Neuromusc. Disord., Vol. 2, No. 2, pp. 75-83. 1992 Printed in Great Britain

096~8966/92 $5.00 + 0.00 ( 1 9 9 2 Pergamon Press Ltd

REVIEW

GENE

THERAPY:

PRESENT

ARTICLE

SITUATION

AND

FUTURE

PROSPECTS

FRANtTOISGROS Institut Pasteur (URA-CNRS 1148), Unit+ de Biochimie, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France (Received 24 February 1992; accepted 8 May 1992)

A~tract--In this review, some of the most characteristic experimental approaches have been surveyed, using in vitro cultivated cell systems or animal models to achieve therapeutical gene treatment of severe monogenic diseases. Great advances have been made in the utilization of viral-mediated gene transfer as well as by direct DNA injection techniques to permit either stable insertion of a "correcting" gene into the chromosomes of a cell or a tissue or its penetration and activity as an episome. In most instances, long-term expression of the newly introduced genetic information could be obtained and certain genetic defects could be compensated for or corrected in cultivated cells or in vivo. Recent approvals on certain protocols concerning somatic gene therapy in humans indicate that the medical world is quite conscious of the importance of this new strategy. Certain ethical considerations are listed and discussed. Key words: Gene therapy, DNA vectors, animal models, human trials, ethics.

In the present review, after having briefly surveyed the main technical strategies utilized in animal models to ensure efficient gene transfer with the aim to correct for a genetic disease, the preliminary results obtained both on in vitro cell systems as well as in vivo on whole animals will be examined, showing that a genetic disease can actually or potentially be corrected. The first trials and future prospects of gene therapy in human beings will then be discussed along with some ethical aspects of these approaches.

INTRODUCTION

There is an increasing body of experimental or preclinical arguments to suggest that gene thera p y - n a m e l y the possibility of curing inheritable h u m a n disorders by introducing foreign genes into the somatic cells of patients--might well represent a new and decisive step in contemporary medicine. This view is supported by: (i) the large sum of data that has been obtained on laboratory animals, some of which are good models for gene therapy in humans, since they display a severe genetic defect that can either be cured or greatly attenuated by appropriate gene transfer; and (ii) present achievements and future prospects of the h u m a n genome p r o g r a m m e (HGP), due to the knowledge, recently acquired at a molecular level, about severe genetic diseases, of which Duchenne muscular dystrophy or cystic fibrosis offer a very significant illustration. A new spectrum of genetic diseases is thus becoming amenable to molecular approaches and could therefore benefit from gene therapy. Moreover, the first trials to apply gene therapy to patients suffering from acute melanoma or from an adenosine deaminase defect (leading to immunodeficiency) have been authorized in the U.S.A., and this might well constitute a decisive step towards a cure for some forms of cancer or some severe monogenic diseases.

MAIN TECHNICAL APPROACHES TO GENE TRANSFER IN ANIMALS--TYPES OF VECTORS/ MODES OF ADMINISTRATION

There are essentially three general "routes" whereby gene therapy can in principle be achieved, as deduced from the work done, so far, on animal models (Fig. 1). Route 1. This can be referred to as " e x vivo gene transfer", an approach which exhibits a formal resemblance to tissue or organ transplantation: cells from an animal that bears the mutation (fibroblasts, bone marrow cells, hepatocytes, etc.) are sampled and gene modification is accomplished in vitro, before reinjecting or regrafting the cells thus engineered into the animal. Several techniques can be utilized to "insert the transgene": one, most often used, 75

76

Review Article

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Fig. 1. Various gene transfer strategies to correct for genetic diseases in an animal.

is by means of a recombinant retrovirus vector, but, with time, many alternative techniques have been found to be equally feasible: electroporation, direct microinjection of DNA into the nucleus, utilization of transferin-polycation DNA particles, etc . . . . In most cases, particularly when a retrovirus vector is utilized, there is random integration of the transgene and correction of the genetic defect relies on the addition o f a compensating gene to the mutated genome. Such an approach would not be appropriate had one to correct or compensate for a dominant mutation. When transfection, electroporation or direct microinjection are used, this might permit, in some circumstances, achievement of homologous recombination, namely replacement of the mutated gene by the normal allele. One can thus select, ex vivo, only those cells in which the replacement has been effective before the reinjection step. Route 2. This consists of infecting the whole animal with harmless viruses carrying the normal gene. Among the virus "vectors" most commonly used so far are: papovaviruses (SV40, P o l y o m a . . . ) or, more often, adenoviruses. This strategy might become compulsory in cases where the mutation hinders the physiology of cells that cannot be manipulated ex vivo, or when the therapy that is required has to be introduced rapidly after birth. Adenoviruses offer many advantages as vectors since they can transfer

exogenous DNA with great efficiency, causing only benign infections to man. Moreover, and contrary to retroviruses, infecting recombinant DNA is not inserted into the cellular genome, which alleviates the risks of stimulating oncogenic sequences. Route 3. This route corresponds to "direct DNA transfer" by physical or chemical methods. For example, DNA can be injected into the whole animal after coupling with an asialoglycoprotein, or by means of a syringe. Recently Jon A. Wolff and his associates, from the University of Wisconsin, have shown that a variety of genes can be injected directly into the extravascular space of skeletal or cardiac muscles. Not only can these genes remain intact for weeks or months, but they have been shown to function at a high rate inside the injected muscles. When a pRSV lac Z-DNA vector carrying an expressible Escherichia coli fl-galactosidase gene was injected into the quadriceps muscle, the gene could direct the synthesis of fl-galactosidase for weeks [1]. DNA vectors carrying genes for chloramphenicol acetyl-transferase (pRSVCAT) and luciferase (pRSVL) were also separately injected into mouse skeletal muscles and the levels of expression from both constructs were comparable with the levels of expression obtained from fibroblasts transiently infected in vitro under optimal conditions [2]. Protein expression from the pRSVCAT vector and the pRSVL vector was stable for at

Review Article

least 2 and 12 months, respectively. Of particular interest is that only muscle tissues appear suited to this, so-called, "direct" gene therapy, a situation that probably stems from the fact that skeletal and cardiac muscle cells have unique microanatomical features, such as T tubules, which may play a critical role in DNA uptake and storage. Independent experiments have shown that the injected DNA was present in the muscle tissue as linear and open circular, extra-chromosomal DNA [3]. A recent illustration of the technique introduced by J. Wolff and his colleagues has been published [4]. A 12 kb human full length human dystrophin cDNA recombined to a RSV vector (PRSV-Dy), or a shorter construct containing a partial Becker type dystrophin cDNA (pCMV Dy-B) was injected intramuscularly into dystrophin-deficient m d x mice and it could be observed that human dystrophin was present in the cytoplasm and sarcolemna of approximately 1% of the myofibers. Although the data are preliminary and their interpretation sometimes suffers from the rather high rate of spontaneous reversion leading to the presence of a background of dystrophin-positive fibers, they suggest that transfer of the dystrophin gene into DMD patient myofibers could be beneficial and emphasize the importance of both the direct DNA transfer approach and the muscle as a target tissue for the delivery of gene-directed products in general. In vivo electroporation has also been reported as an efficient technique to promote the introduction of plasmid DNA into other tissues, such as the skin cells of the mouse [5]. Recently, in the April issue of H u m a n Gene Therapy [6] Curiel et al. at the University of North Carolina and Wagner et al. at the Research Institute of Molecular Pathology in Vienna, have described a new vector which combines potential safety advantages with the ability to carry very large amounts of DNA into the recipient cell. This vector consists of the purified empty shell of an adenovirus at the surface of which DNA carrying the "therapeutic genes" (at the exclusion of any active viral gene) is attached by a linker. This linker comprises an antibody specific for adenovirus (and which hooks onto the envelope protein) covalently bound to a short polypeptide chain made of lysine amino acid units. It is this "lysine tail" that connects DNA (or any nucleic acid) to the viral shell. The protein shell with its attached DNA penetrates the cell via a surface receptor and transports with itself the DNA molecule to the nucleus. Not only does this

77

system permit a very efficient expression of the imported genes, but it was observed that DNA of a size corresponding to 48,000 base pairs could be transported. Experiments are in progress to test whether the appropriate sequences allowing permanent integration into the genome could be added to genes that would be used for therapeutic purposes. This way one could combine potential safety and great transfer efficacy, with long-term expression. Whatever it is, this new composite vector is presently being tested for its ability to permit expression of "therapeutic" genes in vivo (and not only in cultivated cells). There is little doubt that the need to achieve a perfect gene delivery system will induce great progress in the biotechnology of DNA vectors. EXPRESSION OF GENETIC SEQUENCES RELATED TO HUMAN DISEASES AFTER GENE TRANSFER INTO CELLS CULTIVATED I N VITRO

A question that scientists have often addressed before attempting gene therapy on animals is related to the extent of expression of the gene concerned after its transfer into in vitro cultivated cells. Many types of genes related to severe neuropathies, hemoglobinopathies, myopathies, immunodeficiency or hepatic syndromes have thus been investigated. In the majority of cases, retroviral-mediated transfer was used but other viral vectors, such as adenoviruses, or direct DNA transfer techniques, have also been used. In certain cases correction of the abnormal physiology of the cells thus treated could be demonstrated. As shown in Table 1, some of the most significant results with this aproach have been obtained with hematopoietic stem cells as recipients using retroviruses as vectors. The interest in this model system lies in the fact that these cells usually exist in a state of active division, a condition that is a requirement for the retrovirusmediated integration of the foreign gene. Yet, in some instances, the difficulty arising from the quiescent or post-mitotic state of a given tissue could be circumvented by forcing its re-entry into the mitotic cycle. Such was the case, for example, when cells from the differentiated hepatic tissue were induced to regenerate. By perfusing this regenerating liver with a fluid containing the recombined retrovirus, some researchers have succeeded in causing efficient integration of genes related to important liver disorders (for example the disease affecting the human receptor to low

Review Article

78

Table 1. Expression of genetic sequences related to human diseases in cells transfected in vitro with recombinant retroviruses as vectors Enzyme/protein or transcript HPRT* ADAt PRT~ Glucocerebrosidase Human #-globin LDLR§ CFTR I

Disease

Recipient cell

Lesh Nyhan Severe combined immunodeficiency (SCID) SCID-like Gaucher disease Sickle cell anemia Thalassemia Hepatic syndromes Cystic fibrosis

Murine hematopoietic cells Human hematopoietic cells Primate stem cell lines Hematopoietic cell lines Fibroblast- lymphoid cell lines from patients Murine hematopoietic stem cells Primary hepatocytes in tissue culture Human pancreatic adenocarcinoma cells with CF defectql

*HPR'i': hypoxantine phosphoribosyl transferase. t A D A : human adenosine deaminase. SPRT: phosphoribosyl transferase. §LDLR: human receptor for low density lipoproteins. IICFTR: cystic fibrosis transmembrane conductance regulator. ¶!CF: disease causing abnormalities in water and electrolyte transport. Table 2. Expression of genetic sequences related to human diseases in cells transfected in vitro with recombinant adenoviruses or SV40 viruses as vectors Enzyme or protein Adenoviruses ml-AT* (Rosenfeld et al. [23]) Dystrophin (minigene)$ (Quantin et al. [30]) SV40 viruses Dystrophin full length complementary DNA

Disease

Recipient cell

~I-AT deficiency Lung sensitivity to NE-proteaset Duchenne or Becker muscular dystrophy D M D or Becker dystrophy

C H O cells, HeLa cells Rat lung epithelial cells Mouse myoblasts and myotubes

COS cells§

* ~1 -AT: ml-antitrypsin, inhibitor of NE-protease (Ad~I-AT vector: Gilardi et al. F E B S Left. 1990; 2,67:60) [23]. tNE-protease: neutrophil elastase. SDystrophin: the product encoded by the gene whose mutations cause the Duchenne (or Becker) type muscular dystrophy (Quantin et al. International Workshop on Human Gene Transfer, Paris 1991: Use of adenovirus as an expression vector in muscle cells; application to dystrophin). §COS cells (kidney cells): Experiment by Lee et al. [9]. Table 3. Long-term expression following re-introduction into animals of genetically transformed cells (retrovirus) Retroviral-mediated gene transfer

Promoter and transformed tissue

Authors and main conclusion

ADA (adenosine deaminase) ADA

Bone marrow cells re-introduced in irradiated mice Similar protocol Promoters: LTR, f l - a c t i n . . .

ADA

Identical but with SV40 or cytomegalovirus promoter Identical but with PG promoter

Moore et al. [10] Persistent expression for 7 months Wilson et al. [11] Expression for up to 6 months in hematopoietic cells of irradiated mice Osborne et al. [12] Kaleko et al. [I 3] Lira et al. [14] Expression up to 4 months Nolta et al. [15], Correll et al. [16] Long-term expression Palmer et al. [17]

ADA GC (glucocerebrosidase) Factor IX tpA antimitotic agents

Same protocol as for ADA expression Re-introduction of transformed fibroblasts Re-introduction of transformed endothelial cells

density lipoproteins, LDLR, or phenylalanine hydroxylase) [7]. In certain cases previously mentioned, the altered physiology of the cell could be restored. An elegant illustration is afforded by work from Drumm et al. [8] in which these authors have succeeded in complementing the cystic fibro-

Wilson et al. [I 8] Nabel et al. [19]

sis (CF) defect in vitro. Accordingly, using a recombinant retrovirus as vector, they have transferred a functional cystic-fibrosis transmembrane conductance regulator (CFTR) cDNA into CFPAC- 1, a pancreatic adenocarcinoma cell line derived from a patient with CF, that stably displays the chloride transport abnormalities

ReviewArticle characteristic of the disease. Not only could a viral-derived CFTR transcript be detected in the transfected cells, but, a CAMP-dependent stimulated 125Iefflux could also be restored. Whole cell patch clamp measurements performed on transfected clones confirmed that the anion efflux response was actually due to a CAMP-dependent stimulation of C1 conductance. Table 2 indicates that recombinant adenoviruses or SV40 viruses can also direct efficient in vitro expression of genes whose mutations are responsible for severe human pathologies. Even very large structural proteins, such as dystrophin (the membrane bound component of the muscle which is altered or abolished in severe myopathies), can be expressed in the transfected cells. An illustration of this is found in the work of Lee et al. [9]. Using a SV40 recombinant construct, in which a human full length dystrophin cDNA has been inserted, they obtained efficient dystrophin expression in COS cells and showed that the newly expressed dystrophin has a peripheral localization beneath the plasma membrane. However, it is not clear whether this localization corresponds to a correct attachment of dystrophin onto the membranes of these cells.

I N V I V O LONG-TERM EXPRESSION OF GENES

FOLLOWING RE-INTRODUCTION OF RETROVIRAL TRANSFORMED CELLS INTO ANIMALS

Another goal that many groups have attempted to achieve as a prerequisite to efficient gene therapy was to obtain long-term in vivo gene expression, following re-introduction of properly engineered cells or tissues into various animals (Table 3). Many studies have been made along this line mainly with the gene encoding adenosine deaminase (ADA) whose alteration or lack of expression is responsible for the severe combined immunodeficiency disease (SCID). For example, Moore et al. [10] succeeded in showing persistent expression of human ADA in the hematopoietic tissues of recipient mice for as long as 7 months after cocultivating bone marrow cells with cells producing an ADA-retrovirus recombinant and re-introducing the transduced bone marrow tissue into irradiated mice. Similar approaches have been made by Wilson et al. [1 l] who placed the ADA gene under the control of either a viral LTR or an internal chicken fl-actin promoter. As in the previous experiment, bone marrow was transduced by

79

cocultivation and subsequently transplanted into irradiated hosts. The best results were obtained with the fl-actin promoter. All the animals thus treated expressed the gene for up to 6 months following transplantation in their hematopoietic cells. Satisfactory results have also been obtained by using similar protocols with retroviral constructs in which the ADA gene was adjacent to the SV40 or the cytomegalovirus immediate early promoter [12, 13]. Finally, Lim et al. [14] could also demonstrate stable long-term human ADA expression in recipient mice, by making use of a recombinant in which the ADA gene was driven by a phosphoglycerate kinase promoter. Sustained expression was maintained in some animals even after 4 months. All these results reveal encouraging prospects for curing the SCID syndrome by gene transfer into bone marrow stem cells in humans. Another important marker of clinical relevance, the gene encoding human glucocerebrosidase (GC), an enzyme altered in Gaucher disease, has been subject to many experiments with the aim to obtain long-term retroviral-mediated expression, either in vitro (bone marrow cultures) or in vivo by transplanting suitably transformed bone marrow cells into irradiated mice [15, 16]. Similarly, mice or rats transplanted with fibroblasts in which the gene for human factor IX had been transferred have shown long-term expression of this antihemophilic factor [17]. An interesting cell system that seems appropriate for prolonged expression of a variety of genes is that of the vascular endothelial cells [l 8, 19]. These cells can be transformed with genes encoding clotting factors, tpA or antiproliferative agents and, following implantation into animals (dogs), they permit localized delivery of the corresponding products. RESULTS OBTAINED IN ANIMALS EXHIBITING A SPECIFIC DISEASE

Whether the technique consisted of e x vivo gene transfer and re-injection, transfer of cloned genes into prefertilized oocytes or direct DNA transfer into the whole organism, the successful cure of animals suffering from severe pathological syndromes as a consequence of genetic defects could sometimes be demonstrated. This has proved possible in mice, rats, rabbits, dogs, pigs or monkeys. In some cases, when a cure could not be achieved, transient restoration in

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the synthesis of an altered gene product could be demonstrated in the tissues, or circulating fluids of treated animals exhibiting the specific disease. In 1986, Costantini et al. [20] succeeded in correcting a murine/3-thalassemia by the transfer of cloned fl-globin genes into the mouse germ line. The mouse strain was deficient in fl-globin synthesis because of a deletion of the j~maj globin gene. Introduction of either a cloned mouse ~maj globin gene or a cloned human fl-globin gene was able to eliminate or reduce the anemia and the associated abnormalities of the red blood cells. Although it is clear, for obvious ethical reasons, that transfer of genes into the germ line does not constitute a suitable strategy for correcting thalassemia syndromes (or other genetic diseases) in human beings, these data hold some interest in showing that a cloned fl-globin gene can substitute for a defective gene in thalassemic red blood cells. By transferring an OTC c D N A into mouse embryos with an X-linked OTC deficiency (a disease causing liver incapacity to detoxify ammonium ions) it has proved possible to restore the synthesis of the normal enzyme. The same result could be achieved by its cDNA-mediated expression in intestinal mucosa. More specifically, using recombinant adenoviruses in which the OTC cDNA was placed under the control of the viral major late promoter (MLP) of an adenovirus and by infecting new-born mice, Perricaudet, Briand and their colleagues in Paris, succeeded in restoring hepatic OTC activity. This treatment also caused a marked diminution of orotic acid in the urines with a reduction or disappearance of the general symptoms characteristic of the disease. A recombinant adenovirus OTC vector administered intravenously to "~pfash" mice (OTC-deficient) corrected the enzyme deficiency for at least 1 yr, a result showing clearly that long-term expression is possible [21]. Generally speaking considerable work based upon use of retrovirus or adenovirus vector systems, has been devoted to correction of OTC deficiency in the murine species [22]. More recently, scientists from NIH, the Cancer Institute in Villejuif and Perricaudet's group have attempted to challenge the gene therapeutical potential of appropriate adenovirus recombinants that were directly instilled into the respiratory tract of the rat. They used a recombinant adenovirus vector carrying an ~elantitrypsin eDNA with a deletion of a portion of both the E3 region (that permits encapsidation of the recombinant genome containing the exo-

genous gene) and a portion of the E l a coding sequence (that impairs viral replication). The authors were able to show infection of respiratory tract epithelial cells both in vitro and in vivo. Particularly striking was the expression of human ~1 AT transcripts and 0e'! AT protein molecules in the lung tissue from the cotton rats after simple intratracheal instillation [23]. These encouraging results suggest that this technique may be useful for experimental studies with the human CF gene whose alteration causes a severe lung disease. Not only has the gene for the CF transmembrane conductance regulator been cloned [24-26] but, as previously mentioned, a C F T R eDNA was shown to complement the CF chloride transport deficiency in vitro [27, 28].

GENE THERAPY IN MAN: FIRST TRIALS, FUTURE PROSPECTS

The first trials on humans which have been authorized in the U.S.A. have been dealing with two very severe, albeit unrelated diseases. One consisted of an attempt to cure patients carrying metastasic melanoma, while the other concerned the syndrome of immunodeficiency resulting from a mutation in the adenosine deaminase gene. Both projects received definitive approvals in September 1990. Rosenberg and his associates have treated patients suffering from melanoma at an acute phase of the disease. A particular class of lymphocytes, cultivated from the patient's tumors, and endowed with a high tumor infiltrating capacity, or TIL lymphocytes, was used. In a preliminary approach, an antibiotic resistance marker was introduced into these cells and these were re-injected. This permitted demonstration of the penetration and persistence of these particular lymphocytes inside the tumor [28]. In a second series of attempts, the TIL cells were engineered to receive a gene that produces TNF, a growth factor manifesting strong antitumor activity (Memorandum, May 1990, Gene Therapy) in patients with advanced cancer using tumor infiltrating lymphocytes transduced with the gene coding for T N F [29]. Thus far, the results obtained seem encouraging. One should note that this type of therapy does not attempt to compensate for, or to remove the mutated genes (a goal which, in the present case, would be difficult if not impossible to achieve since the disease is a multifunctional one), but to use genetically modified cells as a microfactory

ReviewArticle for releasing a substance displaying a strong pharmacological activity. This particular strategy should probably receive great attention in the future due to its versatility. To this kind of approach belongs, for example, a series of attempts to transfer partial drug resistance genes since a significant limitation to the effectiveness of antineoplastic chemotherapy is precisely related to the amount of drug that can be administered. In the second series of gene therapeutic trials, Anderson and co-workers have tried to restore the adenosine deaminase activity in children suffering from ADA deficiency, a syndrome which is responsible for 15% of all cases of severe combined immunodeficiency (SCID) [29]. Therefore the approach made corresponds to what could be regarded as compensating therapy. In this disease immunodeficiency results from a defect observed in T-lymphocytes. This is because elevated levels of adenosine are converted by a tissue-specific kinase into deoxy ATP which is poisonous to lymphocytes. Transfection of ADA-deficient lymphocytes with a retrovirus recombinant containing the intact ADA gene placed under a SV promotor control greatly diminished their sensitivity to dATP. The results obtained to date support the feasibility of somatic gene therapy of ADA deficiency via the introduction of a retroviralADA recombinant into human hematopoietic stem cells. Stem cell purification procedures are now being combined with the gene transfer procedure to decrease the cell numbers to be manipulated and eventually administered to patients. In particular, light density BMC cells are cocultivated with the virus-producing cells in the presence of recombinant human interleukins 3 and 6. It is clear that much of the future prospects of human gene therapy will depend upon the results of these clinical or preclinical trials. A few months ago the NIH Human Gene Therapy subcommittee granted provisional approval to four new gene therapy experiments involving human subjects. In particular Rosenberg, in trying to go one step further than he had already achieved using TNF carrying TIL cells, has proposed to incorporate two genes encoding T N F or interleukin-2, respectively, into the patient's own tumor cells and to inject the tumor cells, thus engineered, back into the patient. The idea is that the modified tumor cells will stimulate a more powerful immune response. This clinical approach should be made on 30 patients with advanced cancers (kidney, colon, skin, etc . . . . ). The panel also approved an experiment pro-

81

posed by Wilson (University of Michigan) to treat patients with familial hypercholesterolemia. It is well known that an elevated blood level in low density lipoproteins leads to atherosclerosis with a high probability. A large part of the LDL cholesterol is eliminated by specific LDL receptors present at the surface of hepatic cells. In certain patients a genetic disorder causes a defect in, or the absence of the receptor. This is particularly so in homozygotic patients. Wilson's experiment will involve removing a portion of the subject's liver, inserting the gene coding for the LDL receptor into progenitor liver cells and then injecting the modified cells back into the patient. Another protocol that has been approved by the NIH subcommittee concerns a project submitted by Freeman (University of Rochester). The idea is to insert into ovarian cancer cells a gene from the Herpes virus which encodes the enzyme thymidine kinase and to inject these cells back into 16 women with advanced ovarian cancers that are refractory to other therapeutics. The hope is that such ovarian cancer cells will become sensitive to the drug gancyclovir, an antitumor agent known to interact with thymidine kinase. Although these protocols must still be cleared by the full Recombinant Advisory Committee, their description is indicative of some of the general directions being taken by clinicians at the present time.

ETHICALCONSIDERATIONS Gene therapy seems, at first sight, to offer important, and in some instances, very attractive clinical possibilities for the treatment of many human diseases. This stems from the rather large body of results which has been obtained in model laboratory animals carrying specific genetic defects. It stems also from the great variety of techniques which has been developed over the last few years to permit gene transfer into a variety of cells or tissues, including the more recent ones which are based upon direct DNA transfer by intramuscular injection or by other physical means. Finally, a great hope is placed in the preliminary trials, made on human beings and which look, so far, quite encouraging if not yet definitively conclusive. A major advantage offered by somatic gene therapy is that this approach would circumvent, or avoid, in certain instances the technical but also serious ethical problems which are relevant

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to o r g a n t r a n s p l a n t a t i o n (for e x a m p l e i m m u n e rejection, a n d the c o m p l i c a t e d issues due to the s h o r t a g e o f donors). It c o u l d also relieve certain patients from the c o n s t r a i n t o f using, d u r i n g their whole lifespan, large a m o u n t s o f expensive p r o d u c t s (for e x a m p l e the A D A enzyme). Yet, one m u s t b e a r in m i n d that somatic gene t h e r a p y has n o t really entered into clinical practice on a scale sufficient to p e r m i t definitive conclusions r e g a r d i n g its p o t e n t i a l for h u m a n patients. F r o m a m o r e strict ethical s t a n d p o i n t , a m a j o r question is w h e t h e r o r n o t gene t h e r a p y c o u l d be p r a c t i s e d on h u m a n germinal cells, the r a t i o n a l e being to a t t e m p t e r a d i c a t i o n o f a disease within a family at risk. T h e p r o s p e c t o f h u m a n germ line t h e r a p y is raising severe reservations, and, in m o s t cases, strong o b j e c t i o n s p a r t i c u l a r l y in L a t i n countries, an a t t i t u d e t h a t is d i c t a t e d b o t h by m o r a l a n d technical considerations. The m o r a l s t a n d p o i n t , generally speaking, touches, u p o n the status o f the h u m a n e m b r y o which, in the o p i n i o n o f others, should n o t be r e g a r d e d as a biological system that could be m a n i p u l a t e d in vitro, but as a p o t e n t i a l h u m a n being. I n a s m u c h as there is, for the time being, no g u a r a n t e e t h a t g e r m line genetic m o d i f i c a t i o n , even with the intent to cure a h u m a n disease, w o u l d n o t lead to u n i n t e n d e d d a m a g e o f the genetic material, this d a m a g e w o u l d indeed be p e r p e t u a t e d t h r o u g h s u b s e q u e n t generations. R e t r o v i r a l - d i r e c t e d gene transfer results in rand o m i n t e g r a t i o n a n d this m i g h t cause a l t e r a t i o n s in i m p o r t a n t genes which might thus b e c o m e defective. Perhaps, when t a r g e t e d r e p l a c e m e n t o f defective genes by h o m o l o g o u s r e c o m b i n a t i o n is fully mastered, the situation r e g a r d i n g germ line gene t h e r a p y will be c o n t e m p l a t e d with a different attitude. A n o t h e r technical p o i n t often a d v a n c e d , derives from the following a r g u m e n t : to achieve g e r m line gene t h e r a p y one w o u l d have to p r o c e e d at the level o f the first b l a s t o m e r e s after forced o v u l a t i o n a n d in vitro fertilization. Unless the m o t h e r c a r r y i n g the genetic defect is h o m o zygotic for the m u t a t i o n (a s i t u a t i o n r e g a r d e d as very rare), 50% o f the e m b r y o s should, in principle, be n o r m a l . Hence, p r e n a t a l d i a g n o s i s before r e - i m p l a n t a t i o n ( a l t h o u g h it raises o t h e r kinds o f objections) s h o u l d p e r m i t selection o f the healthy e m b r y o s instead o f using gene thera p y to prevent the o u t c o m e o f the disease in those b e a r i n g the m u t a t i o n . In s u m m a r y , there is general a g r e e m e n t that s o m a t i c gene t h e r a p y should be e n c o u r a g e d as

one o f the m o s t p r o m i s i n g a p p r o a c h e s to reduction o f the terrible b u r d e n o f h e r e d i t a r y diseases, a n d even cancers. But reservations still exist a b o u t germ line genetic intervention. M o r e o v e r , it is clear that, until the t e c h n o l o g y has i m p r o v e d a n d the p o t e n t i a l risks have been minimized, s o m a t i c gene t h e r a p y should be restricted to patients with very serious life-threatening conditions, keeping in m i n d that a slippery slope exists leading f r o m the t h e r a p e u t i c a l act to an intervention that w o u l d be d i c t a t e d either by pure individualistic convenience, or by eugenic motives.

Acknowledgments--This work was supported by grants from the Centre National de la Recherche Scientifique, the Pasteur Institute, the Association Franqaise contre les Myopathies, the Institut National de la Sant6 et de la Recherche M6dicale, the Association Franqasie pour la Recherche sur le Cancer and the Foundation pour la Recherche M+dicale. REFERENCES

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