded for the information required to synthesize a protein. One molecule of double-stranded DNA and its genes make up each of a cell’s 46 total chromosomes. Although every cell in the human body contains the same DNA, the expression of individual genes is not the same. Gene expression varies depending on the cell’s function. These differences in gene expression result in the multiple cell and tissue phenotypes that constitute the human body as a whole.

The process by which a gene codes for a specific protein begins with transcription, which is the formation of a single-stranded RNA molecule, which complements a single strand of the DNA subunit (Fig. 2-1). This RNA molecule is subsequently modified to become the messenger, which brings the genetic information or directions from the nucleus to the cell’s cytoplasm where the actual synthesis of the functional protein occurs. After reaching the cytoplasm, the process of translation begins (see Fig. 2-1). During translation, the message from the RNA molecule directs the construction of a protein from its basic subunit, the amino acid. Once a protein is formed, it may require further modification or control steps to enable its designated function. These modifications include the addition of sugars, lipids, or phosphates to the protein backbone. These internal control steps are mediated by existing cytoplasmic enzymes, which, if defective, can also lead to defined diseases. Once a protein is formed within the cell cytoplasm, it can reside in the cell or be released to affect other tissues within the body. Depending on the original genetic program for the protein, it may serve its purpose and be rapidly degraded, or it may enter the circulation or reside in local or distant tissues for an extended period. Although stated simply here, both transcription and translation are complex processes that have many more modification and regulatory steps governed by regulatory genes and their protein products. When these delicate control mechanisms are lost, a disease state or a state of abnormal cell proliferation can ensue, as is exemplified in the development of cancer.

Cell Division

Disorders in cell function can lead either to cell death or proliferation. Abnormal stimulation of cell proliferation is the basis for the development of cancer. For a cell to divide, it must progress through the various phases of growth and reproduction that constitute what is called the cell cycle (Fig. 2-2). The principal components of the cell cycle are the replication nuclear DNA and its distribution among the progeny cells (i.e., daughter cells). The first phase of the cell cycle is called G1, and it is here that all the enzymes, nucleic acids, and other factors are produced that will enable DNA replication or “synthesis,” which occurs in the S phase. Once the DNA is replicated, a period of cell growth and duplication of cellular proteins and structures occurs in a period called G2. After this cell growth, the actual distribution of the replicated DNA and the physical division of the parent cell into two daughter cells occur in the M phase, which takes approximately 1 hour. After cell division, the cell cycle process can begin again, or the cell may enter a state of rest called G0.

The signal to divide can come from either internal factors or exogenous growth factors, which bind to cell surface receptors and stimulate a cascade of events, which lead to division. Important internal control mechanisms exist at the genetic level, which help regulate cell cycling. The “negative regulatory” genes that code for proteins inhibiting abnormal cell proliferation are called tumor suppressor genes. A mutation or loss of a tumor suppressor gene is a basic step in the development of many human cancers. Cell cycling can also be abnormally induced by cellular oncogenes, which naturally exist within cells but are typically kept dormant either by tumor suppressor genes or other regulatory mechanisms. Loss of negative regulation from tumor suppressor genes or amplification or mutation of the actual oncogene has also been associated with cancer formation.

Continued molecular research will provide further understanding of cell cycle regulation and may lead to novel therapies for treating human cancer. A discussion of basic molecular biology techniques, which have provided the means to understand gene expression, regulation, and cell cycling, is found in Chapter 16.

Gene Therapy

The basic principle of gene therapy is that the intrinsic expression of certain genes in body tissues can be modified to treat disease. This may involve replacing a defective gene with a functional version, enhancing the baseline expression level of a gene or, contrastingly, suppressing the expression of genes that may contribute to the pathologic process. As the field of gene therapy continues its rapid advancement, it is likely that gene therapy will eventually become a standard clinical regimen available to both internists and surgeons.

Enhancing Gene Expression

In the past decade, it has become clear that gene therapy may have its most immediate and effective role in treating more common acquired diseases, including various cancers, arthritis, and atherosclerosis.4,5 In order to tackle these conditions, new approaches have been adopted that focus on increasing the expression of beneficial genes beyond normal levels. For example, in a clinical trial for the treatment of melanoma, tumor-infiltrating lymphocytes (TILs) were purified from a tumor at the time of surgical resection.6 The TIL cells were then grown in the laboratory, and the gene for tumor necrosis factor (TNF) was introduced into the cells.7 The genetically engineered cells were subsequently transfused into the patient and preferentially migrated to the site of residual tumor, delivering a therapeutic dose of TNF. As the molecular basis for disease is elucidated, there is likely to be increasing interest in this form of gene therapy.

Suppressing Gene Expression

Most recently, as the molecular pathways that underpin disease—particularly cancers—are unraveled, there has been excitement about the possibility of suppressing key genes known to be involved in the disease process. For example, RET, a proto-oncogene implicated in the pathogenesis of medullary thyroid carcinoma, can be inactivated by inducing expression of a mutant form of the RET gene, resulting in tumor regression.8 This approach is dependent on the “dominant negative” effect where the mutant gene encodes a protein that retains binding ability to other key proteins that normally interact with the wild-type gene. However, the mutant protein is nonfunctional, creating a competitive inhibition of the wild-type functional variant. Dominant negative therapies are presently being translated to clinical trials in the hope of developing novel treatments, particularly within the field of oncology.

A new technique that may have immense potential for the future of gene therapy is RNA interference (RNAi), a method by which genes can be selectively “silenced.” RNAi is a ubiquitous mechanism that is used by the cell to effectively “turn off” specific genes, as described a decade ago in a landmark paper.9 In this study, injection of double-stranded RNA consisting of both sense and antisense strands into the nematode Caenorhabditis elegans profoundly silenced the gene with a sequence complementary to the dsRNA. The potential for RNAi in research and therapeutics was immediately recognized. In effect, any gene, including those involved in cancer, could be silenced and the effect of this loss of function on phenotype could be elucidated. The scientists who discovered RNAi were awarded the Nobel Prize in Medicine in 2006, only 8 years after publication of their initial, landmark paper.

Subsequent studies have partially determined the mechanism for RNAi (Fig. 2-3). In brief, an RNAase enzyme called Dicer cleaves dsRNA to form 21- to 23-nucleotide fragments of RNA. This short-interfering RNA (siRNA) is the actual effector molecule in RNAi.1012 The siRNA is integrated into the RNA-induced silencing complex (RISC), after which the sense strand of the duplex is discarded. The antisense siRNA is retained and acts as the “guide strand,” directing RISC to mRNA molecules with a complementary sequence.13 RISC cleaves the targeted mRNA, thereby silencing gene expression by blocking the translation of the mRNA sequence to protein.10,14

Considerable progress has been made in applying RNAi to mammalian systems. Transfection of mammalian cells with long dsRNA molecules of more than 30 nucleotides was found to induce a significant host antiviral interferon response that produced a general silencing of untargeted genes and often cell death. This hurdle has recently been overcome by chemically synthesizing 21-nucleotide siRNA molecules and using these to induce efficient, targeted gene silencing in mammalian cells without inducing an interferon response.11 These siRNAs can be used in subnanomolar concentrations to achieve greater than 90% reduction in mRNA levels, a highly potent gene-silencing effect.10 Recent animal experiments involving siRNAs have indicated that immune system activation is relatively uncommon, bolstering the safety profile of these novel therapies.

Unfortunately, a persisting problem associated with RNAi techniques are “off-target” effects. This refers to unintended siRNA-mediated silencing of genes that have similar nucleic acid sequences. Even small amounts of sequence homology, as few as seven base pairs, are enough to produce off-target effects. Several computer algorithms have been designed to aid in the selection of siRNAs with minimal off-target effects. It is unclear what effect, if any, off-target effects will have in human trials. In fact, early human trials of RNAi therapies for the treatment of macular degeneration and neurodegenerative diseases have not revealed any significant side effects.12

Somatic versus Germ Cell Gene Therapy

Gene therapy has two possible target cell types. The first and presently used targets are the somatic cells, or those cells that constitute the organs and postnatal tissues of the body. The second potential target is the germ cells, or those cells that produce the sperm or ovum and are passed on to a person’s offspring. Many different organs and cell types are targets for somatic gene therapy, including bone marrow, liver, tumor cells, muscle, skin, endothelium, thyroid, and others (Fig. 2-4). Genetic manipulation and therapy at these sites does not alter the inherited genetic material and raises few novel ethical or social issues.15 Genetic manipulation of the sperm or ovum, however, could prevent inherited diseases by altering the genetic constitution of offspring. Although this is an appealing idea on the surface, there are serious technical and safety issues as well as profound ethical concerns involved. Currently, gene therapy is restricted to somatic cells, and genetic manipulation of human germ cells would be prohibited under existing recombinant DNA guidelines.16

Permanent versus Temporary Gene Therapy

A common perception is that the goal of gene therapy is to “permanently” modify the expression of the therapeutic gene in the patient. Permanent gene therapy, however, may not be necessary or optimal in a clinical setting. As a general principle, permanent gene therapy could prove more desirable when the methods required for introducing the genetic material into the patient involve significant surgical procedures or substantial risk to the patients such as in the case of organ resection, cellular transplantation, or stereotactic injection under anesthesia. To achieve permanent gene therapy, the expression of the target gene should be either enhanced or blocked indefinitely. The gene must also demonstrate appropriate regulation in response to both normal and pathologic situations, and there should be no detrimental consequences of this gene expression in later life. In considering diseases such as growth hormone deficiency or juvenile diabetes, correction by permanent gene transfer would require precise regulation of the gene products to ensure both short-term efficacy and long-term safety. This tight control and regulation of transferred genetic material is not feasible with present techniques.

For many diseases such as cancer, cystic fibrosis, arthritis, and disorders requiring surgery, “temporary” gene regulation in a patient’s tumor, residual tumor, or other target cells could produce a selected beneficial therapy over a limited period of time. For example, the treatment of a tumor or area of residual tumor after surgery may only require a one-time, limited expression of a gene that either produces a substance directly toxic to the cancer cell or a cytokine or other factor that can initiate an antitumor immune response. Instead of permanent gene therapy, repeat gene transfer over a period of time could also prove effective, as in the case of radiation therapy or possible chemotherapy regimens. With respect to surgical procedures, the use of gene therapy to enhance wound healing or tissue regeneration after an injury might only require gene expression for a period of days or weeks. Another new frontier on the near horizon is minimally invasive or noninvasive routes for gene delivery, such as intramuscular, intravenous, oral, or even aerosol administration. This novel future application could allow the establishment of steady-state gene product levels, which parallels present medical regimens and enables the physician to adjust the dose and schedule of administration to the patient’s needs.

Methods of Delivering Gene Therapy

A major focus in the field of gene therapy is the development of vehicles for introducing genetic material into selected target cells. Two general gene therapy delivery methods can be distinguished: (1) direct administration of genetic material, which involves the injection of DNA or RNA into cells in order to modify gene expression, and (2) viral-mediated gene transfer, which involves packaging a therapeutic gene into a defective virus particle and using the natural process of viral infection to introduce the genetic material. The purpose of viral-mediated gene transfer is to exploit the efficient and often complex mechanisms that viruses have evolved to introduce their viral genes into human cells during infections.

The gene that is delivered to a target cell is not itself therapeutic. Rather, it is the product encoded by the gene, which is responsible for the resulting therapeutic effect. The gene product is typically a protein, which has a specific function such as a hormone or cytokine, but could also be a bioactive RNA molecule such as a gene-silencing siRNA that alters the regulation of pathologic processes. Thus, although gene therapy commonly focuses on methods for delivering genes to cells, it is the ability to achieve expression of the gene product at therapeutic levels that ultimately determines the effectiveness and efficacy of therapy.

Direct Injection of Genetic Material

DNA-Mediated Transfer of Therapeutic Genes

The process of DNA-mediated gene transfer is called transfection, and the vehicle through which genetic material is transferred into a cell is called a vector. Functional DNA vectors are circular molecules of DNA called plasmids that contain various additional genetic elements required to achieve expression of the gene product at therapeutic levels (Fig. 2-5). Included in these are the special elements (promoter and enhancer) that direct gene expression and elements that determine the processing and persistence of genetic material within the cell. Plasmid vectors can be used to effectively deliver therapeutic genes to target cells. Plasmids have also been used to prolong the transient, 3- to 7-day gene-silencing effect associated with the transfection of naked siRNAs.

The delivery of DNA vectors into cells is possible via a variety of techniques. One classic method is to simply microinject plasmids directly into the cell nucleus.17 This technique is both time-consuming and inefficient for achieving large numbers of transfected cells. Although this method is common in the laboratory with in vitro studies, its technical limitations prohibit effective application to living animal models or human subjects. A more efficient process, which also is limited to in vitro application, is the process of electroporation, in which cultured cells are exposed to DNA in the presence of a strong electrical pulse.18 The electrical pulse creates pores in the cell membrane, which allows electrophoresis of plasmid DNA into the cell.

It is possible to effectively introduce genes into muscle19 or thyroid20 simply by injecting DNA into these tissues in vivo, where the process of endocytosis enables cellular uptake. Gene transfer into other organs requires special methods to enhance the uptake of DNA into the specific target cells. A common alternative method is the use of cationic lipids, which encase DNA vectors and fuse with the target cell membrane21 to enhance intracellular gene uptake. This process has been termed lipofection. Another method is to couple the DNA vector to proteins that bind to specific receptors on the target cell leading to uptake of the DNA by receptor-mediated endocytosis.22,23 A novel approach to DNA vector delivery involves the use of the “gene gun,” which uses electrical currents to project microscopic gold beads coated with plasmid DNA into target tissues and organs.24

An important point to understand is that DNA-mediated gene transfer typically results in only transient residence of the therapeutic genes in the targeted cell. DNA vectors, which are introduced into cells, are degraded and eliminated from the cell over time. Different cell types eliminate the introduced genetic material at different rates. In muscle, for example, DNA vectors may persist in cells for many months and continue to express gene products.19 In contrast, DNA vectors injected into the thyroid have a shorter half-life and the gene product is eliminated after 2 days.20 Vectors introduced into the liver are eliminated with a half-life of approximately 1 to 2 hours and expression is significantly reduced after 6 to 24 hours.25

Although permanent incorporation of genes into cells occurs rarely after DNA-mediated gene transfer in cultured cells (less than 1 : 105 cells), this phenomenon has not been observed in vivo. DNA vectors are therefore considered “safe” because they do not incorporate into the recipient cell’s chromosome in vivo and thus should not induce the theoretical risks associated with altering the cell’s genome. Moreover, they have not demonstrated significant toxicity to recipient tissues and have not induced any systemic immune response. DNA vectors can thus be delivered repeatedly, which overcomes the potential limitation of transient therapeutic gene expression. The transient nature of gene expression does have an advantage in certain clinical scenarios because the therapeutic gene can be administered by conventional oral, intramuscular, or intravenous routes to provide its beneficial effect over a predictable and extended period. In contrast to conventional medications with short half-lives, DNA-mediated gene therapy could lead to prolonged expression of a gene product at continuous levels, eliminating the need for continuous infusions or enhancing compliance by minimizing the frequency of injections.

Direct Delivery of RNA for Gene Silencing

RNA can also be introduced into cells, opening the door to potential therapeutic implementation of gene silencing. Intravenous administration of siRNA targeting the VEGF gene, for example, has been shown to significantly reduce tumor volume and intratumoral VEGF levels.26 Unfortunately, the gene-silencing effect of synthetic siRNAs lasts only minutes to a few days before they are degraded. Chemical modification of the RNA can improve stability. The plasma half-life of an unmodified siRNA in a mouse model was 0.03 hour, the half-life of a chemically modified siRNA was 0.8 hour, and the half-life of a lipid-conjugated, modified duplex was 6.5 hours.27

Despite the improved stability of modified siRNA, plasma clearance is still rapid and any potential therapeutic benefit will require using chemically modified RNA in combination with some kind of delivery system.10 Liposomes have been successfully used to deliver siRNA in vivo, and significantly improve both the duration and level of gene suppression compared to nonvector delivery methods. More recently, nanotechnology has been used to design compounds consisting of a cationic polymer that binds the RNA cargo, a neutral “stealth” coating to hide the particle from immune surveillance and a targeting ligand. This type of nanoparticle has been used to selectively target neuroblastoma tumors in nude mice with anti-VEGF receptor siRNA, resulting in a dramatic antitumor effect.28 Electroporation and “gene gun” techniques have also been used to effectively deliver siRNA in vivo.10

The most common method used to date for the delivery of siRNA is termed hydrodynamic delivery. This approach involves injecting a large volume of an RNA-containing solution as a rapid intravenous bolus. The hydrostatic pressure generated transiently disrupts the vascular endothelium and achieves siRNA delivery to tissues. To date, hydrodynamic delivery has been successfully used to deliver siRNA to skeletal muscle in rats, dogs, and rhesus macaques.29 Localized delivery can be achieved by application of a tourniquet to isolate a limb before intravenous injection. The potential for this approach in humans is under investigation and will likely transition to clinical trials soon.10

The need for sustained gene silencing has led to the development of novel DNA plasmid expression vectors that direct the synthesis of short RNA over a prolonged period (see Fig. 2-3). The encoded short RNA sequence is transcribed and processed into the effector gene-silencing RNA molecule. One method involves genetically engineering a plasmid to encode a short-hairpin RNA (shRNA). A shRNA molecule consists of a special double-stranded RNA molecule that includes a sharp hairpin turn and is processed in the cell to yield a 21-nucleotide RNA construct capable of gene silencing. Use of DNA plasmid vectors can prolong in vivo mammalian gene silencing for up to several weeks.12 They are, however, limited by their inability to transfect nondividing cells.11 This limitation can be overcome, however, through the use of viral vectors to deliver siRNA, which is discussed in the next section.

Viral-Mediated Gene Transfer

The majority of research to date has focused on developing methods for using viruses as vectors. Viral-mediated gene transfer involves the construction of synthetic virus particles that lack pathogenic functions, are incapable of replication, contain a therapeutic gene within the viral genome, and can deliver this gene to cells by the process of infection. Certain viruses have the property of permanently integrating their genes into the chromosomes of the infected cell; therefore select forms of viral-mediated gene transfer can lead to permanent gene therapy. Furthermore, viral-mediated gene transfer has significant advantages over plasmid-based delivery of siRNA to target tissues, in that constructs encoding siRNA can be introduced to both dividing and nondividing cells including neurons, blood, and bone marrow. By altering the type of viral vector used, siRNA expression can be designed to be transient in, for example, cancer therapy or permanent in the case of genetic diseases.


The original prototypes for viral-mediated gene transfer are retroviral vectors derived from the Moloney murine leukemia virus.30,31 Retroviral vectors were chosen as vehicles because of several useful properties. First, “defective” virus particles can be constructed that contain therapeutic genes and are capable of infecting cells, but which contain no viral genes and express no pathogenic viral gene products. A general scheme for constructing a defective retroviral particle is illustrated in Figure 2-6. Second, retroviral vectors are capable of permanently integrating the therapeutic genes they carry into the chromosomes of the target cell. Because of this property, retroviral vectors are well suited for treating diseases that require permanent gene expression. Third, modifications can be made in retroviral vectors and in the cell lines producing vectors that result in enhanced safety features.

A major limitation for this strategy is that retroviruses will only integrate into actively dividing cells and the efficiency of retroviral infection is relatively low. It is therefore difficult to generate the large numbers of transduced cells that are required for effective gene expression. This shortcoming has been addressed to some extent with the development of lentiviral vectors, which are related to retroviruses but are capable of inserting into the genome of nondividing cells. Perhaps the most serious problem, however, has been the difficulty in achieving stable, regulated gene expression from retroviral vectors in cells despite the permanent genomic integration. Cells are apparently able to shut off expression from retroviral vectors under certain conditions that have not been clearly defined.

Previous experience in animal models32,33 and initial experiences in clinical trials suggest that these vectors are, generally, safe. However, there have been serious complications in clinical trials using retroviral vectors to treat X-linked severe combined immunodeficiency. Although gene therapy reconstituted the immune system and produced excellent clinical outcomes in most children, several cases of T-cell leukemia were reported 2.5 to 5 years after therapy.34 This has been directly attributed to the insertional oncogenesis that can occur when the retroviral vector permanently inserts into the host genome and activates nearby cellular proto-oncogenes. The powerful promoters used to drive expression of the therapeutic gene may also activate nearby genes.35 Consequently, newer generation retroviral vectors are in development that use weaker promoters to minimize trans-activation of genes. Special “insulator” sequences can also be used to block the transcriptional elements of the vector from interacting with cellular genes. Another option is to include “suicide” genes in the vector that will initiate cell death in overproliferating cells.36 These methods have shown early promise but have yet to be used in a clinical trial.


A recent focus of gene therapy has been the development of adenovirus vectors as powerful and effective vehicles for gene transfer.37 An overview for the construction of a replication-defective adenoviral vector is shown in Figure 2-7. Adenoviral vectors differ from retroviral vectors in that they remain episomal; that is, they do not integrate their genes into the target cell’s chromosome. Compared with retroviral vectors, adenoviral vectors demonstrate the significant advantage of infecting a wide variety of both dividing and nondividing cells in vitro and in vivo with a high level of efficiency.3840 Using adenoviral gene transfer, expression of the therapeutic gene is possible for a period of several weeks to months. More recently, adenoviral vectors have been designed to encode and transiently express shRNA, which may be particularly useful in therapeutic strategies against cancers where long-term RNAi is not necessarily desirable.12

First-generation adenoviral vectors contained a deletion of portions of their E1a and E3 regions with the former acting as a “master switch” regulating the expression of other viral genes that are critical for viral replication. Consequently, all first-generation adenoviral vectors are replication-deficient. However, despite these modifications, several of the remaining viral genes are still expressed, markedly increasing immunogenicity.4

Only gold members can continue reading. Log In or Register to continue

Stay updated, free articles. Join our Telegram channel

Jun 5, 2016 | Posted by in OTOLARYNGOLOGY | Comments Off on Fundamentals of Molecular Biology and Gene Therapy

Full access? Get Clinical Tree

Get Clinical Tree app for offline access