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Gene therapy and blood cell transplantation
Gene Therapy and Blood Cell Transplantation Linda J. Cuaron and Betty Gallucci Objectives: To describe the basics of gene transfer and specific applications in marrow ablative therapy. Data sources: Review articles, research studies, and book chapters pertaining to gene therapy. Conclusions: Gene therapy will be a major factor in healthcare options for the 21st century. Genetically engineered biopharmaceuticals will probably have a place in the blood cell transplant regimens of the future.
Implications for nursing practice: Nurses are in a position to guide the successful implementation of gene therapy through their roles as patient educator, counselor, direct-care coordinator, consultant, and through development of resource materials. Copyright © 1997 by W.B. Saunders Company
ROM THE EARLY 1940's to the present, the quest to understand and describe genes has moved from the laboratory to clinical trials in humans. Deoxyribonucleic acid (DNA) was discovered to carry the genetic code and in 1953 Watson and Crick described the double helix structure of DNA. A bacterium, Escherichia coli became an attractive system for examining the relationship between genes and biological functions. 1 In 1977 the first human gene was cloned and in 1982 the first genetically engineered biopharmaceutical (human insulin) gained Federal Drug Administration (FDA) approval for use. The pace of genetic discovery is accelerating. There were almost 8,000 years between the discovery that yeast caused fermentation and the discovery that fermentation was also caused by bacteria. There were approximately 60 years between the discovery of the enzymes in the nucleus of cells, and the discovery of DNA. Only about 12 years passed between the discovery of DNA and the first successful DNA cloning experiments heralding the arrival of recombinant DNA technology. Since the development of recombinant DNA technology, these new advances have led to molecular techniques for diagnosis and new therapeutic products. 2 Between January of 1989
and June of 1996 more than 120 clinical trials were proposed or initiated, worldwide, to study the potential of gene therapy. The impact of biotechnology will continue to advance at a rapid rate and nurses need to prepare for the challenges associated with this type of therapy. 3 The role of the nurse will encompass patient education, development of resource materials, counselor, direct-care coordinator, and consultant for patients undergoing cancer treatments that use gene therapy or biopharmaceutical gene preparations. Nurses are in a position to help guide the successful implementation of this new therapy.4,5
F
From Schering Laboratories, Kenilworth, N J; and University of Washington, School of Nursing, Seattle. Linda J. Cuaron, RN, MN, AOCN: Patient Care Consultant, Schering Laboratories, Kenilworth, N J; Betty Gallucci, RN, PhD: University of Washington, School of Nursing, Seattle, WA. Address reprint requests to Linda J. Cuaron, RN, MN, AOCN, Patient Care Consultant, Schering Laboratories, 2000 Galloping Rd, Kenilworth, NJ 07033. Copyright © 1997 by WB. Saunders Company 0749-2081/97/1303-000755.00/0 200
THE BASICS OF GENE TRANSFER
There are several types of gene therapy, but somatic gene transfer is the only type that is currently used in humans. Somatic cells include all the cells of the body except the reproductive ceils. Germline cells are the sperm and ova. Germline therapy would involve insertion of a healthy gene into the reproductive cells of the patient, so that a disorder would be corrected in the genes passed down to succeeding generations. This type of gene therapy in humans is theoretically feasible, but beyond the realm of current science. 6 Apart from the technical obstacles involved in germline therapy, there are strong ethical arguments that deal with potential clinical risks, changing the gene pool, and the inherent social dangers .7 Somatic gene therapy is the process of delivering a functional gene into any human cell, other than a reproductive or germline cell, to correct a genetic dysfunction or enhance other therapies. The concern that gene therapy may pose health risks has resulted in professional groups adopting guidelines to limit potential hazards. In the United States there
Seminars in Onco/ogy Nursing, Vo113, No 3 (August), 1997: pp 200-207
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are several committees that review research involving gene transfer. The Recombinant DNA Advisory Committee promotes the discussion of social and ethical issues. The FDA focus includes review of the scientific methodology and preclinical safety testing. The National Institute of Health (NIH, Bethesda, MD) review board as well as the institutional review boards from the research site must also scrutinize any proposed research involving gene therapy. 4 Early concerns were addressed in expert panels, with the conclusion that "somatic cell gene therapy is not significantly different from other forms of treatment. ''7 The President's Commission for the Study of Ethical Problems in Medicine and Biomedical Research and the European Medical Research Council concluded " . . . somatic cell gene therapy is not fundamentally different from other therapeutic procedures, such as organ transplantation or blood transfusion. 7'' W. French Anderson, one of the early gene therapy researchers at NIH has stated: "Somatic cell gene therapy for the treatment of severe disease is considered ethical because it can be supported by the fundamental moral principle of beneficence: It would relieve human suffering. 7'' Somatic gene transfer is designed so that it will not target germline cells and will therefore not be passed on to descendants. The ability to achieve this outcome rests in the method and manner of delivery of the desired gene into the cell. Of the currently available systems for gene transfer, the most stable are viral vectors based on retroviruses and adeno-associated viruses (AAV). 8 Retroviruses are ribonucleic acid (RNA) viruses, their genetic component is RNA rather than DNA. Retroviruses have many features desirable in a gene transporter. They bind to the host cell, efficiently integrate into host chromosomes, use the machinery of the cell to replicate and leave the cell fully competent to travel to and infect another cell. Retroviral vectors have been extensively studied and have been used in many clinical trials. The disadvantage of this type of vector is that expression of the transduced gene sometimes diminishes with time or may become inactivated. 9 The adeno-associated viral (AAV) vectors have high transduction frequencies in primary cells and an ability to transfer (transduce) the gene of interest to nondividing cells and primitive hematopoietic progenitor cells. 8 They also do not require the preincubation with growth factors as do most
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retroviral vectors. This is advantageous in the transduction of early progenitor or stem cells because the growth factors could possibly cause the differentiation of stem cells to be less responsive to gene transfer and long-term expression in the target cell. 9 Longitudinal studies will be necessary to understand the long-term safety issues, but both the retroviral vectors and the adeno-associated viral vectors are looking promising. 9 The other methods under study include adenovirus vectors (DNA viruses), liposome-based DNA delivery methods, and plasmid vectors. Finding effective vectors is one of the biggest challenges for successful gene transfer. Additionally, concern was voiced about the finite risk that these vectors could become infectious by combining with undetected viruses in the host. Other risks, such as activation of a proto-oncogene or disruption of an essential gene were also cause for concern. As a result of these issues, rigorous safety trials are required before any vector is used in a human clinical trial.l° To date, these problems have not surfaced in any humans who have been treated with somatic gene therapy. However, it has been a relatively short time since the first patients were treated and long-term follow-up is necessary for a valid assessment. Additionally, these risks are limited to the host recipient and do not pose a health risk to the general public. 7,11 DNA-THE BASICS A brief review of DNA and recombinant DNA technology follows before the discussion of the clinical applications of gene therapy to blood cell transplantation (BCT). The three major types of molecules include DNA, RNA, and protein. DNA is a double-stranded helix whose basic structural units are nucleotides. They are adenine, thymine, guanine, and cytosine and referred to by the letters A, T, G, & C. They form base pairs that are held together by weak hydrogen bonds. The DNA in a human cell is made up of about 3-billion base pairs and would be more than 6-feet long if uncoiled. DNA is organized into chromosomes. Some organisms such as bacteria have circular DNA chains called plasmids, which exist separately from the chromosomes. 12 Plasmids have assumed a role in gene therapy because they are one type of vector or vehicle for delivery of DNA into the cell. Usually genetic information flows in one direction, from DNA to RNA to protein. The first step
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(DNA to RNA) is known as transcription. The second step (RNA to protein) is known as translation. When the process involves information passing from RNA to DNA it is called reverse transcription.l,13 GENES-THE
from the patient's vascular system through a largebore central venous catheter. The patient may have received a mobilization protocol such as cyclophosphamide and growth factors to achieve an optimal level of available progenitor cells. A portion of the harvested stem cells are selected for retroviral transduction. This insures that the engraftment is not jeopardized by the manipulation of all the stem cells. In some protocols, these stem cells are placed in a suspension to be incubated with interleukin-3 (IL-3) and IL-6. It is thought that this enhances the number of cells transduced. A provirus is designed in the laboratory replacing part of the viral gene with the therapeutic gene. Enzymes such as restriction nucleases are used to cleave the desired sequence (see Fig 1). The newly engineered provirus is inserted into the packaging virus (see Fig 2). The resulting vector is incomplete, but will have a normal outer coat that can attach to the target cell and deliver its contents. This therapeutic gene integrates into the cell's DNA and results in
BASICS
The structural intricacy of genes exceeds the simple definition of a " . . . unit of heredity governing a particular trait." 1 One strand of DNA is called the "sense strand." The other is known as the template or antisense strand. The gene is contained on the sense strand and begins with regulatory sequences, followed by a transcription unit made up of exons and introns, and ends with more regulatory sequences. 14 GENE DELIVERY SYSTEMS
One method of somatic gene transfer involves introducing a therapeutic gene into stern cells with a retroviral vector. First, stem cells are removed "Safe" RetroviralVector (Geneticallyengineeredto havetherapeuticgeneincorporated into viral RNA,incapableof formingviral proteinsor virus)
TherapeuticR
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insertionintotargetcell
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..Q~"~ Envelope protein binds to host cell receptor
Retroviralvectoris adsorbedinto hostcell
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convertsRNAto DNA Therapeutic DNA is intearated into host
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can now be generated
Targetcell is ready for transplantation
Fig 1, Insertion of the retroviral vector, now carrying the therapeutic provirus, into the human target cell. (Adapted from: GENES AND THE BIOLOGY OF CANCER by Varmus and Weinberg © 1993 by Scientific American Library. Used with permission of W.H. Freeman and Company,)
GENE THERAPY AND BCT
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Provirus is designed by replacing viral gene with therapeutic gene Restriction nuclease "cuts" the viral DNA RN
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Therapeutic gene inserted into the virus
Fig 2, Restriction endonucleases recognize short D N A base sequences, They can cleave or "cut" the D N A at or near these sequences,
the manufacturing of healthy proteins. These modified cells are returned to the patient through an intravenous infusion. When the cell divides, the daughter cells will express the therapeutic gene for a limited number of replications (Table 1). Overall, the merits of any gene delivery system will be evaluated based on the following factors. • What is the route of gene delivery (ex vivo or in vivo), if in vivo, is delivery by injection, aerosol, or other systemic methods? • What is the nature of the target cell (tumor cell or effector cell)? • What degree of comprehensiveness is required, if it necessary to transduce every cell or a small number of cells? • Is the aim of treatment correction of cell defect, tumor cell death, or protection from toxicity of other drugs? 9 Table 1. Example of Gene Transfer in BCT 1. Stem cells are removed from patient through a largebore central venous catheter. 2. Stem cells are placed in suspension, may be incubated with IL-3 & IL-6. 3. Provirus is designed by replacing viral gene with therapeutic gene (see Fig 1). 4. Therapeutic provirus is inserted into vector (see Fig 2). 5. Resulting vector will be incomplete, but will have a normal outer coat that will allow it to enter cells and deliver its contents. 6. Target cell accepts vector, therapeutic gene enters cellular DNA. Modified cells are returned to patient. When cell divides, progeny will express therapeutic gene. Data from Verma I M Y
GENE THERAPY TECHNIQUES IN MARROW ABLATIVE THERAPY
Gene therapy with stem cell transplantation typically consists of the replication of incompetent retroviral vectors that are modified to carry the gene of interest into specific somatic cells (pluripotent stem cells) of the body. A commonly used strategy involves harvesting the progenitor cells, incubating a portion of these cells with the transducing vector and then reinfusing these cells back in the patient after a regimen of conditioning therapy designed to reduce the number of cancerous cells. 15 This is an ex vivo strategy for gene transfer because the gene transfer took place outside of the patient's body. Previous gene marking and gene therapy studies showed that up to 20% of early precursor ceils can be genetically altered by using retroviral transduction. Other protocols that require the use of suspension transduction, which involves preincubation with growth factors, have modified 2% to 5% of the cells. These modified cells remain in the systemic circulation for up to 15 months, ss There are several gene therapy interventions. These therapies include: gene marking, suicide genes, antisense oligonucleotides, and insertion of therapeutic genes into progenitor cells. Gene marking was the first method used for improving the outcomes of BCT and today still represents the majority of the work done in this area. As of March 1996, 19 of the 22 BCT gene therapy protocols in the United States involved gene marking. 16 Three studies are identified as therapeutic. The future of gene therapy as a first line treatment depends on the success of vectors inserting the genetic information into early hematopoietic cells. The elements of successful gene transfer include: access to target cells, binding to those cells, cytoplasmic transport, DNA delivery, and persistence in the nucleus, and expression of a functional gene product. 9
Gene Marking There are several categories of gene therapy that have been evaluated in clinical trials. The earliest studies involved tumor-infiltrating lymphocytes (TIL) that had been "marked" with a neomycin resistance gene. The purpose of this study was to identify potential problems with gene therapy and to test its feasibility. The impetus for this study was the need to follow the fate of TIL cells over time. 17 The implication for BCT with this type of therapy is that this technique provides a way to
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accurately analyze the efficiency of different purging techniques. If leukemia cells in relapsed patients were found to have originated in the stored stem cells, which would be gene marked, harsher purging techniques would be necessary. If the relapsed leukemia cells were unmarked, thus arising from cells not destroyed by therapy, more strenuous conditioning regemins would be indicated. Although the first gene marking studies were not aimed at a therapeutic result, but rather to identify potential problems with gene transfer, more recent studies do have a therapeutic intent. Additionally, gene marking studies have been combined in conjunction with other therapeutic effects of stem cell transplantation (BCT) to answer questions that will impact future recipients of bone marrow transplantation (BMT) or BCT therapy. A study was initiated in 1994 at Fred Hutchinson Cancer Research Center (Seattle, WA) to examine longterm hematopoietic reconstitution using stem cells marked with retrovirus vectors. Long-term follow-up that includes peripheral blood testing every 6 months for 5 years will provide important information on the extent to which these ceils contribute to long-term hematopoietic reconstitution.18 Another marking study was discussed in March 1996, investigating hematopoietic reconstitution in adult patients undergoing bone marrow and/or blood cell transplant for lymphoma or metastatic breast cancer. 16The major questions being asked by this study are whether stem cells should be used in place of or in addition to bone marrow cells, and how many stem cells are required for engraftment? Also being studied are the optimal conditions for stem cell harvest and autografting, and if harvested hematopoietic stem cells could be retrovirally transduced for persistent gene expression in daughter cells after transplantation. Blood stem cells are most often procured after recovery from high-dose cyclophosphamide and granulocytemacrophage-colony-stimulating factor. The harvested bone marrow and stem cells are preincubated with a mixture of IL-3 and IL-6 to increase the efficiency of gene transduction. One of the major obstacles to effective gene transfer in stem cells is that the cells are resting or quiescent in the Go phase. It is difficult to target nondividing cells with retroviral vectors. It is unknown if cryopreserved stem cells contain viable, early lineage, hematopoietic stem cells (HSC), which might result in long-term engraft-
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ment or if the autograft only provides reconstitution of committed progenitor cells. 16 It is possible that HSC may survive the most intense pretransplant conditioning. Since relapse in lymphoma often occurs at sites of the original disease, two explanations prevail. Either the treatment was insufficient to eradicate the tumor cells, or some tumor cells survived purging and cryopreservation and contributed to relapse. This study should help answer questions about hematopoietic reconstitution that may yield a greater relapse-free response. Umbilical cord blood collected at the birth of a child has been used effectively for hematopoietic reconstitution. Some of the diseases for which this type of transplant has been used include: Fanconi's anemia, aplastic anemia, [3-thalassemia, chronic myelogenous leukemia (CML), acute lymphocytic leukemia, myelodysplastic syndrome, and neuroblastoma. Of the first 50 patients treated with cord blood (either matched or one-antigen mismatched) 70% were surviving 1.5 years after transplant, and 54% were free of disease. Most of the transplants were done with children. Of particular interest is the limited graft versus host disease (GVHD) seen in both related and unrelated transplants. It is thought that this may reflect the low immune reactivity of cord blood T lymphocytes and natural killer cells. A gene marking study has been reported that looks at cord blood transplantation. 19 Questions were asked regarding the ability to transduce the expression of new genetic material into myeloid progenitor cells from cord blood using an adenoassociated vector. Researchers were able to effectively transduce cord blood without preincubation of the cells with growth factors, as is often needed with retroviral vectors. The promising results with cord blood transplantation, an available source that is regarded as waste-product, and the lack of information about specific hematopoietic requirements for engraftment in adults is a prime area for gene therapy research. 2°,21
Gene Therapy and the Epstein-Barr Virus (EBV) For many patients who receive BMT or BCT, a mismatched, nonsibling donor is the only hope if aggressive therapy is desired. Although risks of rejection and GVHD have been reduced by eliminating recipient alloreactive T cells, the risk of death from viral infection has estimates of 5% to 30%. Many times the viral infection appears as a reactivation of latent virus. It has been reported that
GENE THERAPY AND BeT
the EBV is present in more than 90% of normal individuals. Usually a healthy immune system prevents outgrowth of this virus. In immunosuppressed individuals, reactivation of these latent viruses may occur and create active lymphoproliferation. This disease process can be rapidly progressive with no effective treatment known. A study has been proposed to generate cytotoxic T cells specific for EBV and deliver these by genetic transfer to patients at risk for lymphoproliferation due to EBV reactivation.22
"Suicide" Genes and GVHD Most therapies that refer to "suicide" genes are using the herpes simplex virus (HSV) thymidine kinase (tk) gene. This gene is capable of converting the drug gancyclovir to a toxic metabolite. This makes the cells that express this tk gene more sensitive to gancyclovir and normal ceils remain more resistant. 23 One study is examining the use of "suicide" gene therapy in donor peripheral blood lymphocytes as a therapy for GVHD. This study aims to evaluate the safety of increasing doses of donor lymphocytes transduced with a "suicide" retroviral vector. It will also examine the efficacy of the donor lymphocytes that have been activated in vitro and gene transduced. The therapeutic intent of this study is the downregulation of GVHD by the administration of gancyclovir to patients who have had donor lymphocytes tk transduced. By treating the patient with gancyclovir, the number of activated lymphocytes will be reduced. It is hoped that this will lead to amelioration of GVHD by allowing the in vivo selective elimination of cells responsible for severe GVHD while allowing in vivo modulation of the donor antitumor graft versus leukemia response. 24
Therapeutic Gene Transfer One of the novel approaches that involves gene therapy is to transfer the human multidrug resistance gene (MDR-1) into hematopoietic stem cells during autologous transplantation. Patients with metastatic breast cancer who are to receive their own bone marrow and peripheral blood stem cells after very high-doses of ICE chemotherapy (ifosfamide, carboplatin, and etoposide) were studied. They had a portion of their progenitor ceils genetically modified by a retroviral vector that inserted the multidrug resistance gene into these
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cells. The goal of the study was twofold. First, was the study of obtaining engraftment after high-dose chemotherapy with a retrovirally transduced stem cell that expressed the human multidrug resistance gene. Second, to determine if this transduced MDR-1 gene could in fact pass drug resistance to hematopoietic ceils. If the breast cancer recurred after BMT or BCT they could treat with paclitaxel, a chemotherapy drug that is responsive to the MDR-1 gene. Hopefully this would cause the MDR-1 gene modified hematopoietic cells to have a protective mechanism. That mechanism is the function induced by the MDR-1 gene, which would cause those modified cells to pump out the paclitaxel, sparing the hematopoietic cells from cell death. The paclitaxel would work in its usual manner to stop the growth of the cancerous cells. This study was approved in 1993 for investigation at the NIH, but is still pending accrual of its first patient. 25 GENETICALLY ENGINEERED BIOPHARMACEUTICALS
New genetic medicines are being developed that target DNA or RNA. There are two main strategies employed by these therapies. Triplex-forming drugs hinder the production of an unwanted protein by inhibiting transcription whereas antisense therapies aim to selectively impede translation, thereby preventing the cell from manufacturing deleterious proteins. 26 By using drugs that are capable of attaching selected segments of DNA (which is the triplex strategy) or mRNA (the antisense strategy) it is hoped that transcription of DNA or translation mRNA will be disrupted. This would then block the cell's process of producing aberrant proteins that contribute to disease56 The agents being investigated for this therapy are nucleic acid binding agents. The most promising group is known as DNA oligonucleotides. These short strands of nucleotides are known to combine with other nucleotides in a predictable manner. This allows for design of a drug that can recognize a unique site on a particular gene. Although this type of therapy involves delivery of genetically altered substances to target cells, it is different than standard gene therapies. As described previously, most gene therapies substitute healthy genes for versions that are missing or unable to direct the appropriate synthesis of a needed protein.
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Oligonucleotides are too small to participate in the manufacture of a new protein, they can only block the expression of existing genes so they do not manufacture a specific protein. Although there are barriers to the therapeutic use of antisense oligonucleotides, there has been some progress from current clinical trials. One study that is examining cancer-fighting oligonucleotides is being performed with patients with acute myelogenous leukemia. Researchers at University of Nebraska Medical Center (Omaha) are testing the ability of an antisense oligonucleotide to destroy cancer cells in ex vivo blood cell purging. The oligonucleotide of interest is targeted to RNA that carries the p53 gene. This gene, when functioning normally, is a tumor suppressor gene, but it is over expressed in individuals with AML. A different trial is also underway at the University of Pennsylvania (Philadelphia) for the treatment of CML. This study is evaluating the ability of an antisense drug to eliminate cancer cells during ex vivo blood cell purging. The target is mRNA, which is transcribed from the c-myb gene. Under normal, healthy circumstances c-myb promotes normal proliferation of blood cells. Abnormal regulation of this gene may be responsible for the development of leukemias such as CML. Although these therapies are still in the process of being perfected, the success that has been seen in recent years suggests that antisense and triplex agents will one day become common treatments for diseases that today have no effective therapy, and foi) patients in relapse. Genetically engineered biopharmaceuticals may also impact the health care practices in novel ways. Nonviral pharmaceutical formulations of genes are being developed that do not require delivery using a vector such as a retrovirus. New methods of particulate drug delivery are being designed that will deliver DNA to the desired somatic cell in need of repair or alteration without transplantation. Genetically altered biopharmaceuticals may have a future in controlling some of the adverse sequelae that accompany BCT and BMT such as infection, inflammation, and organ toxicity. By using DNA complexes that contain lipid, protein, peptide, or polymeric carriers, as well as ligands capable of binding to cell-surface receptors on the target cell, genes can be delivered to somatic cells. This type of system has been used to transfer genes to the lung, liver, endothelium, epithelium,
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and tumor cells. These products are known as "gene medicines," "genes in a bottle," or "gene drugs."27 Methods of delivery for these genes include injection, aerosol spray, and the "gene gun" or "needle-free injection devices." The "gene gun" has been used in experimental models to enhance wound healing in a porcine partial-thickness w o u n d . 27 The disadvantage of the "gene gun" is that effective gene delivery is commonly seen in only the superficial layers of the injected tissues. The injection delivery method is being studied in diseased cardiac tissue due to myocardial infarction. An injection of DNA leads to the production of growth factors that stimulate angiogenesis causing a direct therapeutic effect. Injection or implantation of genetically altered myoblasts is a method that will result in muscle that secretes missing circulatory proteins. The aerosol method of delivery can be used for several therapeutic goals. Studies are currently underway that involve using a liposomal aerosol to replace a defective product. In patients with cystic fibrosis the cystic fibrosis transmembrane conductance regulator (CFTR) gene comes in direct contact with the airway epitheliumY The findings from this type of research may have implications for treatment of other pulmonary complications, wound healing, and cardiac muscle damage because of chemotoxicity. These could someday impact the therapeutic options for patients who are undergoing BCT. In all nonviral, in vivo studies to date, DNA is gradually eliminated from the target cell leading to a decrease in the level of the therapeutic gene over time. The kinetics of this type of delivery provide the physician with several advantages. Treatment of both acute and chronic disease is possible, as well as the ability to alter the dose andfrequency of administration in response to the patient's changing clinical picture. Studies to date indicate that the safety profile for this class of drugs is comparable to conventional drugs and biological products. There are no reports of significant toxicity from direct injection of DNA into muscle, thyroid, or other organs. Additionally, there is no evidence of development of anti-DNA antibodies after single or repetitive administration. Further, there has been no evidence of antinuclear antibodies in human subjects using a variety of delivery systems including "naked" (or plasmid) DNA, cationic lipid formulations, or protein-DNA complexes. 27
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CONCLUSION Nurses will n e e d to be as c o m f o r t a b l e with the l a n g u a g e and basic processes o f genetic engineering as they n o w are with understanding the processes o f the i m m u n e system. H i g h - t e c h bio t e c h n o l o g y will c o n t i n u e to expand. It is estimated that by the y e a r 2020 genetically e n g i n e e r e d biopharrnaceuticals will m a k e up 50% o f all drugs prescribed in the U n i t e d States, Japan, and Europe.
G e n e therapy will be a m a j o r factor in healthcare options for the 21st century. Questions n e e d to be addressed that e n c o m p a s s not o n l y the scientific, but also the sociopolitical and v a l u e issues. R e s o u r c e allocation will w e i g h against ethical and h u m a n rights for the individual. S c i e n c e is a social institution and sharing o f scientific i n f o r m a t i o n is vital for i n f o r m e d discussions. 7 Nurses are p o i s e d to p r o v i d e guidance, education, and leadership in all o f these areas.
REFERENCES
1. Berg R Singer M: Dealing With Genes; The Language of Heredity. Mill Valley, CA, University Science Books, 1992 2. Mulligan R: The basic science of gene therapy. Science 260:926-931, 1993 3. Payne IY: Will gene therapy revolutionize medicine? Nursing interventions in oncology. MD Anderson Case Rep Rev 8:9-12, 1996 4. Jenkins J, Wheeler V, Albright L: Gene therapy for cancer. Cancer Nuts 17:447-456, 1994 5. Wheeler V: Gene therapy: Current strategies and future applications. Oncol Nurs Forum 22:20-26, 1995 (suppl 12) 6. Sedivy J, Joyner A: Gene Targeting. New York, NY, Oxford University, 1992 7. Elias S, Annas G: Somatic and germline gene therapy, in Annas G, Elias S (eds): Gene Mapping, Using Law and Ethics as Guides. New York, NY, Oxford University, 1992, pp 142-154 8. Chatterjee S, Lu D, Podsakoff G, et al: Strategies for efficient gene transfer into hematopoietic cells. Ann NY Acad Sci 220:79-89, 1995 9. Blaese M, Blankenstein T, Brenner M, et al: Vectors in cancer therapy: How will they deliver? Cancer Gene Ther 2:291-297, 1995 I0. Thompson L: Stem-cell gene therapy moves toward approval. Science 255:1072, 1993 11. Verma IM: Gene therapy. Sci Am Special Issue 4:78-85, 1993 12. Varmus H, Weinberg R: Genes and the Biology of Cancer. New York, NY, Freeman 1993 13. Schwartz R: Molecular medicine, jumping genes. Mol Med 332:941-944, 1995 14. Rosenthal N: Molecular medicine, recognizing DNA. Mol Med 333:925-927, 1995 15. Giles R, Hanania E, Fu S, et al: Genetic therapy using bone marrow transplantation, in Buckner CD (ed): Technical and Biological Components of Marrow Transplantation. Boston, MA, KluwerAcademic, 1995, pp 271-280 16. Douer D, Levine A, Anderson WF, et al: High-dose chemotherapy and autologous bone marrow plus peripheral blood stem cell transplantation for patients with lymphoma or metastatic breast cancer: Use of marker genes to investigate
hematopoietic reconstitution in adults. Hum Gene Ther 7:669-684, 1996 17. Anderson WF: Human gene therapy. Science 256:808813, 1992 18. Schuening E Miller D, Torok-Storb B, et al: Study on contribution of genetically marked peripheral blood repopulating cells to hematopoietic reconstitution after transplantation. Hum Gene Ther 5:1523-1534, 1994 19. Broxmeyer H, Cooper S, Etienne-Julan M, et al: Cord blood transplantation and the potential for gene therapy. Ann NY Acad Sci 220:105-113, 1996 20. Almici C, Carlo-Stella C, Wagner J, et al: Umbilical cord blood as a source of hematopoietic stem cells: From research to clinical application. Haematologica 80:473-479, 1995 21. Thompson C: Umbilical cords: Turning garbage into clinical gold, Science 268:805-806, 1995 22. Heslop H, Brenner M, Rooney C, et al: Administration of neomycin resistance gene marked EBV specific cytotoxic T lymphocytes to recipients of rnismatched-related or phenotypically similar unrelated donor marrow grafts. Hum Gene Ther 5:381-397, 1994 23. Beck C, Cayeux S, Lupton S, et al: The thymidine kinase/ganciclovir-mediated "suicide" effect is variable in different tumor cells. Hum Gene Ther 6:1525-1530, 1995 24. Bordignon C, Bonini C, Verzeletti S, et al: Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. Hum Gene Ther 6:813-819, 1995 25. O'Shaughnessy J, Cowan K, Niehuis A, et al: Retroviral mediated transfer of the human multidrug resistance gene (MDR-1) into hematopoietic stem cells during autologous transplantation after intensive chemotherapy for metastatic breast cancer. Hum Gene Ther 5:891-911, 1994 26. Cohen J, Hogan M: The new genetic medicines. Sci Am 271:76-82, 1994 27. Ledley F: Nonviral gene therapy: The promise of genes as pharmaceutical products. Hum Gene Ther 6:1129-1144, 1995 28. Blan R, Springer M: Gene therapy--A novel form of drug delivery. N Engl J Med 333:1204-1207, 1995
Implications for nursing practice: Nurses are in a position to guide the successful implementation of gene therapy through their roles as patient educator, counselor, direct-care coordinator, consultant, and through development of resource materials. Copyright © 1997 by W.B. Saunders Company
ROM THE EARLY 1940's to the present, the quest to understand and describe genes has moved from the laboratory to clinical trials in humans. Deoxyribonucleic acid (DNA) was discovered to carry the genetic code and in 1953 Watson and Crick described the double helix structure of DNA. A bacterium, Escherichia coli became an attractive system for examining the relationship between genes and biological functions. 1 In 1977 the first human gene was cloned and in 1982 the first genetically engineered biopharmaceutical (human insulin) gained Federal Drug Administration (FDA) approval for use. The pace of genetic discovery is accelerating. There were almost 8,000 years between the discovery that yeast caused fermentation and the discovery that fermentation was also caused by bacteria. There were approximately 60 years between the discovery of the enzymes in the nucleus of cells, and the discovery of DNA. Only about 12 years passed between the discovery of DNA and the first successful DNA cloning experiments heralding the arrival of recombinant DNA technology. Since the development of recombinant DNA technology, these new advances have led to molecular techniques for diagnosis and new therapeutic products. 2 Between January of 1989
and June of 1996 more than 120 clinical trials were proposed or initiated, worldwide, to study the potential of gene therapy. The impact of biotechnology will continue to advance at a rapid rate and nurses need to prepare for the challenges associated with this type of therapy. 3 The role of the nurse will encompass patient education, development of resource materials, counselor, direct-care coordinator, and consultant for patients undergoing cancer treatments that use gene therapy or biopharmaceutical gene preparations. Nurses are in a position to help guide the successful implementation of this new therapy.4,5
F
From Schering Laboratories, Kenilworth, N J; and University of Washington, School of Nursing, Seattle. Linda J. Cuaron, RN, MN, AOCN: Patient Care Consultant, Schering Laboratories, Kenilworth, N J; Betty Gallucci, RN, PhD: University of Washington, School of Nursing, Seattle, WA. Address reprint requests to Linda J. Cuaron, RN, MN, AOCN, Patient Care Consultant, Schering Laboratories, 2000 Galloping Rd, Kenilworth, NJ 07033. Copyright © 1997 by WB. Saunders Company 0749-2081/97/1303-000755.00/0 200
THE BASICS OF GENE TRANSFER
There are several types of gene therapy, but somatic gene transfer is the only type that is currently used in humans. Somatic cells include all the cells of the body except the reproductive ceils. Germline cells are the sperm and ova. Germline therapy would involve insertion of a healthy gene into the reproductive cells of the patient, so that a disorder would be corrected in the genes passed down to succeeding generations. This type of gene therapy in humans is theoretically feasible, but beyond the realm of current science. 6 Apart from the technical obstacles involved in germline therapy, there are strong ethical arguments that deal with potential clinical risks, changing the gene pool, and the inherent social dangers .7 Somatic gene therapy is the process of delivering a functional gene into any human cell, other than a reproductive or germline cell, to correct a genetic dysfunction or enhance other therapies. The concern that gene therapy may pose health risks has resulted in professional groups adopting guidelines to limit potential hazards. In the United States there
Seminars in Onco/ogy Nursing, Vo113, No 3 (August), 1997: pp 200-207
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are several committees that review research involving gene transfer. The Recombinant DNA Advisory Committee promotes the discussion of social and ethical issues. The FDA focus includes review of the scientific methodology and preclinical safety testing. The National Institute of Health (NIH, Bethesda, MD) review board as well as the institutional review boards from the research site must also scrutinize any proposed research involving gene therapy. 4 Early concerns were addressed in expert panels, with the conclusion that "somatic cell gene therapy is not significantly different from other forms of treatment. ''7 The President's Commission for the Study of Ethical Problems in Medicine and Biomedical Research and the European Medical Research Council concluded " . . . somatic cell gene therapy is not fundamentally different from other therapeutic procedures, such as organ transplantation or blood transfusion. 7'' W. French Anderson, one of the early gene therapy researchers at NIH has stated: "Somatic cell gene therapy for the treatment of severe disease is considered ethical because it can be supported by the fundamental moral principle of beneficence: It would relieve human suffering. 7'' Somatic gene transfer is designed so that it will not target germline cells and will therefore not be passed on to descendants. The ability to achieve this outcome rests in the method and manner of delivery of the desired gene into the cell. Of the currently available systems for gene transfer, the most stable are viral vectors based on retroviruses and adeno-associated viruses (AAV). 8 Retroviruses are ribonucleic acid (RNA) viruses, their genetic component is RNA rather than DNA. Retroviruses have many features desirable in a gene transporter. They bind to the host cell, efficiently integrate into host chromosomes, use the machinery of the cell to replicate and leave the cell fully competent to travel to and infect another cell. Retroviral vectors have been extensively studied and have been used in many clinical trials. The disadvantage of this type of vector is that expression of the transduced gene sometimes diminishes with time or may become inactivated. 9 The adeno-associated viral (AAV) vectors have high transduction frequencies in primary cells and an ability to transfer (transduce) the gene of interest to nondividing cells and primitive hematopoietic progenitor cells. 8 They also do not require the preincubation with growth factors as do most
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retroviral vectors. This is advantageous in the transduction of early progenitor or stem cells because the growth factors could possibly cause the differentiation of stem cells to be less responsive to gene transfer and long-term expression in the target cell. 9 Longitudinal studies will be necessary to understand the long-term safety issues, but both the retroviral vectors and the adeno-associated viral vectors are looking promising. 9 The other methods under study include adenovirus vectors (DNA viruses), liposome-based DNA delivery methods, and plasmid vectors. Finding effective vectors is one of the biggest challenges for successful gene transfer. Additionally, concern was voiced about the finite risk that these vectors could become infectious by combining with undetected viruses in the host. Other risks, such as activation of a proto-oncogene or disruption of an essential gene were also cause for concern. As a result of these issues, rigorous safety trials are required before any vector is used in a human clinical trial.l° To date, these problems have not surfaced in any humans who have been treated with somatic gene therapy. However, it has been a relatively short time since the first patients were treated and long-term follow-up is necessary for a valid assessment. Additionally, these risks are limited to the host recipient and do not pose a health risk to the general public. 7,11 DNA-THE BASICS A brief review of DNA and recombinant DNA technology follows before the discussion of the clinical applications of gene therapy to blood cell transplantation (BCT). The three major types of molecules include DNA, RNA, and protein. DNA is a double-stranded helix whose basic structural units are nucleotides. They are adenine, thymine, guanine, and cytosine and referred to by the letters A, T, G, & C. They form base pairs that are held together by weak hydrogen bonds. The DNA in a human cell is made up of about 3-billion base pairs and would be more than 6-feet long if uncoiled. DNA is organized into chromosomes. Some organisms such as bacteria have circular DNA chains called plasmids, which exist separately from the chromosomes. 12 Plasmids have assumed a role in gene therapy because they are one type of vector or vehicle for delivery of DNA into the cell. Usually genetic information flows in one direction, from DNA to RNA to protein. The first step
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CUARON AND GALLUCCI
(DNA to RNA) is known as transcription. The second step (RNA to protein) is known as translation. When the process involves information passing from RNA to DNA it is called reverse transcription.l,13 GENES-THE
from the patient's vascular system through a largebore central venous catheter. The patient may have received a mobilization protocol such as cyclophosphamide and growth factors to achieve an optimal level of available progenitor cells. A portion of the harvested stem cells are selected for retroviral transduction. This insures that the engraftment is not jeopardized by the manipulation of all the stem cells. In some protocols, these stem cells are placed in a suspension to be incubated with interleukin-3 (IL-3) and IL-6. It is thought that this enhances the number of cells transduced. A provirus is designed in the laboratory replacing part of the viral gene with the therapeutic gene. Enzymes such as restriction nucleases are used to cleave the desired sequence (see Fig 1). The newly engineered provirus is inserted into the packaging virus (see Fig 2). The resulting vector is incomplete, but will have a normal outer coat that can attach to the target cell and deliver its contents. This therapeutic gene integrates into the cell's DNA and results in
BASICS
The structural intricacy of genes exceeds the simple definition of a " . . . unit of heredity governing a particular trait." 1 One strand of DNA is called the "sense strand." The other is known as the template or antisense strand. The gene is contained on the sense strand and begins with regulatory sequences, followed by a transcription unit made up of exons and introns, and ends with more regulatory sequences. 14 GENE DELIVERY SYSTEMS
One method of somatic gene transfer involves introducing a therapeutic gene into stern cells with a retroviral vector. First, stem cells are removed "Safe" RetroviralVector (Geneticallyengineeredto havetherapeuticgeneincorporated into viral RNA,incapableof formingviral proteinsor virus)
TherapeuticR
N
A
insertionintotargetcell
-
f
~
~
ReverseTranscriptase
~
..Q~"~ Envelope protein binds to host cell receptor
Retroviralvectoris adsorbedinto hostcell
\ Reverse transcriptase
convertsRNAto DNA Therapeutic DNA is intearated into host
~k
Therapeutic proteins
can now be generated
Targetcell is ready for transplantation
Fig 1, Insertion of the retroviral vector, now carrying the therapeutic provirus, into the human target cell. (Adapted from: GENES AND THE BIOLOGY OF CANCER by Varmus and Weinberg © 1993 by Scientific American Library. Used with permission of W.H. Freeman and Company,)
GENE THERAPY AND BCT
203
Provirus is designed by replacing viral gene with therapeutic gene Restriction nuclease "cuts" the viral DNA RN
RN
|
l
m
e
|
Therapeutic gene inserted into the virus
Fig 2, Restriction endonucleases recognize short D N A base sequences, They can cleave or "cut" the D N A at or near these sequences,
the manufacturing of healthy proteins. These modified cells are returned to the patient through an intravenous infusion. When the cell divides, the daughter cells will express the therapeutic gene for a limited number of replications (Table 1). Overall, the merits of any gene delivery system will be evaluated based on the following factors. • What is the route of gene delivery (ex vivo or in vivo), if in vivo, is delivery by injection, aerosol, or other systemic methods? • What is the nature of the target cell (tumor cell or effector cell)? • What degree of comprehensiveness is required, if it necessary to transduce every cell or a small number of cells? • Is the aim of treatment correction of cell defect, tumor cell death, or protection from toxicity of other drugs? 9 Table 1. Example of Gene Transfer in BCT 1. Stem cells are removed from patient through a largebore central venous catheter. 2. Stem cells are placed in suspension, may be incubated with IL-3 & IL-6. 3. Provirus is designed by replacing viral gene with therapeutic gene (see Fig 1). 4. Therapeutic provirus is inserted into vector (see Fig 2). 5. Resulting vector will be incomplete, but will have a normal outer coat that will allow it to enter cells and deliver its contents. 6. Target cell accepts vector, therapeutic gene enters cellular DNA. Modified cells are returned to patient. When cell divides, progeny will express therapeutic gene. Data from Verma I M Y
GENE THERAPY TECHNIQUES IN MARROW ABLATIVE THERAPY
Gene therapy with stem cell transplantation typically consists of the replication of incompetent retroviral vectors that are modified to carry the gene of interest into specific somatic cells (pluripotent stem cells) of the body. A commonly used strategy involves harvesting the progenitor cells, incubating a portion of these cells with the transducing vector and then reinfusing these cells back in the patient after a regimen of conditioning therapy designed to reduce the number of cancerous cells. 15 This is an ex vivo strategy for gene transfer because the gene transfer took place outside of the patient's body. Previous gene marking and gene therapy studies showed that up to 20% of early precursor ceils can be genetically altered by using retroviral transduction. Other protocols that require the use of suspension transduction, which involves preincubation with growth factors, have modified 2% to 5% of the cells. These modified cells remain in the systemic circulation for up to 15 months, ss There are several gene therapy interventions. These therapies include: gene marking, suicide genes, antisense oligonucleotides, and insertion of therapeutic genes into progenitor cells. Gene marking was the first method used for improving the outcomes of BCT and today still represents the majority of the work done in this area. As of March 1996, 19 of the 22 BCT gene therapy protocols in the United States involved gene marking. 16 Three studies are identified as therapeutic. The future of gene therapy as a first line treatment depends on the success of vectors inserting the genetic information into early hematopoietic cells. The elements of successful gene transfer include: access to target cells, binding to those cells, cytoplasmic transport, DNA delivery, and persistence in the nucleus, and expression of a functional gene product. 9
Gene Marking There are several categories of gene therapy that have been evaluated in clinical trials. The earliest studies involved tumor-infiltrating lymphocytes (TIL) that had been "marked" with a neomycin resistance gene. The purpose of this study was to identify potential problems with gene therapy and to test its feasibility. The impetus for this study was the need to follow the fate of TIL cells over time. 17 The implication for BCT with this type of therapy is that this technique provides a way to
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accurately analyze the efficiency of different purging techniques. If leukemia cells in relapsed patients were found to have originated in the stored stem cells, which would be gene marked, harsher purging techniques would be necessary. If the relapsed leukemia cells were unmarked, thus arising from cells not destroyed by therapy, more strenuous conditioning regemins would be indicated. Although the first gene marking studies were not aimed at a therapeutic result, but rather to identify potential problems with gene transfer, more recent studies do have a therapeutic intent. Additionally, gene marking studies have been combined in conjunction with other therapeutic effects of stem cell transplantation (BCT) to answer questions that will impact future recipients of bone marrow transplantation (BMT) or BCT therapy. A study was initiated in 1994 at Fred Hutchinson Cancer Research Center (Seattle, WA) to examine longterm hematopoietic reconstitution using stem cells marked with retrovirus vectors. Long-term follow-up that includes peripheral blood testing every 6 months for 5 years will provide important information on the extent to which these ceils contribute to long-term hematopoietic reconstitution.18 Another marking study was discussed in March 1996, investigating hematopoietic reconstitution in adult patients undergoing bone marrow and/or blood cell transplant for lymphoma or metastatic breast cancer. 16The major questions being asked by this study are whether stem cells should be used in place of or in addition to bone marrow cells, and how many stem cells are required for engraftment? Also being studied are the optimal conditions for stem cell harvest and autografting, and if harvested hematopoietic stem cells could be retrovirally transduced for persistent gene expression in daughter cells after transplantation. Blood stem cells are most often procured after recovery from high-dose cyclophosphamide and granulocytemacrophage-colony-stimulating factor. The harvested bone marrow and stem cells are preincubated with a mixture of IL-3 and IL-6 to increase the efficiency of gene transduction. One of the major obstacles to effective gene transfer in stem cells is that the cells are resting or quiescent in the Go phase. It is difficult to target nondividing cells with retroviral vectors. It is unknown if cryopreserved stem cells contain viable, early lineage, hematopoietic stem cells (HSC), which might result in long-term engraft-
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ment or if the autograft only provides reconstitution of committed progenitor cells. 16 It is possible that HSC may survive the most intense pretransplant conditioning. Since relapse in lymphoma often occurs at sites of the original disease, two explanations prevail. Either the treatment was insufficient to eradicate the tumor cells, or some tumor cells survived purging and cryopreservation and contributed to relapse. This study should help answer questions about hematopoietic reconstitution that may yield a greater relapse-free response. Umbilical cord blood collected at the birth of a child has been used effectively for hematopoietic reconstitution. Some of the diseases for which this type of transplant has been used include: Fanconi's anemia, aplastic anemia, [3-thalassemia, chronic myelogenous leukemia (CML), acute lymphocytic leukemia, myelodysplastic syndrome, and neuroblastoma. Of the first 50 patients treated with cord blood (either matched or one-antigen mismatched) 70% were surviving 1.5 years after transplant, and 54% were free of disease. Most of the transplants were done with children. Of particular interest is the limited graft versus host disease (GVHD) seen in both related and unrelated transplants. It is thought that this may reflect the low immune reactivity of cord blood T lymphocytes and natural killer cells. A gene marking study has been reported that looks at cord blood transplantation. 19 Questions were asked regarding the ability to transduce the expression of new genetic material into myeloid progenitor cells from cord blood using an adenoassociated vector. Researchers were able to effectively transduce cord blood without preincubation of the cells with growth factors, as is often needed with retroviral vectors. The promising results with cord blood transplantation, an available source that is regarded as waste-product, and the lack of information about specific hematopoietic requirements for engraftment in adults is a prime area for gene therapy research. 2°,21
Gene Therapy and the Epstein-Barr Virus (EBV) For many patients who receive BMT or BCT, a mismatched, nonsibling donor is the only hope if aggressive therapy is desired. Although risks of rejection and GVHD have been reduced by eliminating recipient alloreactive T cells, the risk of death from viral infection has estimates of 5% to 30%. Many times the viral infection appears as a reactivation of latent virus. It has been reported that
GENE THERAPY AND BeT
the EBV is present in more than 90% of normal individuals. Usually a healthy immune system prevents outgrowth of this virus. In immunosuppressed individuals, reactivation of these latent viruses may occur and create active lymphoproliferation. This disease process can be rapidly progressive with no effective treatment known. A study has been proposed to generate cytotoxic T cells specific for EBV and deliver these by genetic transfer to patients at risk for lymphoproliferation due to EBV reactivation.22
"Suicide" Genes and GVHD Most therapies that refer to "suicide" genes are using the herpes simplex virus (HSV) thymidine kinase (tk) gene. This gene is capable of converting the drug gancyclovir to a toxic metabolite. This makes the cells that express this tk gene more sensitive to gancyclovir and normal ceils remain more resistant. 23 One study is examining the use of "suicide" gene therapy in donor peripheral blood lymphocytes as a therapy for GVHD. This study aims to evaluate the safety of increasing doses of donor lymphocytes transduced with a "suicide" retroviral vector. It will also examine the efficacy of the donor lymphocytes that have been activated in vitro and gene transduced. The therapeutic intent of this study is the downregulation of GVHD by the administration of gancyclovir to patients who have had donor lymphocytes tk transduced. By treating the patient with gancyclovir, the number of activated lymphocytes will be reduced. It is hoped that this will lead to amelioration of GVHD by allowing the in vivo selective elimination of cells responsible for severe GVHD while allowing in vivo modulation of the donor antitumor graft versus leukemia response. 24
Therapeutic Gene Transfer One of the novel approaches that involves gene therapy is to transfer the human multidrug resistance gene (MDR-1) into hematopoietic stem cells during autologous transplantation. Patients with metastatic breast cancer who are to receive their own bone marrow and peripheral blood stem cells after very high-doses of ICE chemotherapy (ifosfamide, carboplatin, and etoposide) were studied. They had a portion of their progenitor ceils genetically modified by a retroviral vector that inserted the multidrug resistance gene into these
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cells. The goal of the study was twofold. First, was the study of obtaining engraftment after high-dose chemotherapy with a retrovirally transduced stem cell that expressed the human multidrug resistance gene. Second, to determine if this transduced MDR-1 gene could in fact pass drug resistance to hematopoietic ceils. If the breast cancer recurred after BMT or BCT they could treat with paclitaxel, a chemotherapy drug that is responsive to the MDR-1 gene. Hopefully this would cause the MDR-1 gene modified hematopoietic cells to have a protective mechanism. That mechanism is the function induced by the MDR-1 gene, which would cause those modified cells to pump out the paclitaxel, sparing the hematopoietic cells from cell death. The paclitaxel would work in its usual manner to stop the growth of the cancerous cells. This study was approved in 1993 for investigation at the NIH, but is still pending accrual of its first patient. 25 GENETICALLY ENGINEERED BIOPHARMACEUTICALS
New genetic medicines are being developed that target DNA or RNA. There are two main strategies employed by these therapies. Triplex-forming drugs hinder the production of an unwanted protein by inhibiting transcription whereas antisense therapies aim to selectively impede translation, thereby preventing the cell from manufacturing deleterious proteins. 26 By using drugs that are capable of attaching selected segments of DNA (which is the triplex strategy) or mRNA (the antisense strategy) it is hoped that transcription of DNA or translation mRNA will be disrupted. This would then block the cell's process of producing aberrant proteins that contribute to disease56 The agents being investigated for this therapy are nucleic acid binding agents. The most promising group is known as DNA oligonucleotides. These short strands of nucleotides are known to combine with other nucleotides in a predictable manner. This allows for design of a drug that can recognize a unique site on a particular gene. Although this type of therapy involves delivery of genetically altered substances to target cells, it is different than standard gene therapies. As described previously, most gene therapies substitute healthy genes for versions that are missing or unable to direct the appropriate synthesis of a needed protein.
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Oligonucleotides are too small to participate in the manufacture of a new protein, they can only block the expression of existing genes so they do not manufacture a specific protein. Although there are barriers to the therapeutic use of antisense oligonucleotides, there has been some progress from current clinical trials. One study that is examining cancer-fighting oligonucleotides is being performed with patients with acute myelogenous leukemia. Researchers at University of Nebraska Medical Center (Omaha) are testing the ability of an antisense oligonucleotide to destroy cancer cells in ex vivo blood cell purging. The oligonucleotide of interest is targeted to RNA that carries the p53 gene. This gene, when functioning normally, is a tumor suppressor gene, but it is over expressed in individuals with AML. A different trial is also underway at the University of Pennsylvania (Philadelphia) for the treatment of CML. This study is evaluating the ability of an antisense drug to eliminate cancer cells during ex vivo blood cell purging. The target is mRNA, which is transcribed from the c-myb gene. Under normal, healthy circumstances c-myb promotes normal proliferation of blood cells. Abnormal regulation of this gene may be responsible for the development of leukemias such as CML. Although these therapies are still in the process of being perfected, the success that has been seen in recent years suggests that antisense and triplex agents will one day become common treatments for diseases that today have no effective therapy, and foi) patients in relapse. Genetically engineered biopharmaceuticals may also impact the health care practices in novel ways. Nonviral pharmaceutical formulations of genes are being developed that do not require delivery using a vector such as a retrovirus. New methods of particulate drug delivery are being designed that will deliver DNA to the desired somatic cell in need of repair or alteration without transplantation. Genetically altered biopharmaceuticals may have a future in controlling some of the adverse sequelae that accompany BCT and BMT such as infection, inflammation, and organ toxicity. By using DNA complexes that contain lipid, protein, peptide, or polymeric carriers, as well as ligands capable of binding to cell-surface receptors on the target cell, genes can be delivered to somatic cells. This type of system has been used to transfer genes to the lung, liver, endothelium, epithelium,
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and tumor cells. These products are known as "gene medicines," "genes in a bottle," or "gene drugs."27 Methods of delivery for these genes include injection, aerosol spray, and the "gene gun" or "needle-free injection devices." The "gene gun" has been used in experimental models to enhance wound healing in a porcine partial-thickness w o u n d . 27 The disadvantage of the "gene gun" is that effective gene delivery is commonly seen in only the superficial layers of the injected tissues. The injection delivery method is being studied in diseased cardiac tissue due to myocardial infarction. An injection of DNA leads to the production of growth factors that stimulate angiogenesis causing a direct therapeutic effect. Injection or implantation of genetically altered myoblasts is a method that will result in muscle that secretes missing circulatory proteins. The aerosol method of delivery can be used for several therapeutic goals. Studies are currently underway that involve using a liposomal aerosol to replace a defective product. In patients with cystic fibrosis the cystic fibrosis transmembrane conductance regulator (CFTR) gene comes in direct contact with the airway epitheliumY The findings from this type of research may have implications for treatment of other pulmonary complications, wound healing, and cardiac muscle damage because of chemotoxicity. These could someday impact the therapeutic options for patients who are undergoing BCT. In all nonviral, in vivo studies to date, DNA is gradually eliminated from the target cell leading to a decrease in the level of the therapeutic gene over time. The kinetics of this type of delivery provide the physician with several advantages. Treatment of both acute and chronic disease is possible, as well as the ability to alter the dose andfrequency of administration in response to the patient's changing clinical picture. Studies to date indicate that the safety profile for this class of drugs is comparable to conventional drugs and biological products. There are no reports of significant toxicity from direct injection of DNA into muscle, thyroid, or other organs. Additionally, there is no evidence of development of anti-DNA antibodies after single or repetitive administration. Further, there has been no evidence of antinuclear antibodies in human subjects using a variety of delivery systems including "naked" (or plasmid) DNA, cationic lipid formulations, or protein-DNA complexes. 27
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CONCLUSION Nurses will n e e d to be as c o m f o r t a b l e with the l a n g u a g e and basic processes o f genetic engineering as they n o w are with understanding the processes o f the i m m u n e system. H i g h - t e c h bio t e c h n o l o g y will c o n t i n u e to expand. It is estimated that by the y e a r 2020 genetically e n g i n e e r e d biopharrnaceuticals will m a k e up 50% o f all drugs prescribed in the U n i t e d States, Japan, and Europe.
G e n e therapy will be a m a j o r factor in healthcare options for the 21st century. Questions n e e d to be addressed that e n c o m p a s s not o n l y the scientific, but also the sociopolitical and v a l u e issues. R e s o u r c e allocation will w e i g h against ethical and h u m a n rights for the individual. S c i e n c e is a social institution and sharing o f scientific i n f o r m a t i o n is vital for i n f o r m e d discussions. 7 Nurses are p o i s e d to p r o v i d e guidance, education, and leadership in all o f these areas.
REFERENCES
1. Berg R Singer M: Dealing With Genes; The Language of Heredity. Mill Valley, CA, University Science Books, 1992 2. Mulligan R: The basic science of gene therapy. Science 260:926-931, 1993 3. Payne IY: Will gene therapy revolutionize medicine? Nursing interventions in oncology. MD Anderson Case Rep Rev 8:9-12, 1996 4. Jenkins J, Wheeler V, Albright L: Gene therapy for cancer. Cancer Nuts 17:447-456, 1994 5. Wheeler V: Gene therapy: Current strategies and future applications. Oncol Nurs Forum 22:20-26, 1995 (suppl 12) 6. Sedivy J, Joyner A: Gene Targeting. New York, NY, Oxford University, 1992 7. Elias S, Annas G: Somatic and germline gene therapy, in Annas G, Elias S (eds): Gene Mapping, Using Law and Ethics as Guides. New York, NY, Oxford University, 1992, pp 142-154 8. Chatterjee S, Lu D, Podsakoff G, et al: Strategies for efficient gene transfer into hematopoietic cells. Ann NY Acad Sci 220:79-89, 1995 9. Blaese M, Blankenstein T, Brenner M, et al: Vectors in cancer therapy: How will they deliver? Cancer Gene Ther 2:291-297, 1995 I0. Thompson L: Stem-cell gene therapy moves toward approval. Science 255:1072, 1993 11. Verma IM: Gene therapy. Sci Am Special Issue 4:78-85, 1993 12. Varmus H, Weinberg R: Genes and the Biology of Cancer. New York, NY, Freeman 1993 13. Schwartz R: Molecular medicine, jumping genes. Mol Med 332:941-944, 1995 14. Rosenthal N: Molecular medicine, recognizing DNA. Mol Med 333:925-927, 1995 15. Giles R, Hanania E, Fu S, et al: Genetic therapy using bone marrow transplantation, in Buckner CD (ed): Technical and Biological Components of Marrow Transplantation. Boston, MA, KluwerAcademic, 1995, pp 271-280 16. Douer D, Levine A, Anderson WF, et al: High-dose chemotherapy and autologous bone marrow plus peripheral blood stem cell transplantation for patients with lymphoma or metastatic breast cancer: Use of marker genes to investigate
hematopoietic reconstitution in adults. Hum Gene Ther 7:669-684, 1996 17. Anderson WF: Human gene therapy. Science 256:808813, 1992 18. Schuening E Miller D, Torok-Storb B, et al: Study on contribution of genetically marked peripheral blood repopulating cells to hematopoietic reconstitution after transplantation. Hum Gene Ther 5:1523-1534, 1994 19. Broxmeyer H, Cooper S, Etienne-Julan M, et al: Cord blood transplantation and the potential for gene therapy. Ann NY Acad Sci 220:105-113, 1996 20. Almici C, Carlo-Stella C, Wagner J, et al: Umbilical cord blood as a source of hematopoietic stem cells: From research to clinical application. Haematologica 80:473-479, 1995 21. Thompson C: Umbilical cords: Turning garbage into clinical gold, Science 268:805-806, 1995 22. Heslop H, Brenner M, Rooney C, et al: Administration of neomycin resistance gene marked EBV specific cytotoxic T lymphocytes to recipients of rnismatched-related or phenotypically similar unrelated donor marrow grafts. Hum Gene Ther 5:381-397, 1994 23. Beck C, Cayeux S, Lupton S, et al: The thymidine kinase/ganciclovir-mediated "suicide" effect is variable in different tumor cells. Hum Gene Ther 6:1525-1530, 1995 24. Bordignon C, Bonini C, Verzeletti S, et al: Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. Hum Gene Ther 6:813-819, 1995 25. O'Shaughnessy J, Cowan K, Niehuis A, et al: Retroviral mediated transfer of the human multidrug resistance gene (MDR-1) into hematopoietic stem cells during autologous transplantation after intensive chemotherapy for metastatic breast cancer. Hum Gene Ther 5:891-911, 1994 26. Cohen J, Hogan M: The new genetic medicines. Sci Am 271:76-82, 1994 27. Ledley F: Nonviral gene therapy: The promise of genes as pharmaceutical products. Hum Gene Ther 6:1129-1144, 1995 28. Blan R, Springer M: Gene therapy--A novel form of drug delivery. N Engl J Med 333:1204-1207, 1995