Applied Radiology

Dr. Chen is a postgraduate year 4 radiology resident at Baystate Medical Center in Springfield, MA. She graduated from Tufts Medical School in 1994 and completed several years of surgery residency at Rhode Island Hospital before switching to radiology.

The Eagle Pub on Bene't Street in Cambridge, England, has the distinction of once being the haunt of a brilliant, brash young Englishman named Francis Crick and a brilliant, even brasher and younger American named James Watson. On the last day of February 1953, Crick burst through the pub's doorway and announced to all within earshot that he and Watson had found the secret of life. The secret that Watson and Crick (or Crick and Watson, as they are referred to on the Eagle's side of the Atlantic) had uncovered was the shape of a now familiar molecule called deoxyribonucleic acid (DNA). ¹ A Swiss scientist had isolated the chemical from the white blood cells of pus in 1869, but neither he nor anyone else had any idea of its importance to life. It was just before Watson and Crick began their collaboration that an American bacteriologist named Oswald Avery demonstrated that DNA was the stuff of genes--discrete, inheritable packets of information aligned along the chromosomes inside the cell's nucleus. ^2,3

DNA is the basic genetic material that determines our genetic makeup and can be reproduced and passed on to subsequent generations. Within the nucleus, the DNA is packaged into 23 pairs of chromosomes, half of which are derived from each parent. Chromosomal DNA is wrapped tightly around histone proteins, which allows it to remain condensed within the nucleus. ^4,5

A gene is a stretch of DNA that contains the blueprint for the sequence of amino acids making up a particular protein. Cellular homeostasis is maintained by genes encoded for structural proteins and cytoskeletal elements along with genes responsible for cellular replication, protein, and nucleic acid. Mammalian genes consist of multiple noncontiguous segments of DNA coding sequences (exons) separated by varying lengths of noncoding intervening sequence DNA (introns). Both upstream and downstream regulatory elements control which genes are actively expressed and in which cell type (figure 1). These elements, called promoters or enhancers, are species-, tissue-, and cell-type specific and can be a target for the disruption of regulated cell growth. Such disruption can lead to neoplastic transformation of the cell. ^5,6

If a particular gene is mutated, it may not be able to produce protein or it may function poorly or even too aggressively. This disrupts cell and tissue functions that depend on the normal gene product and causes abnormal cell behavior, leading to symptoms of disease. Simply put, gene therapy is the deliberate transfer of DNA for correction of mutated genes, which can be carried out by introduction of a normal gene into the cell or by direct repair. ^3,7

Science of the construct

DNA cloning

Certain viruses have evolved to exploit cellular nucleic acid and protein synthesis to ensure their own replication and survival. Viruses are ideal vectors, being naturally suited for delivery of genetic material into target cells. DNA viruses may exist extrachromsomally as self-replicating episomes or may integrate into the host genome. Ribonucleic acid (RNA) viruses, termed retroviruses, reverse the normal sequence of events by converting their RNA genome back into DNA by using a viral enzyme called reverse transcriptase. The DNA thus produced is integrated into the genome; host transcription and translation machinery is then usurped for viral production. Discovery of reverse transcriptase was a Nobel prize-winning landmark in molecular biology and allowed DNA coding sequences to be derived from cellular messenger RNA (mRNA), thus paving the way for gene cloning. ^8,9

Gene cloning exploits the fact that coding sequences have been separated or spliced from intervening sequences during the process of transcription and mRNA production. After purification of mRNA from the cell, single-stranded mRNA molecules are reverse transcribed back into double-stranded DNA by using retroviral reverse transcriptase. Reverse transcriptase then synthesizes a second complementary DNA (cDNA) strand by using the first as a fresh template. This process yields a double-stranded DNA molecule that contains all of the relevant coding sequences present in the original genomic DNA gene sequence, but without the often millions of bases of noncoding intervening sequence DNA. Polymerase chain reaction is currently the method of choice for amplifying the cloned DNA into sufficient quantities (figure 2).

Preparation of the reverse transcription-polymerase chain reaction cDNA for cloning into an expression vector is performed by adding specific linker sequences onto the cDNA. Vectors are DNA constructs (usually derivatives of bacterial plasmids ["naked DNA"]) that allow sequences contained within them to be shuttled about between various cell types (figure 3). After cloning in the expression vector, transfection into bacteria such as Escherichia coli allows large-scale manufacture of milligram-to-gram quantities of the cDNA-vector complex. The cloned cDNA must first be purified from the bacteria, after which it is suitable for transfer into the gene delivery vehicle of choice (figure 4). ^8-11

Vectors: A Trojan horse

An ideal vector should theoretically have the following properties. It should provide ease of penetration and sustained expression. It should deliver an adequate amount of therapeutic genes with high enough concentration to allow many cells to be infected. It should be able to integrate within the active regions of the genome that replicate and should be responsive to manipulation of its regulatory elements. Naturally, it should pose no hazard to the patient. It should be efficient and selective; it should be able to target the desired type of cell and have no components to elicit an immune response. Finally, it should be conveniently produced in large quantities.

Genes in the form of DNA are typically delivered to a target cell by one of three methods: (1) packaging them into a virus (adenovirus or DNA virus, retrovirus or RNA virus); (2) directly applying them in the form of a plasmid or a plasmid in a synthetic delivery system ("nonviral vector"); or (3) by delivering them through physical means such as direct injection, electric pulse discharge (electroporation), gene guns, or radiofrequency pulses. Once inside the cell, transgenes can be expressed in different locations: within the cytoplasm, in the nucleus, or episomally integrated into the host genome. Cytoplasmic expression is a useful technique of gene transfer since the construct does not need to be transported into the cell nucleus. However, the co-delivery of RNA polymerase is required to initiate transcription. All other categories of transgene expression necessitate DNA penetration into the nucleus. Once the gene is incorporated into the genome, the duration and level of foreign gene expression is usually long. ^11,12

Viral vectors-- Current gene technology has focused primarily on the use of viral vectors, which can provide highly efficient transduction and high levels of gene expression, as several viruses have efficient mechanisms for transferring genetic material to target cells. The precise design of a particular viral vector depends largely on the type of virus used. ^12-14 Table 1 lists specific disease applications of various viral delivery systems.

Adenovirus: Adenoviruses contain double-stranded DNA and enter the cell by receptor-mediated endocytosis. Their genomes do not integrate into the host and therefore have no oncogenic potential. Adenoviral vectors are capable of high-level transduction in cells of multiple lineages, including hepatocytes. ^15-18 Because of the relative safety of replication-deficient infectious adenoviruses and the ability to produce high titers in vitro, adenoviruses are widely used in gene transfer. They are easy to construct and are generally not perceived as toxic to cells. Nonetheless, adenovirus is quite immunogenic, and antiviral immunity is the primary factor limiting successful re-administration. Also, because adenoviruses do not integrate into the host genome, an important limitation is unstable expression resulting from the loss of the recombinant gene from the transduced cell. ^19-21 In addition, hepatic side effects have been reported after in vivo administration. These side effects are apparently due to expression of vector-encoded viral sequences and production of associated viral proteins. ¹³

Retrovirus: Retroviruses contain a single-stranded RNA genome. They are RNA viruses that replicate through a DNA intermediate synthesized by viral reverse transcriptase. Retroviral-cDNA constructs, like adenovirus, are replication-deficient and must be transfected into packaging cell lines to allow encapsulation of viral sequences into an infectious retroviral virion. ^22,23 These viruses enter the cell by direct fusion to the plasma membrane and then integrate in stable form into the host chromosome during cell division. The high efficiencies of retroviruses in transduction and stable gene delivery make them attractive vehicles for gene delivery. ²⁴ However, retroviruses infect only dividing cells, a property that limits potential delivery of therapeutic cells to nondividing cells, slowly growing tumors, or nonmitotic fractions of the tumor populations. Retroviral vectors also have a restricted size capacity of approximately 8 kilobases (8000 bases), which limits its use with large genes. Further important limitations include inactivation by serum complement, low serum titers of vector after in vivo administration, and the appearance of replication-competent retroviruses during large-scale production. ²⁵

Amplicon: More recently, herpes simplex virus (HSV)-derived amplicons have been used as an alternative method of gene delivery. ²⁶ Amplicons are plasmids that can be packaged into recombinant viral particles and used to transfer anticancer genes in vivo and in vitro, although the vector itself does not possess oncolytic functions. Amplicons can be packaged as multiple copies into each viral particle, making it a more efficient method of plasmid delivery. Another advantage of amplicons is that they are maintained extrachromosomally (episomally) in replicating cells, assuring passage to dividing daughter tumor cells. Finally, amplicons, like adenoviruses, show little toxicity.

Other viral delivery systems: A number of other viral delivery systems are currently under development. These include poxviruses, such as the Sindbis and Semliki Forest viruses. These allow viral protein expression without viral replication and would therefore be useful for vaccination. Lentiviruses, including human immunodeficiency virus and simian immunodeficiency virus, have the advantages of high efficiency of expression, trophism for immune cells, and stable expression in nondividing cells. ^26,27 Lastly, adeno-associated viruses (AAV) are simple, single-stranded DNA viruses; AAV vectors can infect nondividing cells, do not elicit an immune response, have long-term expression, and can integrate into chromsomes at specific sites. Their disadvantages are: they are smaller viruses with restricted usefulness because they can carry only a small genetic payload; they insert themselves randomly into chromosomes with risk of disrupting functional genes; and, finally, they are difficult to manufacture in large quantities. ²⁴

Nonviral, artificial vectors-- Despite the ability to transfect cells efficiently, viral methods for human gene therapy have several limitations. Viral particles are often unstable and can have low titers. Secondly, viral particles are often immunogenic. Thirdly, a pathologic replication-competent virus may be generated. Finally, oncogene activation and cancer development may occur. To circumvent these potential problems, transfection methods using nonviral artificial vectors carrying plasmid DNA (naked DNA) have been developed. Other nonviral synthetic vectors for gene therapy have included a variety of compounds--for example, cationic liposomes, poly-L-lysine-DNA complexes, DNA-coated microprojectiles (gold particles), dendrimers, or even free-plasmid DNA. Liposome-mediated gene transfer has been utilized extensively in in vitro studies ("lipofection"). Liposomes are formed from phospholipids similar to those found on cell membranes of living organisms. These synthetic, lipid bubbles act as carriers for plasmids whose original genes have been replaced by therapeutic ones. Because liposomes consist of a double layer of lipid molecules similar to those of animal cells, a liposome can fuse with a cell and deliver its contents into the cellular interior. ^28,29 A drawback is that the internal diameter of a liposome is much less than the longest DNA plasmid. This presents a size restriction for incorporation of "long" DNA plasmids into the carrier liposome and limits this method of transfer. ^30-32 Finally, ionic poly-L-lysine DNA complexes have been attached to target molecules such as asialoglycoprotein or transferrin. Although these nonviral vector model systems seem to work some degree ex vivo, gene transfer is generally lower in vivo. ³³

Gene therapy applications

Genetic deficiencies

In the setting of genetic deficiences, the goal of therapy is restoration of a particular function in cases in which a genetic mutation has led to deficient or absent expression of a critical gene product. This has been applied to several diseases (e.g., cystic fibrosis [transmembrane conductance regulator], adenosine deaminase deficiency, or Gaucher's diseases [beta-glucocerebroside deficiency]). ³⁴ Table 2 further elaborates candidate diseases for gene therapy and corresponding approaches for treatment. Additionally, chronic pathologic processes may be amenable to gene therapy with the goal being delay or cessation of chronic progressive disease, i.e., peripheral vascular disease and vascular gene therapy. ³⁵ Fischer has presented an extensive review of vascular gene therapy application and delivery. ⁶

Gene therapy for cancer

Many gene therapy protocols to date have concentrated upon treatments for cancer. Experts predict that the major emphasis in human gene therapy experimentation over the next few years will be directed toward the treatment of cancer. ^34,36 Though many cancers have a genetic predisposition, they all involve acquired mutations, and as they progress, their cells become less differentiated and more heterogenous with the respect to the mutations they carry. In general, cancers have one mutation to a proto-oncogene (yielding an oncogene) and at least one to a tumor suppressor gene, allowing the cancer to proliferate. The range of different cancers and the mutations they carry have led to a variety of strategies for gene therapy, namely oncogene inactivation, tumor suppressor gene replacement, immunopotentiation, molecular chemotherapy, and drug resistance genes. Emphasis will be on genetically modifying cells of the body's own immune system to fight tumors. In this way, the same genes may be used to treat many different forms of cancer. ³⁶

Cell growth versus apoptosis: Shifting the balance­­ Cellular proliferation is an intricate balance between signals driving cell cycle progression, signals maintaining quiescence, and signals initiating the pathway for cellular destruction (apoptosis). Signal transduction is the process of converting extracellular signals into cellular function and is tightly regulated. Apoptosis is a normal process by which senescent cells undergo programmed cell death. Apoptosis is critical for the body to ensure that aged or potentially damaged cells are targeted for elimination from the system (figure 5). Immunologic protection often involves induction of apoptosis (e.g., in infected or neoplastic cells). Development of malignancy has been shown by numerous studies to involve aberrant or absent apoptosis of transformed neoplastic cells. ^37-39 One signal transduction approach to therapy involves shifting the balance back in favor of cell death by inserting genes, such as the fas ligand gene, that are involved in triggering apoptosis. Antibodies directed against fas ligand expressed on the tumor cell may also mimic fas/fas ligand interactions and trigger the apoptotic death of the tumor (figures 6 and 7). ³⁸

Gene therapy approaches to cancer-- Oncogene inactivation: Oncogenes are cellular genes that play a role in regulating normal cell growth and proliferation. Expression of certain oncogenes related to normal growth factors or their receptors, such as the erb-2B and ras oncogenes, may result in disregulated intracellular signaling, which leads to uncontrolled neoplastic proliferation. Oncogene activity can also be inhibited with antisense RNA, intracellular antibodies, catalytic RNA molecules (ribo-zymes) that inactivate or digest oncogene RNA, or antioncoprotein antibodies designed to block aberrant signal transduction. ⁴⁰

Tumor suppressor gene replacement: Anticoncogenes, also known as tumor suppressor genes, are implicated in the neoplastic transformation of many tumors. These genes are termed antioncogenes because it is their absence rather than their presence that leads to malignancy. ^41-43 Figure 8 illustrates the role that one particularly ubiquitous tumor suppressor gene, p53, plays in regulating tumor cell apoptosis. ^44-46 Preliminary results from one phase I trial in which retroviral-p53 gene complexes were injected into refractory, p53-deficient, primary non­small-cell lung carcinoma showed tumor regression in one-third of patients and stabilization of disease in an additional one-third. ⁴⁷ Moreover, expression of p53 is synergistic with chemotherapeutic drugs, such as cisplatin, ⁴⁴ and adjacent tumor cells that have not been transduced are killed. This is termed the bystander effect . ⁴⁷ Other tumor suppressor genes include BRCA1sv, the retinoblastoma (RB) gene (also found in prostate and bladder tumors), and the Wilms' tumor (WT) gene, also implicated in acute myelogenous leukemia. ^48-50

Immunopotentiation: The aim of immunopotentation is to enhance the response of the immune system to cancers, thereby leading to their destruction. Passive immunotherapy aims to increase the pre-existing immune response to the cancer, while active immunotherapy initiates an immune response against an unrecognized or poorly antigenic tumor. ^49-52

Passive immunotherapy usually involves harvesting tumor-infiltrating lymphocytes and treating them to express increased cytokines. Various immunostimulatory cytokines have been inserted into tumors and shown to increase antitumor immunity. ⁵³ These cytokines include IL-2, GM-CSF, Il-4, Il-12, tumor necrosis factor (TNF), and interferon (INF). Paracrine secretion of cytokine by the tumor elicits local immune activation, which enhances antitumor immunity. Alternative strategies modify the host immune cells directly. Lymphocytes are known to infiltrate tumors but somehow remain suppressed and anergic. Gene transfer approaches are being explored to enhance the activity of tumor-infiltrating lymphocytes, as well as peripheral blood lymphocytes, by inserting immunostimulatory cytokine genes such as IL-2, TNF, and INF into the lymphocytes, thus allowing both autocrine and paracrine activation of antitumor immunity. Data from early clinical trials have indicated augmented antitumor immune reactivity in the gene-modified lymphocytes, although few cases of sustained tumor remission have been documented. ^53-55

Active immunotherapy genetically modifies tumor cells to increase expression of antigen presenting molecules/costimulatory molecules, local concentrations of cytokines, or tumor antigens. The cells are then irradiated prior to being returned to the patient, preventing the reintroduction of replication-competent tumor cells. These approaches have been termed cancer vaccines . Tumor vaccines are genetically modified tumor cells or peptide products of tumor that are used to "vaccinate" patients against their own tumors. ⁵⁶

A strategy currently in clinical trials for metastatic melanoma is based on the observation that creating an immunostimulatory environment in and around a tumor can activate lymphocytes that have infiltrated the tumor but have remained quiescent. This strategy involves partial tumor resection and insertion of a gene that codes for an immunostimulatory cytokine (e.g., IL-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF) into the resected tumor cells. After several cycles of cell division in the laboratory to allow for expression of the cytokine gene, the modified tumor cells are irradiated to prevent further cell division and returned to the patient with a depot injection. The modified tumor cells are still able to synthesize and secrete cytokine, which produces paracrine effects on infiltrating immune cells. ⁵⁴ To circumvent difficulties in maintaining tumor cells in the laboratory, autologous fibroblasts have also been transduced with cytokine genes and injected together with irradiated tumor cells. ⁵⁷

Costimulation: Another way tumor cells are genetically modified for active immunotherapy is increasing the expression of costimulatory molecules. One way in which tumors evade the immune system is by means of loss or alteration of these important costimulatory molecules. ⁵⁸ The tumor-lymphocyte interaction is facilitated by the costimulatory ligand-receptor (B7-CD28) interaction. B7 is absent in many tumors, a condition that leads to tumor-specific anergy and thus deficient antitumor immunity. Re-establishing B7 expression by insertion of the B7 gene into B7-deficient tumors has been shown to facilitate immune recognition by tipping the balance away from anergy and back in favor of protective immunity. ⁵⁹ An alternative approach to increasing tumor immunogenicity is to express foreign HLA antigens in the tumor by means of direct intratumoral transfer, which causes the tumor to be "rejected" by the host immune systems just as a foreign-tissue transplant might be rejected. Phase I trials in which this approach has been used in patients with metastatic melanoma have demonstrated significant antitumor activity and, thus, the feasbility of this approach. In some cases, complete regression of residual disease has been documented. ^58,59

Molecular chemotherapy and drug resistance genes: Molecular chemotherapy and drug resistance genes direct chemotherapy drug production by the tumor itself. A suicide gene that encodes a prodrug converting enzyme, such as herpes simplex virus-thymidine kinase (HSV-TK), is inserted into tumor cells. ⁶⁰ A specific nontoxic prodrug (ganciclovir) is then administered systemically. On uptake by suicide gene-expression tumor cells, the prodrug is converted into a toxic antimetabolite, which leads to tumor cell death. ⁶¹

Two systems have been extensively studied. The first is HSV-TK, in which intratumoral expression of the HSV-TK allows ganciclovir to be converted into a toxic triphosphate derivative, a process that leads to early DNA chain termination. The second system involves cytosine deaminase in which the cytosine deaminase gene expressed by tumor cells allows 5-fluorocytosine to be converted into the antimetabolite 5-fluorouracil. Local release and bystander effects on adjacent cells offer a theoretical solution to often inefficient gene transfer into cells. Indeed, Ogawa et al ⁴⁴ reported significant antitumor activity when a cytosine deaminase-based gene transfer system was used in colorectal cancer. The bystander effects include tumor lysis, which changes the tumor microenvironment from inhibitory to immunostimulatory and leads to infiltration of the tumor by immune cells. Preclinical data obtained with an experimental model of hepatic metastasis from colorectal carcinoma have demonstrated that combinations of suicide gene therapy and cytokine gene therapy may produce synergistic antitumor effects. ^44,58,60

Gene delivery

Irrespective of the specific type of the gene delivery vehicle used, it must be delivered efficiently to its intended target in order for the gene to be expressed at therapeutic concentrations. This may include ex vivo, in situ, and systemic in vivo routes of therapy. Therefore, the interventional radiologist will play a crucial role as part of the gene therapy team. We are uniquely positioned to offer expertise in choosing approaches and sites for gene delivery. Although systemic IV administration of vectors have been performed, it has not proven to be as efficacious as local delivery. Angiographic guidance will be of use in localizing local tumor blood supply and directing the targeted intra-arterial delivery of genes of interest so that vector-DNA complexes can be delivered with accuracy and specificity. This approach offers considerable advantages over systemic approaches in which much of the vector-DNA complex may be lost in the first pass through the pulmonary and hepatic reticuloendothelial systems. Embolization techniques may also aid in prolonging vector contact with the target cells by delaying washout and thereby further enhancing target cell uptake. Other delivery strategies include image-guided delivery to target tissues or tumor through stereotactically placed needles and cavitary administration using US, CT, or MR guidance. ^6,7

Using ultrasound, the process of liposomal vector delivery can be monitored directly because lipid vesicles are echogenic. Such monitoring is of considerable importance in demonstrating accurate delivery to sites of interest. After injection, acoustically activatable carriers (ultrasound "contrast agents" such as microbubbles carrying genes and/or drugs) can be monitored by ultrasound or MR (liposomes encapsulating gadopentetate), and once at the region of interest, high-frequency ultrasound pulses can be applied to increase the vascular permeability and rupture the microbubbles. It also can increase the permeability of cell membrane and facilitate the diffusion of foreign DNA into the cell. Lastly, guided biopsy of transduced tissues for histopathologic analysis after gene delivery should improve confidence in the evaluation of gene expression. The radiological applications of this technique are self-evident and could be incorporated into the clinical practice of ultrasound imaging. ^62-64

Radiotherapeutic uses of ionizing radiation for gene therapy are also in development. With radiation-sensitive inducible promoters of gene transcription, the use of targeted gene delivery to a tumor followed by activation of expression of the therapeutic gene with stereotactically guided radiation therapy may be possible. One such system has linked expression of the tumor necrosis factor gene to a radiation-inducible promoter. Ionizing radiation is thus used in both temporal and spatial regulation of gene therapy. Other examples of radiation-sensitized genes include p53 and fas, both of which play a role in promoting radiation-triggered apoptosis. ^65-67

More recently, additional interventional strategies have been employed to improve gene delivery, including electroporation, microinjection, particle bombardment or gene gun therapy, high-frequency ultrasound, and other mechanical or chemical disruptions that improve local delivery. Electroporation creates small temporary openings in the cell membrane, allowing introduction of DNA through application of an electrical field for a few microseconds to milliseconds. Microinjection is an old technique performed manually under a microscope. A syringe is used to pierce the cellular membrane and insert the genetic material into the cytoplasm. Lastly, particle bombardment or "gene gun" technology involves the direct bombardment of DNA-coated gold particles through the cell membrane into the cytoplasm. ⁶⁵

Conclusion

Since Francis Crick "winged" into the Eagle in Febrary 1953, the spiraling double helix has become one of the most famous scientific images of our times. With the discovery of DNA, the genetic code was cracked, thereby paving the way for gene therapy. Gene therapy places us at a vital moment in human as well as medical history. Genetics is to our generation what nuclear physics was to the last: only the atomic bomb is an appropriate analogy for the explosive and implosive implications of gene therapy. Increasingly, gene therapy is used experimentally and clinically to replace defective genes and/or impart new functions to cells and tissues. With the recent advances in vector design, and improvements in transgene and prodrug activation strategies, gene therapy has been applied to a wide variety of diseases, tissues, and organ systems. The contemporary explosion in molecular probe design, imaging technology, and genetic engineering is expected to generate new imaging concepts and new affinity ligands, which will most likely drive the development of novel imaging technology. The new frontier of "molecular imaging" will expand our current capabilities and will complement advances in other fields of molecular medicine. It will revolutionize current approaches to screening, detecting, characterizing, and treating different diseases.

Finally, it has always been clear that our specialty will play a critical role in gene therapy and its clinical applications, particularly in delivering genes and vector products by minimally invasive interventional techniques. Radiology will be involved at every level of gene therapy. We will characterize the disease and its process, choose sites for targeting therapy, contribute to gene delivery, monitor uptake in tissues of interest, and, finally, evaluate levels of target gene expression and clinical response. Consequently, it is especially important that we have an understanding of the molecular biology and basic techniques used to clone a gene, current applications for gene therapy, and contemporary delivery systems in order to ensure our integration into the gene therapy team as this field rapidly and furiously evolves. *