- Email: [email protected]
Clinical potential for gene therapy
OBSTETRICS & GYNAECOLOGY
,~:
~i®~
~yi ~
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Mini-symposium: Molecular genetics in obstetrics and gynaecology
Clinical potential for gene therapy
M. C o n n o r Gene therapy has become a clinical reality due to scientific progress in isolating human genes and elucidating their function in health and disease. The first such treatment (gene supplementation for adenosine deaminase deficiency) was administered in 1990, and subsequently numerous protocols for gene therapy have been approved by the regulatory authorities. These early proposals can be broadly divided into gene supplementation for single gene disorders and therapeutic strategies in cancer. Amongst the single gene disorders, attention has particularly focussed on adenosine deaminase deficiency, cystic fibrosis, familial hypercholesterolaemia and haemophilia B. In cancer, gene therapy is being explored for tumour marking for residual disease, for tumour cell destruction (by immune stimulation or by targetted drug delivery) and with anti-sense strategies. However, gene therapy with administration of exogenous genes and manipulation of function of endogenous genes promises to have much wider applications. Strategies to treat a wide range of cardiovascular and neurological disorders and to curtail H I V infection have already been proposed and as the remaining human genes are cloned and their function elucidated, the list of potential therapies is bound to grow. These developments will be guided by the level of success achieved in the early trials in progress and there will need to be close regulation of this early work and prolonged follow-up of treated patients to ensure that the promise is realised.
does not assume prior expertise in molecular biology and outlines normal gene regulation, the pathophysiology of the various subtypes of genetic disease, general principles of gene therapy, progress towards clinical application in single gene disorders, cancer and other conditions and ethical considerations. At present, there are over 300 publications per annum on gene therapy and hence only key review articles have been provided for each section which should guide the reader to relevant original articles if required.
Gene therapy, which includes the administration of exogenous genes and the manipulation of endogenous genes, has become a clinical reality in the past few years due to scientific progress in gene cloning and elucidation of the complexities of gene regulation and function. This has led to new treatment strategies for a wide range of conditions and although the earliest focus was on rare genetic disorders, this has been quickly overtaken by work on gene therapy for cancer and in the future this is likely to be exceeded by treatments for common cardiovascular and neurological conditions. This is intended as a general review of progress so far and difficulties yet to be surmounted for clinicians who want to identify which patients are likely to benefit and to advise them accordingly. It
Normal gene regulation~,2 The first human gene, (chorionic sommatomamotrophin) was cloned in 1977 and to date, over 25 000 coding genes (representing 25% of the estimated total) have been partially or completely isolated (cloned). Some (an estimated 20% of the total) of these genes
M. Connor Institute o f Medical Genetics, School of Clinical Medicine, Yorkhill, Glasgow, U K
Current Obstetrics & Gynaecology (1993-)5, 206-211
© 1995PearsonProfessionalLtd
206
CLINICAL POTENTIAL FOR GENE THERAPY
are active (expressed) in a wide variety of tissues. These are called 'housekeeping genes' as they fulfill common basic functions and usually account for 90% or more of those expressed in any particular cell type. The other genes are used only at specific times and places. Thus, although every nucleated cell of an individual has the same genetic composition, the relative pattern of gene expression varies widely. Thus, for example, haemoglobin genes are only expressed in red cell precursors and their levels of activity are tightly controlled to avoid imbalance between the amounts of alpha and beta globin chains which combine to form haemoglobin. Thus, not only must control mechanisms determine which genes are active, they must also be able to adapt the level of activity to suit local requirements. Regions adjacent to each gene are involved in this process and these undergo complex and still incompletely understood interactions with multiple general and specific transcription factors in determining the level of each gene's activity. When active, many genes produce messenger RNA which passes to the cytoplasm and is decoded (translated) into a protein product which may be used in the cell or be transported to a different location. The proteins may function as enzymes in metabolic pathways or have structural roles (e.g. collagen, elastin) or act as carrier molecules (e.g. haemoglobin, albumin). The pattern of gene activity varies from moment to moment in every cell, but the overall pattern is similar in cells of a given type. Hence the overall pattern of gene activity determines the cell's characteristics (i.e. its differentiation) and manipulation of the pattern of gene expression thus has the potential to alter either a single function (e.g. ectopic production of a nonhousekeeping protein) or to alter its appearance and potential functions (e.g. specialised tissue repair by non-specialised precursor cells). Pathophysiology of genetic disease 3
There are four broad subtypes of genetic disease (Table 1). Chromosomal disorders, where there is a visible abnormality of chromosomal structure using the light microscope, are best known but are actually the rarest type of genetic disease. Affected individuals
Table 1--Types and lifetime cumulative frequencies of genetic
disease Type
% of population
Chromosomal disorders
0.7%
Singlegene disorders (autosomal dominant, autosomal recessive and sex-linked)
1.0%
Cumulative genetic disorders (cancer)
30%
Part-genetic disorders (congenital malformations, chronic disorders of adulthood)
60%
207
have imbalance at multiple gene loci with corresponding reductions (for deletions) and increases (for duplications) in activity of involved genes. The mechanism whereby such imbalances produce particular clinical features is not yet understood and hence gene therapy to ameliorate or rectify chromosomal disorders is a distant prospect at the present time. Single gene disorders arise as a result of mutations in one or both members of a pair of genes on an autosome or sex chromosome. There are a large number of subtypes with over 6000 already known and many more suspected and their collective frequency exceeds that of the chromosomal disorders. Autosomal recessive single gene disorders commonly involve gene loci for enzymatic proteins and these enzymes can often function adequately with quite low levels of residual activity. In an autosomal recessive condition, the affected person has two copies of the mutant gene and has close to 0% residual activity whereas their healthy parents with one normal copy and one underactive copy have enzyme activities of 50% of normal. Commonly, restoration of activity to 10% or more will allow the biochemical pathway to function adequately and this large safety margin has meant that they have been obvious targets for early gene therapy experiments. For some autosomal recessive disorders (e.g. phenylketonuria) the metabolic block can be circumvented by dietary modifications and for some (e.g. adult type of Gaucher's disease) it is possible to purify and replace the missing enzyme. For many autosomal recessive disorders, treatment by dietary modification or enzyme replacement is not possible or fully effective and these are candidates for gene therapy by addition of an extra normal copy of the altered gene to the target tissue (supplementation gene therapy). In contrast, autosomal dominant traits commonly involve structural and carrier proteins rather then enzymes. When they encode enzymes (e.g. acute intermittent porphyria) or receptor proteins (e.g. the low density lipoprotein receptor in familial hypercholesterolaemia) the reduced enzyme activity to 50% or 50% reduction in receptor activity exceeds the safety margin and symptoms result. (This appearance of symptoms in the individual with one mutant and one normal gene of a pair (a heterozygote) is the hallmark of dominant inheritance). In this situation, addition of a supplementary normal gene to the target tissue is a valid approach and has already been employed in familial hypercholesterolaemia. In many dominant traits, the disease pathogenesis and approach to gene therapy is less straightforward. Often, the gene product interacts with proteins from the partner gene or other genes to make the final structural protein. Thus, for example, type I collagen is a trimer of two alpha 1 and one alpha 2 polypeptide chains which are products of separate paired genes. Mutation in one alpha 2 gene causes 50% of type I collagen to be abnormal and mutation in one alpha 1 gene causes
208
CURRENT OBSTETRICS AND GYNAECOLOGY
75% to be abnormal. Hence, a single gene can have a disproportionate effect (called protein suicide) and addition of a further normal gene would not rectify the problem. It is technically possible to repair genes in vitro by site-directed mutagenesis, but such a direct approach in vivo is not possible at the present time. Hence, many dominant traits are not likely to be early candidates for gene therapy. Sex-linked genes may encode either enzymes (e.g. clotting factors for haemophilia A and B) or structural proteins (e.g. dystrophin whose deficiency causes Duchenne muscular dystrophy). Where the enzyme has a large safety margin (e.g. severe haemophiliacs have less than 5% clotting factor activity) supplementation gene therapy is being attempted but where gene repair would be required or where the function of the protein is currently not understood (e.g. fragile X syndrome) early attempts at gene therapy are unlikely. Part-genetic disorders are common and include many congenital malformations and common chronic disorders of adulthood (e.g. hypertension, arthritis, prematul"e vascular disease and dementia). In contrast to the single gene disorders of the preceding paragraphs, the family tree for part-genetic disorders is not striking and they are identified by the increased recurrence risks for family members in proportion to the degree of genetic similarity. The genes act singly or in combination to render the individual more liable to the influence of environmental factors and hence prevention could be achieved either by avoidance of the environmental influence or by modifying the genetic susceptibility. At the present time, the number and nature of the genes which are involved in most part-genetic disorders is largely unknown and similarly, relatively little is known about their environmental interactions. (Hence, gene therapy for prevention of part-genetic disorders is not imminent but strategies are being considered to influence disease progression/complication). Cumulative genetic disorders are also common and include all cancers. Cancer results from individual cells which have escaped from normal regulatory mechanisms and these control mechanisms are exerted by a series of genes. For cancer to occur, several paired tumour suppressor genes usually need to be inactivated. The first inactivating mutation may be inherited and this would be associated with a family history of early onset of cancer and a tendency to multiple tumours in an affected individual (e.g. inherited breast and ovarian cancer due to BRCA1 mutations). In the progression to cancer in addition to loss of function of a series of tumour suppressor genes, there are also commonly activating mutations of oncogenes. The precise pattern of inactivation/activation will differ from patient to patient with the same cancer and differs for different tumour types and contributes to the variation in prognosis and responsiveness to standard therapies. The simplest gene therapy strategy of supplementation with a normal copy of a key tumour suppressor gene is limited by the difficulties in
targetting delivery to every malignant cell, but a variety of alternatives are being evaluated in on-going trials. Basic principles of gene therapy ~6
Current approaches to gene therapy may be broadly considered under addition of exogenous genes and manipulation of endogenous gene function. There are two basic requirements for exogenous gene transfer and expression: 1. The gene and its adjacent regulatory regions need to have been cloned and characterised. 2. The gene needs to be transferred to the target tissue site and needs to function appropriately. As indicated earlier rapid progress has been made with cloning human genes although progress with understanding their regulation has been slower. These genes vary widely in size and some such as the globins (at 1500 base pairs) are tiny whereas others such as dystrophin (at 2 400 000 base pairs) are enormous. Many transfer methods can only accommodate relatively small genes and for the larger genes, the identification of key functional regions for transfer has been important. Multiple methods have been developed to transfer genes (Table 2). Retrovirus vectors were widely used in early experiments as their biology was relatively simple compared to other viral vectors and they had the ability to efficiently enter target host cells and integrate their genome into the host DNA with the consequent potential for long-term expression. Their main disadvantages are the relatively small amount of exogenous DNA which they can accommodate and their dependence on active DNA replication for efficient integration. This restriction of infection to actively dividing cells means that this approach is not applicable to terminally differentiated cells (e.g. neurones). Apart from the need for cellular division, retrovirus vectors are also relatively non-specific and this (and their relative instability in vivo) means that the target cells usually need to be isolated and infected ex vivo with the retrovirus containing the corrective gene before return to the patient. Other disadvantages Table 2
Methods for transfer of exogenous genes
Viral vectors Retroviruses Adenoviruses Herpes viruses Adeno-associated viruses Non-viral vectors Cationic liposomes Mammalian artificial chromosomes Mechanical methods Direct injection Particle bombardment - 'gene gun'
CLINICAL POTENTIAL FOR GENE THERAPY
are the poor long-term expression of human DNA from the viral promoter and the random nature of integration into host genomic DNA which has the potential for unintentional insertional mutagenesis. Adenovirus vectors can accommodate larger amounts of exogenous DNA and can infect nondividing cells. The exogenous DNA is introduced into infected cells but does not integrate into the host genome and thus repeated delivery will be required with consequent potential for immune stimulation and reduced clinical effect in long-term treatment. Liposomes are membranous lipid vesicles which enclose an aqueous volume. Cationic liposomes form complexes with DNA and can accommodate large amounts of exognous DNA. Cellular targetting is a problem as is cellular entry. The former is being approached by incorporating proteins for specific cellular receptors (e.g. transferrin for cells with transferrin receptors) and the latter is being approached by incorporating bacterial proteins internalin and invasin which specifically bind to cell surfaces and allow bacterial entry into cells by receptor mediated endocytosis or phagocytosis. Direct injection to single cells is tedious but useful for transgenic animals. Another approach which is being explored is to use a 'gene gun' which introduces tiny DNA-coated gold or silver particles directly into skin or deeper tissues. The alternative strategy is to modify the function (increase or decrease) or endogenous genes. Oligonucleotide probes will bind specifically to complementary sequences and these are being studied to block transcription (anti-gene approach), to block translation (the anti-sense strategy) or as molecular decoys to sequester low concentration transcription factors and to act as ribozymes in cleavage of target RNA species. Gene supplementation for single gene disorders 7-9
Gene supplementation aims to provide a functional copy of a gene which can then circumvent the effect of the single or pair of mutant genes in that target cell. Several criteria need to be considered in selecting single gene disorders for gene supplementation. The condition must be serious with poor or no alternative therapy and as indicated it has to be potentially correctible by addition of an extra normal gene copy. The gene must have been cloned and its regulatory sequences identified and ideally the gene should be small and not require highly precise regulation. The target tissue needs to be accessible and before proceeding to human gene therapy correction with low levels of expression in vivo in an animal model and/or in vitro using human target tissue is desirable. Given these criteria, relatively few single gene disorders were ideal candidates for early attempts at gene therapy and of these adenosine deaminase (ADA) deficiency was the clear favourite.
209
ADA deficiency causes a serious immunodeficiency for which treatment options are limited. The gene is a small 'housekeeping' gene and treatment with allogeneic bone marrow transplantation (i.e. by the introduction of genetically normal haemopoeitic cells) is effective if the level of ADA is restored to 10% or more of normal. There is also evidence for in vivo selection in favour of corrected cells with allogeneic bone marrow transplantation. The main drawback (apart from the rarity of affected patients) is that the optimal target cells namely the marrow stem cells constitute only 0.01% of all nucleated bone marrow cells and are not actively dividing. The first two children commenced treatment in 1990. In these patients, the white cells were separated and treated with recombinant ADA retrovirus in vitro and then the transfected cells were returned to the patients. As peripheral white cells are the target repeated treatments have been required, but these have resulted in a sustained return of immune function. Cystic fibrosis results from a deficiency of the protein cystic fibrosis transmembrane conductance regulator (CFTR). In vitro a single copy of CFTR can correct the metabolic defect in cystic fibrosis cells and it has also been shown that the presence of less than 10% corrected cells in a monolayer can restore function in the entire cell monolayer, the so-called bystander effect. Adenoviruses naturally infect the respiratory epithelium and they and cationic liposomes have been the usual transfer methods for trials of gene therapy in cystic fibrosis. Progress has been steady but not without setbacks (e.g. generalised adenovirus infection) and repeated administration is envisaged given that neither approach allows integration into the host genome. Gene therapy for cancer l°,n
Because the pathophysiology of most cancers appears to involve, at some stage, the activation of dominant oncogenes and/or the loss of function of tumour suppressor genes, the most obvious approach to gene therapy of cancer would be to inhibit the function of the activated oncogene(s) or to introduce a normal copy of a tumour suppressor gene(s). The main limitations of these approaches is our current inability to target very efficiently and specifically malignant cells in vivo for gene transfer. Other forms of gene therapy, however, would not require such high efficiency of gene transfer. These include genetic immunomodulation, specific activation of prodrugs in tumours and normal tissue protection. Adoptive immunotherapy involves the transfer to the tumour-bearing host of immune cells that have antiturnout reactivity and can mediate direct or indirect antitumour effects. Adoptive immunotherapy of cancer has been attempted with lymphokine-activated killer (LAK) cells and with tumour-infiltrating lymphowtes (TIL). TIL are isolated by culturing
210 CURRENT OBSTETRICS AND GYNAECOLOGY single cell suspensions from tumours under the influence of recombinant interleukin-2. Gene therapy has been used both to study and to improve adoptive immunotherapy. The first approved gene transfer experiment in humans involved retroviral gene marking of TILs in patients with advanced melanoma. In this experiment a neomycin resistance gene was delivered to autologous TIL by retroviral infection so genetically marking these cells and allowing study of their long-term distribution and survival. TIL produce a measurable anti-tumour effect but are unable to eradicate all turnout. Attempts have thus been made to enhance the antitumour activity of TIL by transducing them with a vector for tumour necrosis factor (TNF). TNF is a cytokine that possesses a wide variety of biologic activities including potent antitumour activity and immunomodulatory properties but systemic administration of adequate doses is limited by side-effects and the idea is for TIL to achieve high local concentrations of TNF. Another approach to genetic immunomodulation is the introduction of genes (e.g. exogenous cytokines or MHC class I expression vectors) into tumour cells that will induce an immune reaction against modified and unmodified turnout cells. Genes that result in the conversion of non-toxic prodrugs to toxic active forms can be directed to cancer cells in two ways: the 'suicide' gene could be placed under control of a promoter/enhancer that would be active in tumour cells specifically (e.g. erb B2 promoter in breast cancer) or the suicide gene could be specifically delivered to the tumour cells (e.g. by intratumoural injection). The activation of a prodrug by such a transgene that is specifically expressed in turnout cells has been called virally directed enzyme prodrug therapy (VDEPT). For example vectors expressing the Herpes simplex thymidine kinase (HSV-tk) gene under the control of an alphafetoprotein promoter will be specifically expressed in hepatoma cells. Thymidine kinase can convert the non-toxic prodrug 6-methoxypurine arabinonucleoside to potent cytotoxic products and thus provide cytotoxicity specifically to hepatoma cells. HSV-tk can also convert ganciclovir to toxic metabolites and this approach is being explored for glioblastoma multiforme by intratumoural injection of the transgene in replication-defective adenovirus or liposomes. In these early experiments a clinically important 'bystander' effect has been observed. In a mixture of tumour cells with only one-half transfected over 90% will die when treated with the prodrug thus improving the prospects for turnout eradication without the need to target every malignant cell. The mechanism is unclear and might be due to transfer of toxic metabolites through tumour cell gap junctions. Somatic gene transfer could also be used in the treatment of cancer to confer protection against the toxicities of anticancer drugs. Because haematotoxicity is often dose-limiting in cancer treatment, the
haematopoetic system would be a prime target for this approach and this might be achieved by transferring drug resistance genes. Other applications 12-~7
As indicated there has been a rapid evolution in the field of gene therapy from the initial focus on rare single gene disorders to cancer and already a host of strategies are being suggested for a wide range of common disorders of adulthood (e.g. prevention of restenosis in arterial grafts, induction of new cardiac muscle or revascularisation of ischaemic areas, antiviral approaches or immune restoration for HIV disease and focal gene expression in Parkinson's disease). This list is growing rapidly and it would be a safe prediction that further developments in this area are inevitable as the remaining genes are cloned and their functions elucidated. Ethical aspect of gene therapy is
The use of tissue or organ transplants (including their component genes) to treat disease has a long history and treatment with protein products from genetic engineering has been available since 1982 but treatment at the level of the gene using recombinant DNA technology is a very recent development. In 1989, the first clinical protocol was approved and in 1990, the first treatment (gene supplementation for adenosine deaminase deficiency) was administered. There has since been substantial and wide ranging public debate and the consensus view is that gene therapy is ethically no different from other forms of therapy but careful assessment of therapeutic trials is essential and at present only gene therapy directed at somatic cells (as opposed to germ cells) can be considered. References 1. Lewin B. Genes V. Oxford: Oxford University Press, 1994 2. Barsh GS, Epstein CJ Gene structure and function. In: Rimoin D J, Connor JM, Pyeritz RE, eds. Principles and Practice of Medical Genetics, 3rd Edn. Edinburgh: Churchill Livingstone, 1996 3. Connor JM, Ferguson-Smith MA. Essential Medical Genetics. 5th edn. Oxford: Blackwell Science, 1996 4. Gordon EM, Anderson W E Gene therapy using retroviral vectors. Curr Opin Biotechnol 1994; 5:611-6 5. Trapnell BC, Gorziglia M. Gene therapy using adenoviral vectors. Curr Opin Biotechnol 1994; 5:617-25 6. Ledley FD. Non-viral gene therapy. Curr Opin Biotechnol 1994; 5:626-36 7. O'Neal WK, Beaudet AL. Somatic gene therapy for cystic fibrosis. Hum Molec Gene 1994; 3:1497-1502 8. Kay MA, Woo SLC. Gene therapy for metabolic disorders. Trends Genet 1994; 10: 2 5 3 ~ 9. Morgan JE. Cell and gene therapy in Duchenne muscular dystrophy. Hum Gene Ther 1994; 5:165-73 10. Culver KW, Blaese RM. Gene therapy for cancer. Trends Genet 1994; 10:174-8 11. Huber BE. Gene therapy strategies for treating neoplastic disease. Ann N Y Acad Sci 1994; 716:6-11 12. Nabel EG. Gene therapy for cardiovascular disease. Circulation 1994; 91:541 8
CLINICAL POTENTIAL FOR GENE THERAPY
13. Isner JM, Feldman LJ. Gene therapy for arterial disease. Lancet 1994; 344:16534 14. Friedmann T. Gene therapy for neurological disorders. Trends Geneti 1994;10: 210M 15. Bridges SH, Sarver N. Gene therapy and immune restoration for HIV disease Lancet 1995; 345:427-32 16. Evans C, Robbins PD. Prospects for treating arthritis by
211
gene therapy. Journal of Rheumatology 1994; 21: 779-82 17. Riley DJ, Lee WH. The potential of gene therapy for treatment of kidney diseases. Seminars in Nephrology 1995; 15:57 69 18. Bolton RG. The ethics of gene therapy. J Roy Soc Med 1994; 87:3024
,~:
~i®~
~yi ~
~
Mini-symposium: Molecular genetics in obstetrics and gynaecology
Clinical potential for gene therapy
M. C o n n o r Gene therapy has become a clinical reality due to scientific progress in isolating human genes and elucidating their function in health and disease. The first such treatment (gene supplementation for adenosine deaminase deficiency) was administered in 1990, and subsequently numerous protocols for gene therapy have been approved by the regulatory authorities. These early proposals can be broadly divided into gene supplementation for single gene disorders and therapeutic strategies in cancer. Amongst the single gene disorders, attention has particularly focussed on adenosine deaminase deficiency, cystic fibrosis, familial hypercholesterolaemia and haemophilia B. In cancer, gene therapy is being explored for tumour marking for residual disease, for tumour cell destruction (by immune stimulation or by targetted drug delivery) and with anti-sense strategies. However, gene therapy with administration of exogenous genes and manipulation of function of endogenous genes promises to have much wider applications. Strategies to treat a wide range of cardiovascular and neurological disorders and to curtail H I V infection have already been proposed and as the remaining human genes are cloned and their function elucidated, the list of potential therapies is bound to grow. These developments will be guided by the level of success achieved in the early trials in progress and there will need to be close regulation of this early work and prolonged follow-up of treated patients to ensure that the promise is realised.
does not assume prior expertise in molecular biology and outlines normal gene regulation, the pathophysiology of the various subtypes of genetic disease, general principles of gene therapy, progress towards clinical application in single gene disorders, cancer and other conditions and ethical considerations. At present, there are over 300 publications per annum on gene therapy and hence only key review articles have been provided for each section which should guide the reader to relevant original articles if required.
Gene therapy, which includes the administration of exogenous genes and the manipulation of endogenous genes, has become a clinical reality in the past few years due to scientific progress in gene cloning and elucidation of the complexities of gene regulation and function. This has led to new treatment strategies for a wide range of conditions and although the earliest focus was on rare genetic disorders, this has been quickly overtaken by work on gene therapy for cancer and in the future this is likely to be exceeded by treatments for common cardiovascular and neurological conditions. This is intended as a general review of progress so far and difficulties yet to be surmounted for clinicians who want to identify which patients are likely to benefit and to advise them accordingly. It
Normal gene regulation~,2 The first human gene, (chorionic sommatomamotrophin) was cloned in 1977 and to date, over 25 000 coding genes (representing 25% of the estimated total) have been partially or completely isolated (cloned). Some (an estimated 20% of the total) of these genes
M. Connor Institute o f Medical Genetics, School of Clinical Medicine, Yorkhill, Glasgow, U K
Current Obstetrics & Gynaecology (1993-)5, 206-211
© 1995PearsonProfessionalLtd
206
CLINICAL POTENTIAL FOR GENE THERAPY
are active (expressed) in a wide variety of tissues. These are called 'housekeeping genes' as they fulfill common basic functions and usually account for 90% or more of those expressed in any particular cell type. The other genes are used only at specific times and places. Thus, although every nucleated cell of an individual has the same genetic composition, the relative pattern of gene expression varies widely. Thus, for example, haemoglobin genes are only expressed in red cell precursors and their levels of activity are tightly controlled to avoid imbalance between the amounts of alpha and beta globin chains which combine to form haemoglobin. Thus, not only must control mechanisms determine which genes are active, they must also be able to adapt the level of activity to suit local requirements. Regions adjacent to each gene are involved in this process and these undergo complex and still incompletely understood interactions with multiple general and specific transcription factors in determining the level of each gene's activity. When active, many genes produce messenger RNA which passes to the cytoplasm and is decoded (translated) into a protein product which may be used in the cell or be transported to a different location. The proteins may function as enzymes in metabolic pathways or have structural roles (e.g. collagen, elastin) or act as carrier molecules (e.g. haemoglobin, albumin). The pattern of gene activity varies from moment to moment in every cell, but the overall pattern is similar in cells of a given type. Hence the overall pattern of gene activity determines the cell's characteristics (i.e. its differentiation) and manipulation of the pattern of gene expression thus has the potential to alter either a single function (e.g. ectopic production of a nonhousekeeping protein) or to alter its appearance and potential functions (e.g. specialised tissue repair by non-specialised precursor cells). Pathophysiology of genetic disease 3
There are four broad subtypes of genetic disease (Table 1). Chromosomal disorders, where there is a visible abnormality of chromosomal structure using the light microscope, are best known but are actually the rarest type of genetic disease. Affected individuals
Table 1--Types and lifetime cumulative frequencies of genetic
disease Type
% of population
Chromosomal disorders
0.7%
Singlegene disorders (autosomal dominant, autosomal recessive and sex-linked)
1.0%
Cumulative genetic disorders (cancer)
30%
Part-genetic disorders (congenital malformations, chronic disorders of adulthood)
60%
207
have imbalance at multiple gene loci with corresponding reductions (for deletions) and increases (for duplications) in activity of involved genes. The mechanism whereby such imbalances produce particular clinical features is not yet understood and hence gene therapy to ameliorate or rectify chromosomal disorders is a distant prospect at the present time. Single gene disorders arise as a result of mutations in one or both members of a pair of genes on an autosome or sex chromosome. There are a large number of subtypes with over 6000 already known and many more suspected and their collective frequency exceeds that of the chromosomal disorders. Autosomal recessive single gene disorders commonly involve gene loci for enzymatic proteins and these enzymes can often function adequately with quite low levels of residual activity. In an autosomal recessive condition, the affected person has two copies of the mutant gene and has close to 0% residual activity whereas their healthy parents with one normal copy and one underactive copy have enzyme activities of 50% of normal. Commonly, restoration of activity to 10% or more will allow the biochemical pathway to function adequately and this large safety margin has meant that they have been obvious targets for early gene therapy experiments. For some autosomal recessive disorders (e.g. phenylketonuria) the metabolic block can be circumvented by dietary modifications and for some (e.g. adult type of Gaucher's disease) it is possible to purify and replace the missing enzyme. For many autosomal recessive disorders, treatment by dietary modification or enzyme replacement is not possible or fully effective and these are candidates for gene therapy by addition of an extra normal copy of the altered gene to the target tissue (supplementation gene therapy). In contrast, autosomal dominant traits commonly involve structural and carrier proteins rather then enzymes. When they encode enzymes (e.g. acute intermittent porphyria) or receptor proteins (e.g. the low density lipoprotein receptor in familial hypercholesterolaemia) the reduced enzyme activity to 50% or 50% reduction in receptor activity exceeds the safety margin and symptoms result. (This appearance of symptoms in the individual with one mutant and one normal gene of a pair (a heterozygote) is the hallmark of dominant inheritance). In this situation, addition of a supplementary normal gene to the target tissue is a valid approach and has already been employed in familial hypercholesterolaemia. In many dominant traits, the disease pathogenesis and approach to gene therapy is less straightforward. Often, the gene product interacts with proteins from the partner gene or other genes to make the final structural protein. Thus, for example, type I collagen is a trimer of two alpha 1 and one alpha 2 polypeptide chains which are products of separate paired genes. Mutation in one alpha 2 gene causes 50% of type I collagen to be abnormal and mutation in one alpha 1 gene causes
208
CURRENT OBSTETRICS AND GYNAECOLOGY
75% to be abnormal. Hence, a single gene can have a disproportionate effect (called protein suicide) and addition of a further normal gene would not rectify the problem. It is technically possible to repair genes in vitro by site-directed mutagenesis, but such a direct approach in vivo is not possible at the present time. Hence, many dominant traits are not likely to be early candidates for gene therapy. Sex-linked genes may encode either enzymes (e.g. clotting factors for haemophilia A and B) or structural proteins (e.g. dystrophin whose deficiency causes Duchenne muscular dystrophy). Where the enzyme has a large safety margin (e.g. severe haemophiliacs have less than 5% clotting factor activity) supplementation gene therapy is being attempted but where gene repair would be required or where the function of the protein is currently not understood (e.g. fragile X syndrome) early attempts at gene therapy are unlikely. Part-genetic disorders are common and include many congenital malformations and common chronic disorders of adulthood (e.g. hypertension, arthritis, prematul"e vascular disease and dementia). In contrast to the single gene disorders of the preceding paragraphs, the family tree for part-genetic disorders is not striking and they are identified by the increased recurrence risks for family members in proportion to the degree of genetic similarity. The genes act singly or in combination to render the individual more liable to the influence of environmental factors and hence prevention could be achieved either by avoidance of the environmental influence or by modifying the genetic susceptibility. At the present time, the number and nature of the genes which are involved in most part-genetic disorders is largely unknown and similarly, relatively little is known about their environmental interactions. (Hence, gene therapy for prevention of part-genetic disorders is not imminent but strategies are being considered to influence disease progression/complication). Cumulative genetic disorders are also common and include all cancers. Cancer results from individual cells which have escaped from normal regulatory mechanisms and these control mechanisms are exerted by a series of genes. For cancer to occur, several paired tumour suppressor genes usually need to be inactivated. The first inactivating mutation may be inherited and this would be associated with a family history of early onset of cancer and a tendency to multiple tumours in an affected individual (e.g. inherited breast and ovarian cancer due to BRCA1 mutations). In the progression to cancer in addition to loss of function of a series of tumour suppressor genes, there are also commonly activating mutations of oncogenes. The precise pattern of inactivation/activation will differ from patient to patient with the same cancer and differs for different tumour types and contributes to the variation in prognosis and responsiveness to standard therapies. The simplest gene therapy strategy of supplementation with a normal copy of a key tumour suppressor gene is limited by the difficulties in
targetting delivery to every malignant cell, but a variety of alternatives are being evaluated in on-going trials. Basic principles of gene therapy ~6
Current approaches to gene therapy may be broadly considered under addition of exogenous genes and manipulation of endogenous gene function. There are two basic requirements for exogenous gene transfer and expression: 1. The gene and its adjacent regulatory regions need to have been cloned and characterised. 2. The gene needs to be transferred to the target tissue site and needs to function appropriately. As indicated earlier rapid progress has been made with cloning human genes although progress with understanding their regulation has been slower. These genes vary widely in size and some such as the globins (at 1500 base pairs) are tiny whereas others such as dystrophin (at 2 400 000 base pairs) are enormous. Many transfer methods can only accommodate relatively small genes and for the larger genes, the identification of key functional regions for transfer has been important. Multiple methods have been developed to transfer genes (Table 2). Retrovirus vectors were widely used in early experiments as their biology was relatively simple compared to other viral vectors and they had the ability to efficiently enter target host cells and integrate their genome into the host DNA with the consequent potential for long-term expression. Their main disadvantages are the relatively small amount of exogenous DNA which they can accommodate and their dependence on active DNA replication for efficient integration. This restriction of infection to actively dividing cells means that this approach is not applicable to terminally differentiated cells (e.g. neurones). Apart from the need for cellular division, retrovirus vectors are also relatively non-specific and this (and their relative instability in vivo) means that the target cells usually need to be isolated and infected ex vivo with the retrovirus containing the corrective gene before return to the patient. Other disadvantages Table 2
Methods for transfer of exogenous genes
Viral vectors Retroviruses Adenoviruses Herpes viruses Adeno-associated viruses Non-viral vectors Cationic liposomes Mammalian artificial chromosomes Mechanical methods Direct injection Particle bombardment - 'gene gun'
CLINICAL POTENTIAL FOR GENE THERAPY
are the poor long-term expression of human DNA from the viral promoter and the random nature of integration into host genomic DNA which has the potential for unintentional insertional mutagenesis. Adenovirus vectors can accommodate larger amounts of exogenous DNA and can infect nondividing cells. The exogenous DNA is introduced into infected cells but does not integrate into the host genome and thus repeated delivery will be required with consequent potential for immune stimulation and reduced clinical effect in long-term treatment. Liposomes are membranous lipid vesicles which enclose an aqueous volume. Cationic liposomes form complexes with DNA and can accommodate large amounts of exognous DNA. Cellular targetting is a problem as is cellular entry. The former is being approached by incorporating proteins for specific cellular receptors (e.g. transferrin for cells with transferrin receptors) and the latter is being approached by incorporating bacterial proteins internalin and invasin which specifically bind to cell surfaces and allow bacterial entry into cells by receptor mediated endocytosis or phagocytosis. Direct injection to single cells is tedious but useful for transgenic animals. Another approach which is being explored is to use a 'gene gun' which introduces tiny DNA-coated gold or silver particles directly into skin or deeper tissues. The alternative strategy is to modify the function (increase or decrease) or endogenous genes. Oligonucleotide probes will bind specifically to complementary sequences and these are being studied to block transcription (anti-gene approach), to block translation (the anti-sense strategy) or as molecular decoys to sequester low concentration transcription factors and to act as ribozymes in cleavage of target RNA species. Gene supplementation for single gene disorders 7-9
Gene supplementation aims to provide a functional copy of a gene which can then circumvent the effect of the single or pair of mutant genes in that target cell. Several criteria need to be considered in selecting single gene disorders for gene supplementation. The condition must be serious with poor or no alternative therapy and as indicated it has to be potentially correctible by addition of an extra normal gene copy. The gene must have been cloned and its regulatory sequences identified and ideally the gene should be small and not require highly precise regulation. The target tissue needs to be accessible and before proceeding to human gene therapy correction with low levels of expression in vivo in an animal model and/or in vitro using human target tissue is desirable. Given these criteria, relatively few single gene disorders were ideal candidates for early attempts at gene therapy and of these adenosine deaminase (ADA) deficiency was the clear favourite.
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ADA deficiency causes a serious immunodeficiency for which treatment options are limited. The gene is a small 'housekeeping' gene and treatment with allogeneic bone marrow transplantation (i.e. by the introduction of genetically normal haemopoeitic cells) is effective if the level of ADA is restored to 10% or more of normal. There is also evidence for in vivo selection in favour of corrected cells with allogeneic bone marrow transplantation. The main drawback (apart from the rarity of affected patients) is that the optimal target cells namely the marrow stem cells constitute only 0.01% of all nucleated bone marrow cells and are not actively dividing. The first two children commenced treatment in 1990. In these patients, the white cells were separated and treated with recombinant ADA retrovirus in vitro and then the transfected cells were returned to the patients. As peripheral white cells are the target repeated treatments have been required, but these have resulted in a sustained return of immune function. Cystic fibrosis results from a deficiency of the protein cystic fibrosis transmembrane conductance regulator (CFTR). In vitro a single copy of CFTR can correct the metabolic defect in cystic fibrosis cells and it has also been shown that the presence of less than 10% corrected cells in a monolayer can restore function in the entire cell monolayer, the so-called bystander effect. Adenoviruses naturally infect the respiratory epithelium and they and cationic liposomes have been the usual transfer methods for trials of gene therapy in cystic fibrosis. Progress has been steady but not without setbacks (e.g. generalised adenovirus infection) and repeated administration is envisaged given that neither approach allows integration into the host genome. Gene therapy for cancer l°,n
Because the pathophysiology of most cancers appears to involve, at some stage, the activation of dominant oncogenes and/or the loss of function of tumour suppressor genes, the most obvious approach to gene therapy of cancer would be to inhibit the function of the activated oncogene(s) or to introduce a normal copy of a tumour suppressor gene(s). The main limitations of these approaches is our current inability to target very efficiently and specifically malignant cells in vivo for gene transfer. Other forms of gene therapy, however, would not require such high efficiency of gene transfer. These include genetic immunomodulation, specific activation of prodrugs in tumours and normal tissue protection. Adoptive immunotherapy involves the transfer to the tumour-bearing host of immune cells that have antiturnout reactivity and can mediate direct or indirect antitumour effects. Adoptive immunotherapy of cancer has been attempted with lymphokine-activated killer (LAK) cells and with tumour-infiltrating lymphowtes (TIL). TIL are isolated by culturing
210 CURRENT OBSTETRICS AND GYNAECOLOGY single cell suspensions from tumours under the influence of recombinant interleukin-2. Gene therapy has been used both to study and to improve adoptive immunotherapy. The first approved gene transfer experiment in humans involved retroviral gene marking of TILs in patients with advanced melanoma. In this experiment a neomycin resistance gene was delivered to autologous TIL by retroviral infection so genetically marking these cells and allowing study of their long-term distribution and survival. TIL produce a measurable anti-tumour effect but are unable to eradicate all turnout. Attempts have thus been made to enhance the antitumour activity of TIL by transducing them with a vector for tumour necrosis factor (TNF). TNF is a cytokine that possesses a wide variety of biologic activities including potent antitumour activity and immunomodulatory properties but systemic administration of adequate doses is limited by side-effects and the idea is for TIL to achieve high local concentrations of TNF. Another approach to genetic immunomodulation is the introduction of genes (e.g. exogenous cytokines or MHC class I expression vectors) into tumour cells that will induce an immune reaction against modified and unmodified turnout cells. Genes that result in the conversion of non-toxic prodrugs to toxic active forms can be directed to cancer cells in two ways: the 'suicide' gene could be placed under control of a promoter/enhancer that would be active in tumour cells specifically (e.g. erb B2 promoter in breast cancer) or the suicide gene could be specifically delivered to the tumour cells (e.g. by intratumoural injection). The activation of a prodrug by such a transgene that is specifically expressed in turnout cells has been called virally directed enzyme prodrug therapy (VDEPT). For example vectors expressing the Herpes simplex thymidine kinase (HSV-tk) gene under the control of an alphafetoprotein promoter will be specifically expressed in hepatoma cells. Thymidine kinase can convert the non-toxic prodrug 6-methoxypurine arabinonucleoside to potent cytotoxic products and thus provide cytotoxicity specifically to hepatoma cells. HSV-tk can also convert ganciclovir to toxic metabolites and this approach is being explored for glioblastoma multiforme by intratumoural injection of the transgene in replication-defective adenovirus or liposomes. In these early experiments a clinically important 'bystander' effect has been observed. In a mixture of tumour cells with only one-half transfected over 90% will die when treated with the prodrug thus improving the prospects for turnout eradication without the need to target every malignant cell. The mechanism is unclear and might be due to transfer of toxic metabolites through tumour cell gap junctions. Somatic gene transfer could also be used in the treatment of cancer to confer protection against the toxicities of anticancer drugs. Because haematotoxicity is often dose-limiting in cancer treatment, the
haematopoetic system would be a prime target for this approach and this might be achieved by transferring drug resistance genes. Other applications 12-~7
As indicated there has been a rapid evolution in the field of gene therapy from the initial focus on rare single gene disorders to cancer and already a host of strategies are being suggested for a wide range of common disorders of adulthood (e.g. prevention of restenosis in arterial grafts, induction of new cardiac muscle or revascularisation of ischaemic areas, antiviral approaches or immune restoration for HIV disease and focal gene expression in Parkinson's disease). This list is growing rapidly and it would be a safe prediction that further developments in this area are inevitable as the remaining genes are cloned and their functions elucidated. Ethical aspect of gene therapy is
The use of tissue or organ transplants (including their component genes) to treat disease has a long history and treatment with protein products from genetic engineering has been available since 1982 but treatment at the level of the gene using recombinant DNA technology is a very recent development. In 1989, the first clinical protocol was approved and in 1990, the first treatment (gene supplementation for adenosine deaminase deficiency) was administered. There has since been substantial and wide ranging public debate and the consensus view is that gene therapy is ethically no different from other forms of therapy but careful assessment of therapeutic trials is essential and at present only gene therapy directed at somatic cells (as opposed to germ cells) can be considered. References 1. Lewin B. Genes V. Oxford: Oxford University Press, 1994 2. Barsh GS, Epstein CJ Gene structure and function. In: Rimoin D J, Connor JM, Pyeritz RE, eds. Principles and Practice of Medical Genetics, 3rd Edn. Edinburgh: Churchill Livingstone, 1996 3. Connor JM, Ferguson-Smith MA. Essential Medical Genetics. 5th edn. Oxford: Blackwell Science, 1996 4. Gordon EM, Anderson W E Gene therapy using retroviral vectors. Curr Opin Biotechnol 1994; 5:611-6 5. Trapnell BC, Gorziglia M. Gene therapy using adenoviral vectors. Curr Opin Biotechnol 1994; 5:617-25 6. Ledley FD. Non-viral gene therapy. Curr Opin Biotechnol 1994; 5:626-36 7. O'Neal WK, Beaudet AL. Somatic gene therapy for cystic fibrosis. Hum Molec Gene 1994; 3:1497-1502 8. Kay MA, Woo SLC. Gene therapy for metabolic disorders. Trends Genet 1994; 10: 2 5 3 ~ 9. Morgan JE. Cell and gene therapy in Duchenne muscular dystrophy. Hum Gene Ther 1994; 5:165-73 10. Culver KW, Blaese RM. Gene therapy for cancer. Trends Genet 1994; 10:174-8 11. Huber BE. Gene therapy strategies for treating neoplastic disease. Ann N Y Acad Sci 1994; 716:6-11 12. Nabel EG. Gene therapy for cardiovascular disease. Circulation 1994; 91:541 8
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13. Isner JM, Feldman LJ. Gene therapy for arterial disease. Lancet 1994; 344:16534 14. Friedmann T. Gene therapy for neurological disorders. Trends Geneti 1994;10: 210M 15. Bridges SH, Sarver N. Gene therapy and immune restoration for HIV disease Lancet 1995; 345:427-32 16. Evans C, Robbins PD. Prospects for treating arthritis by
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gene therapy. Journal of Rheumatology 1994; 21: 779-82 17. Riley DJ, Lee WH. The potential of gene therapy for treatment of kidney diseases. Seminars in Nephrology 1995; 15:57 69 18. Bolton RG. The ethics of gene therapy. J Roy Soc Med 1994; 87:3024