Eur. J. Biochem. 208, 211 -225 (1992) 0FERS 3 992

Review Concepts and strategies for human gene therapy Klaus ROEMER and Theodore FRIEDMANN Cenier for Molecular Genetics, University of California, San Diego, USA (Received April 2, 1992) - EJB 92 0462

Methods of' modern molecular genetics have been developed that allow stable transfer and expression of foreign DNA sequences in human and other mammalian somatic cells. It is therefore no surprise that the methods have been applied in attempts to complement genetic defects and correct disease phenotypes. Two decades of research have now led to the first clinically applicable attempts to introduce genetically modified cells into human beings to cure diseases caused at least partially by genetic defects. We discuss here some of the strategies being followed for both in vitro and in vivo application of therapeutic gene transfer and summarize some of the technical and conceptual difficulties associated with somatic-cell gene therapy.

Beginning in the late 1960s and early 1970s, the availability of new molecular-biological tools and concepts began to suggest that designed genetic changes may eventually provide a new and effective form for the therapy of human diseases. In 1972, the medical need, as well as the technical and conceptual issues involved in efficient virally mediated gene transfer for therapeutic purposes, were outlined (Friedmann and Roblin, 1972). In the 20 years since that time, progress has been so great that human experiments are now underway to test the efficacy of foreign genes in defective human cells as a form of gene therapy. There are a very large number of human diseases that represent potential targets for this kind of manipulation. Morc than 4500 human diseases are currently classified as genetic (McKusick, 1988). Until now, only a very small minority of these diseases has been associated with specific mutations in the human genome, Recessive genetic diseases like cystic fibrosis and adenosine deaminase (ADA) deficiency require mutations to be present in both alleles of a gene in order to generate the disease phenotype, while dominant diseases like Huntington's disease can be caused by the presence of only one mutated copy of a gene. The mere presence of a mutant allele overrides the remaining normal allele and leads to disease. With the application of the tools of molecular genetics, and with new approaches to the identification and characterization of disease-related defects, therapy through the correction of genetic defects has come within reach (Friedmann, 1989; Anderson 1992). Most modern gene-therapeutic approaches are based on the introduction of functional copies of defective genes into cells. They really represent gene Correspondence to T. Friedmann, Center Por Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634, USA Abbreviations. HIV, human immunodeficiency virus; AIDS, acquired immune-dcficiency syndrome; ADA, adenosine deaminase; LDL, low-dcnsity lipoproteins; CFTR, cystic-fibrosis transmembrane-conductance regulator; LTR, long tcrminal repeats; MDR, multidrug resistance; HSV, herpcs simplex virus,

augmentation rather than genetic correction. These appoaches are therefore most applicable for the correction of recessive diseases and may be applicable for dominant diseases in those cases where a dominant phenotype can be overridden by expression of the funtional copy of a wild-type gene. Some dominant disorders, which involve gain-of-function mutations, however, may need more elaborate strategies like substitution of defective sequences by genetic recombination or the delivery of genetic information that encodes functions that inactivate or otherwise interfere with the action of the dominant-acting allele. The concept of correction of genetic defects by introduction of functional sequences into cells is, in principle, valid for both monogenetic and polygenetic disorders, although the latter obviously require the knowledge and availability of all genetic sequences affected in the polygenetic disease. For the purpose of this review, gene therapy may be defined as the correction of a disease phenotype through the introduction of new genetic information into the affected organism. Such foreign genetic information might either restore or supplement defective functions or alternatively, interfere with the expression of a mutant genetic function. The foreign genetic material can be introduced directly into the appropriate tissue in vivo, or can first be introduced into appropriate cells in vitro which are subsequently grafted to a physiologically relevant site in the organism. Most genetic diseases are individually relatively rare. Nevertheless, we now recognize that some of the most common diseases of our society such as most human cancers, forms of cardiovascular disease, neuropsychiatric disease and degenerative disease have important genetic components and, therefore, for the purpose of the design of new forms of therapy, should be considered genetic disorders. We wish to suggest that approaches to the therapy of many of these disorders, including degenerative disorders such as Parkinson's and Alzheimer's disease, even infectious diseases such as acquired immune-deficiency syndrome (AIDS), will come to rely on gene-therapy techniques. However, our current technical

212 capacities and knowledge of the fundamental biological functions are still too rudimentary to suggest wide-scale application of gene therapy. The methods and concepts of gene therapy must improve and evolve, and physiological characteristics of the target tissues and organs must be better understood than they are now. Nevertheless, without doubt, many techniques satisfying the unique demands of the many different types of potential target cells will be developed. Tn this review, we focus on basic concepts and currently available techniques for gene therapy as they are now coming to be applied to the correction of several model diseases. We present here a description of the approaches being taken toward somatic-cell gene therapy. We will not discuss in detail the more complicated and much more controversial issue of germ-line gene therapy. Gene-transfer strategies Gene therapy requires not only the introduction of foreign DNA sequences into eukaryotic cells, but also their stable and appropriately regulated expression in the new environment. Ideally, this need would be best achieved by replacement of the defective sequences with normal sequences through homologous recombination. Only in this way can one ensure that the foreign genes will be regulated faithfully and appropriately as the endogenous gene would be. While there have been considerable advances in making such specific genetic corrections by methods of homologous recombination, particularly in mouse embryonal stem cells (Frohman and Martin, 1989), those techniques have not yet been applied efficiently to many other cell types including those most likely to be target cells for gene therapy. Newer developments may eventually make such specific modifications possible by increasing the frequency of homologous recombination or allowing efficient site-specific integration of sequences in mammalian cells (O’Gorman et al., 1991; Buerstedde and Takeda, 1991). Such methods are not yet available. Until now, current gene therapeutic approaches have relied mainly on the complementation of the defects through the introduction of additional functional genes to a defective genome, i.e. gene augmentation. During the past several decades, techniques have developed to introduce DNA into mammalian cells with great efficiency. Many of these techniques have involved the use of transducing viruses. Other methods for gene transfer into mammalian cells, generally referred to as transfection, have taken advantage of procedures to overcome electrochemical barrier between the negatively charged cell membrane and the negatively charged DNA macromolecule. Such physical methods for DNA transfer have included the introduction into target cells of DNA compexed with DEAE-dextran (Pagan0 et al., 1967), the use of calcium phosphate coprecipitation (Graham and van der Eb, 1973), and the transfer of complete metaphase chromosomes (McBride and Ozer, 1973; Spandidos and Siminovitch, 1977). Genes also have been transferred into cells through fusion of recipient cells with bacterial spheroblasts (Schaffner, 1980), liposomes (Fraley et al., 1980), erythrocyte-membrane vesicles (Sugawa et al., 1985), fusion of cells with Sendai-virus envelopes (Volsky et al., 1984), whole-cell fusion (Stanbridge, 1976), and through uptake of DNA complexed with nonhistone nuclear proteins (Kaneda et al., 1989) or with polylysine-carrying receptor ligands (Wu et al., 1991). Still other methods of gene transfer have included the use of a complex between DNA and lipids (Felgner et al., 1987), microinjection of DNA (Capecchi, 1980), targeting of cells with micropro-

jectiles (Klein et al., 1987), or introduction of DNA through cell membranes damaged by high-voltagc electric fields (Shigekawa and Dower, 1988; Keating and Toneguzzo, 1990). Finally, the use of transducing viruses has come to be one of the most powerful tools for gene transfer into mammalian cells (Eglitis and Anderson, 1988; Friedmann, 1989; McLachlin et al., 1990). The efficiency of DNA transfer by any of these methods vanes greatly, depending on the target-cell type and the state of cellular replication at the time of DNA delivery. Most of the methods mentioned above allow stable integration of the transgenes in some cell types, albeit at a very low frequency, ranging from one transformation event in 10’ - 10’ cells. In most cases, stable integration of the transferred sequences seems to occur into random sites of the recipient genome. Unfortunately, the integration process following most physical methods of gene transfer is frequently accompanied by amplification and rearrangement of the transferred sequences, resulting in insertion of tandem arrays of the transgene. The newly introduced sequences are also sometimes modified by methylation or other epigenetic and genetic changes that can down-regulate or suppress their expression (Gebara et al., 1987). In all cases, the expression of a given transgene depends partly on the site of integration. The same transgene in different genomic sites can be expressed at vastly different levels and can also show markedly different stability properties in individual cell clones (Jolly et al., 1986). Most of the physical methods of gene transfer are most easily applicable to models in which defective cells are genetically modified in vitro and subsequently introduced into the organism by transplantation. Due to these severe limitations of gene-transfer efficiency, and the resulting difficulties for in vivo application, many workers have recently emphasized the use of the highly efficient gene-transfer strategies made feasible through the use of viral vectors, in particular those derived from murine retroviruses. In principle, viral vectors might be administered directly to whole animals in vivo or to cells in vitro for subsequent grafting into recipient organisms. Retroviral vectors in particular have the advantage over other vectors of being small, their life cycle very well understood, being capable of introducing single copies of integrated sequences into their recipient cell genome, and of being non-injurious to the recipient cell. With sufficiently high-titer virus preparations, it is possible, at least in vitro, to infect permissive cells with extremely high efficiency (McLachlin et al., 1990) approaching 100% of exposed cells. Nevertheless, despite the thorough understanding of the structure and life cycle of retroviruses, some difficulties remain before these agents can be used in broad-scale application for clinically relevant gene therapy. Retroviral vectors The principles of retroviral structure and vector design have been reviewed thoroughly (Varmus, 1982, 1988; McLachlin et al., 1990). Retroviruses contain RNA genomes that are reverse transcribed after infection to produce a doublestranded cDNA copy of the genome (provirus) flanked by identical elements called long terminal repeats (LTR). These regions of the provirus contain the regulatory sequences needed for the expression of the intervening genes, including a promoter, an enhancer and transcription-termination and polyadenylation signals. The double-stranded proviral DNA integrates stably and heritably into random sites of the host genome as a single copy colinear with the original viral genome. To produce a retroviral vector, viral genes are deleted

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Fig. 1. Construction of a retroviral vector and its infection of a target cell at the nucleic acid level. (a) A plasmid carrying a transgene situated between the two LTR is transfected into a packaging cell line. E, cnhancer, and P, promoter, in U3. An RNA transcript is produced as indicated; cp indicates capping of the 5' nucleotide; (A)t, symbolizes the poly(adeny1ic acid) tail at the 3' end of the transcript. R indicates repeat sequences at the ends of the RNA genome. Unique sequences duplicated during each cycle of replication are denoted U3 and U5. A host-cell tRNA is annealed close to the U5 region (t). The RNA and the viral structural proteins Gag, Pol and Env assemble into a virus particle which is then released from the cell. (b) Initiation of minus strand (-) synthesis at the tRNA primer by the reverse transcriptase. RNA is shown as wavy lines, cDNA as solid lines. cDNA synthesis proceeds to the 5' end of viral RNA genome. (c) The 5' end of the RNA template is removed and the newly synthesized cDNA base-pairs with the R scquences at the 3' end carrying the poly(adeny1ic acid) tail. (d) As minus-strand synthesis proceeds, synthesis of the plus strand (+) of the double-stranded cDNA is initiated at the plus-strand RNA primer site close to U3 and terminates within the tRNA primer-binding sequence. The plus strand is synthesized by using the minus strand as template. RNA is degraded by RNaseH activity of the reverse transcriptase. (e) Minus-strand synthesis from the RNA template is completed, and basepairing between the progressing minus strand and the terminated plus strand leads to a template shift and allows the completion of minusstrand synthesis on the plus-strand template. (0 The complete double-stranded cDNA contains the directly repeated LTR with identical duplications of U3, R and U5. In infected cells, the viral cDNA appears in linear or in closed circular forms with either one or two LTR. Both the double-LTR linear and the circular DNA seem to be capable of integration into the host-cell genome. (6) Integration of the viral cDNA (provirus) into the host-cell genome (dotted lines). The R region (filled box) separates U3 and U5 in the LTR. The transprotein is synthesized from a capped and polyadenylated RNA transcript. Figure is not drawn to scale.

from the provirus and replaced with a gene(s) of therapeutic interest. Fig. 1 illustrates the mechanisms involved in the production of retroviral vectors and their fate in infected cells. Many different forms of retroviral vectors have been designed, as summarized in Fig. 2. Parent viruses that have been used for the production of vectors include Rous sarcoma (Sorge and Hughes, 1982), Harvey murine sarcoma (Ueda et

al., 1987), murine myeloproliferative sarcoma (Laker et al., 1987), murine mammary tumor (Giinzburg and Salmons, 1986) and Moloney murine leukemia virus (McLachlin et al., 1990). In most cases, the amount of foreign DNA that can be incorporated into the vectors between the LTR is limited to approximately 7 - 8 kb, permitting incorporation of most cDNA but very few full-length genomic genes. Vectors have

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Fig..?. Basic designs of rctrnviral vectors. LTR are shown as filled boxes. SDand SAarc splice donor and acceptor sites, respectively. Psi indicates the packaging signal sequence. Hcterologous promoters are outlined as hatched boxes. Transcription-start sites and directions in the proviruses are indicated by arrows. A, B, C, specify different transgenes, one may be a selectable marker gene. PO symbolizes the translation-reinitiationregion of the picomavirus 5’ untranslated region. (a-c) Single-gene retroviral vectors with (b,c) or without (a) internal promoters. (c) Prototype of a self-inactivatingvector with a deletion of the regulatory sequences in the 3’ LTR, a mutation which is copied onlo the 5’ LTR during reverse transcription (Fig. 1 j. (d) The same principle leads to the duplication of a gene with its promoter introduced in the 3’ LTR in double-copy vectors. The transgene in the 5’ LTR is situuted outside the transcriptional unit of the provirus and therefore less influenced by viral transcription. Vectors (c - k j are multi-genevectors with (f- h) or without (e, i-k) internal promoters. Anti-sense orientation of the transgene with respect to proviral transcription (g, h) may improve the expression of the transgene, yet often reduces the virus titer. Vectors (i, j and k) are viruses that encode an unspliced polycistronic transcript (for further discussion. see text).

been made containing only the single therapeutic gene of interest, o r containing and expressing several genes including dominant selectable markers such as that encoded by the transposons (Tn) neomycin-resistance phosphotransphase-expressing marker gene (neo‘), which confers resistance of cells to the neomycin analog G418 (Eglitis, 1991). Such multiplegene vectors, containing a selectable marker in addition to the potentially therapeutic transgene, are frequently used to take advantage of the ability to select producer cells that generate

high titers of virus and later to detect and isolate target cells infected with the vector. Single-gene and multiple-gene vectors have been used for the infection of many different mammalian cell types, including cells of the bone marrow, fibroblasts, hepatocytes, leukemic cells, endophelial cells, keratinocytes and others. There are several major approaches being used to transfer and express transgenes by multiple-gene vectors (Fig. 2). The first approach uses the same genetic strategy found in wild-type virus. I n this class of vectors, the several genes in the vectors are expressed only by the 5’ LTR through differential RNA splicing (Fig. 2a and e). A second approach is that in which one gene is expressed from the retroviral LTR and the second from a n internal heterologous promoter (Fig. 2bd and f-h). Vectors of this type have been used in many laboratories for many model studies, in most cases employing promoters such as those of Simian virus 40, human cytomegalovirus immediate-early gene, the Rous sarcoma LTR, metallothionein, as well as other promoter types. Vectors with internal promoters have been successfully used for the transfer and expression of genes in many cell types (McLachlin et al., 1990), and have also been used in early human applications such as the initial studies in patients with severe combined immunodeficiency disease resulting from deficiency in A D A (Anderson, 1992). However, a number of in viva and in vitro studies have shown that these kinds of vectors are often hampered by interactions between the retroviral LTR promoter and the internal promoter, leading to structural and functional instability of the integrated vector and in many cases to excision or rearrangement of the transgenes and/or shutdown of gene expression (Xu et a]., 1989). As a result of these technical difficulties, a number of workers have begun to develop a third class of vectors in which the several genes in the vector are expressed from the same retroviral regulatory sequences that d o not include internal promoters (Fig. 2i k). Such polycistronic vectors have been produced by the introduction of picornavirus 5’ non-translated sequences bctween the transgenes to allow internal ribosomal attachment and thereby efficient translation of those genes from one dicistronic template (Adam et al., 1991; Ghattas et al., 1991; Fig. 29. Still another method for construction of a polycistronic vector has used the concept that internal translational reinitiation at several sites on a polycistronic message occurs with reasonable efficiency, if the individual cistrons are separated by only short intervening segments which contain no spurious potential AUG translation-initiation codons (Levine et al., 1991; Fig. 2j,k). Recent successful approaches to prevent functional instability and shutdown of transgene expression from integrated vectors also include the use of cell-type-specific enhancers, like the muscle creatine-kinase enhancer replacing the viral LTR enhancer sequences.

Conceptual and technical problems with retrovirus vectors There are several difficulties associated with retroviral vectors that complicate their use for therapeutic genetic modification of cells. One of the major concerns is the fact that titers that can be generated from most retroviral vector producer cells are limited in most cases to 1O6 infectious virus particles/ ml tissue-culture medium, or often lower. Virus titers are influenced by the nature of the transgene(s), the presence of internal promoters in the vector, the relative positions and orientations of the different genetic elements in a defined vector, and the producer-cell type. Even through the use of mechanical and physical methods for virus concentration, such as sedimen-

215 tation and selective filtration, it is not possible in most systems to produce virus stocks with titers greater than lo7 infectious units/ml. For most applications that involve in vitro genetic modification of cells followed by grafting, such titers are acceptable. However, direct in vivo applications would require much higher titers, and several procedures have been reported recently to produce virus at much higher titers than previously possible (Bestwick et al., 1988; Bodine et al., 1990). The usefulness of such methods has yet to be established. One of the major concerns of retroviral-mediated gene transfer involves the level and stability of transgene expression in the infected cell. It is well recognized that gene expression from integrated proviruses depends on the position in the genome and that retroviruses integrate in a quasi-random fashion into the target-cell genome (Shih et al., 1988). Bulkinfected cell populations therefore contain individual cells with vastly different levels of transgene expression and stability. Although this important effect of flanking sequences and chromatin structure on the provirus is still largely uncharacterized, there are a number of potential solutions to this problem of position effect and its impact on the levels and stability of gene expression from retroviral proviruses. The most useful, but unfortunately the most long-term and difficult approach involves the design of retroviral and other vectors that are targeted to specific sites of the cell genome. In the event that it would be possible to target a transgene to a specific gcnomic site, the foreign sequences should come under faithful cell regulation. Attempts to construct such vectors have begun (Ellis and Bernstein, 1989), but have not yet led to reproducible techniques for vector targeting. Along these same lines, there has recently been considerable interest in the small adcno-associated virus (AAV), which scems, at least in some cell types, to integrate preferentially into a region of human chromosome 19. The general applicability of this site for integration oftherapeutic genes or the role that this system can play in elucidating mechanisms of site-specific integration have yct to be demonstrated. Technical difficulties with the adeno-associated virus system include a relatively low coding capacity (less than 4kb). low virus titers and a limited ability to infect important potential target cell types such as fibroblasts and hematopoietic cells (Lebkowski et al., 1988). Another approach to confer position independence onto the expression of a transgene may make use of the newly discovered matrix-attachment sequences which flank transcriptionally active regions in the mammalian genome (Stief et a]., 1989). Since it seems likely that these scyuences must be positioned between or within the retroviral LTR, and since they also may not permit transcriptional read-through, the presence of such sequences may interfere with the generation of full-length transcripts, thereby limiting their use in retroviral vectors. Despite these technical and conceptual problems, the goal of producing a truly targetable vector that contains all of the regulatory elements necessary for faithful gene regulation remains of paramount importance. Relativcly long-term expression of genes transduced by retroviral vectors of various designs has been well documented in a number of systems such as bone marrow, fibroblasts, hepatocytes, muscle cells, keratinocytes and others (McLachlin et al., 1990). However, it has been commonly found that the expression of a retroviral-vector-transduced gene in an infected cell may be transient, for instance when genetically modified cells are grafted to recipicnt animals. Even though the organization of the provirus remains structurally intact, expression of the transgene may be unstable and in some cases completely shut off(Jol1y et al., 1986; Xu et al., 1989; Palmer

et al., 1991;Roman et al., 1992).Occasionally, gene shut-down is associated with epigenetic changes such as methylation of the DNA (Jahner and Jaenisch, 1985). In some cases, provirus sequences are lost or rearranged (Jolly et al., 1986; Xu et al., 1989). Together, these issues of structural and functional instability of the provirus suggest that care must be taken to select appropriate regulatory sequences and combinations of promoters and enhancers to assure prolonged stable transgene expression in vivo. Additional occasional problems with retroviral gene expression come from the tendency of the regulatory sequences of the LTR to interact with the internal promoter and other internal regulatory elements. Vectors that express transgenes from internal promoters may be relatively unstable, a fact that has led several workers to develop selfinactivating vectors which contain LTR devoid of promoters or enhancers (Yu et al., 1986; Yee et al., 1987; Fig. 2). While such vectors are attractive in principle, they often are made in only low titers. Fortunately, additional approaches to the same problem have led to the development of polycistronic retroviral vectors, as described above. These vectors seem to be produced at acceptably high titers and to be more stable than their multipromotor counterpart vectors. Mechanisms leading to loss of the transduced gene function may also include immune response of the host organism to the new gene product. If the recipient organism has not had immunological experience with the gene product in order to become tolerant to it, or if the wild-type therapeutic gene product presents epitopes not present in the mutant forms of the protein in the patient, the protein will become the target of immune attack, and its function will then rapidly be lost (St. Louis and Verma, 1988). This issue remains one of the most important and least thoroughly studied aspects of gene therapy. While replication-defective retroviral vectors integrate at random into the host genome and therefore will, on occasion, inactivate essential cellular genes, the problems posed by these rare instances of insertional mutagenesis are thought by most workers not to pose major safety problems. Nevertheless, the integration of a replication-defective vector may represent a mutational effect that triggers a preneoplastic cell to become neoplastic through the activation of a previously silent protooncogene or the inactivation of a tumor-suppressor gene. The fact that most human cancers in all likelihood result from multiple mutations, together with the fact that such target genes are very rare provide some comfort that the risk of neoplasia resulting from the retroviral integration, will be extremely low. The use of self-inactivating vectors, missing enhancing elements from their LTR, could further reduce the likelihood of activation of cellular protooncogenes during proviral integration. Additional safety issues arise through the potential for recombination between the defective viral vector and cellular elements, a process that can occasionally generate replication-competent virus by genetic recombination. A recent unpublished study by Nienhuis and his colleagues at the National Heart, Lung and Blood Institute of the NIH has shown that such helper virus is produced in sufficiently large amounts by some virus preparation methods to lead with vcry high frequency to malignancies in immunosuppressed monkeys that have received grafts of bone-marrow stem cells infected with such vectors (Kolberg, 1992). These results are in contrast to work recently reported by other investigators who have followed monkeys for up to six years after exposure to replication-defective vectors (Cornetta et a]., 1991). In the latter studies, no apparent deleterious effects were noted. With the use of currently available kinds of producer cells that

21 6 markedly reduce the opportunity for rescue of replicationcompetent virus by recombination, the chances for the emergence of wild-type replication-competent virus are generally agreed to be very low (Miller and Buttimore, 1986). All these reservations notwithstanding, it is obvious that the use of these and other vectors for gene transfer is not a trivial or whimsical undertaking and should be reserved for those situations of serious disease without alternative effective treatments. The possibility that germ-line gene therapy may be an alternative to somatic-cell gene therapy for the correction of certain disorders remains a very contentious and inadequately understood issue. In contrast to somatic-cell gene therapy, which involves genetic modifications of a subset of cells only in a single host organism, germ-line therapy involves the transmission of the foreign genetic information to subsequent generations by introduction of the genes into the host germline cells. Such germ-line modifications have been successfully applied to mice and other domestic animals by introducing transgenes into an early embryo. It is obvious that while somatic-cell genetic modifications are confined to a single organism, germ-line gene therapy has the potential to affect the genetic make-up of future generations in a population and therefore raises significant ethical and political concerns. The quality of scientific and public debate on this subject has lagged far behind that of somatic-cell gene therapy, but now must become subject for more serious and enlightened discussion.

Disease models and applications Bone marrow Hematopoietic stem cells in the mammalian bone marrow have long been considered ideal targets for gene therapy, since cells of the bone marrow are readily accessible, can be manipulated and genetically modified in v i m and returned to the patient by standard and established methods of bonemarrow transplantation. Several studies have documented successful gene transfer and long-term gene expression in hematopoietic stem cells of adult mice employing retroviral vectors (Dick et al., 1985; Keller et al., 1985; Dzierzak et al., 1988). Until recently, it has been difficult to identify and culture totipotential bone-marrow stem cells in large animal systems and to demonstrate that they are susceptible to infection with retroviral or other vectors (Kantoff et al., 1987; Stead et al., 1988; Bodine et al., 3991). In some studies, the infection of hematopoietic stem cells from adult human and non-human primates by amphotropic retroviral vectors has been found to be inefficient. Also, vectors which allowed gene expression in vitro were inactive in vivo (Williams et al., 1986; Magli et al., 1987). However, in the past several years, very impressive progress has been made toward solving these problems, and several investigators have developed procedures which allow stem cells to be infected more stably and efficiently and to express transgenes for prolonged periods of time in vivo. Advances in the field have not only come through the identification and enrichment of authentic bone-marrow stem cells (Spangrude et al., 1988), but through improvements in bone-marrow-culture methods through the introduction of the additional growth factors such as interleukins 3 and 6, and granulocyte colony-stimulating factor in bone-marrowculture systems (Bodine et al., 1991; Einerhand et al., 3992, Hamada et al., 2991). Also, recent results have shown that the inability to infect primate hematopoietic stem cells with

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murine retroviruses can be overcome by expression of cloned retroviral receptors on the surface of these cells, indicating that one problem with the infectibility of stem cells is at the level of entrance of the viral vector. Diseases that represent prime targets for genetic manipulation of hematopoietic stem cells include disorders characterized by defective biosynthesis of the globins, such as the thallassemias and sickle-cell anemia. In addition, since stem cells are the progenitors for all the cells of the immune system, macrophages and other cell types, genetic disorders of these cells might also be approached by repopulation of the bone marrow with genetically modified stem cells. In fact, the severe combined immunodeficiency disease caused by deficiency of the purine-biosynthetic enzyme ADA represents one of the most popular target diseases for an early attack by gene-

21 7 therapy methods (van Beusechem et al., 1989). In the design of gene-therapy approaches to the correction of disorders of globin gene expression, it will probably be vital to maintain the exquisite tissue-specific and developmental regulation of these genes. A major advance toward this goal has recently been made by the identification of regulatory sequences involved in globin gene expression, which lie far from the traditional regulatory elements adjacent to or within the genes themselves. These sequences, constituting the locus-control region, are scattered over large distances in the genome, as far as 50 kb or more distant from the genes themselves. Fortunately, these regulatory elements are relatively small and could be introduced together with other known regulatory sequences into vectors to allow efficient and position-independent expression of 8-globin after infection of erythroleukemia cells with a suitable retroviral vector (Novak et al., 1990). Another potentially important application of retrovirally mediated gene transfer into hernatopoietic stem cells would be the expression of the multidrug-resistance gene ( MD R ) to protect a repopulated bone marrow from the toxicity of cancer chemotherapeutic agents (Guild et al., 1988). Human MDR-I cDNA encodes a cell-surface glycoprotein that acts as an efflux pump for cytotoxic drugs, including many drugs commonly used in cancer chemotherapy. Unfortunately, many of these drugs have devastating side effects on the rapidly proliferating cells of the hematopoietic system and other organs. In patients with a wide variety of non-hematopoietic malignancies, implantation of stem-cell populations expressing a virally transduced MDR-1 gene prior to chemotherapy may therefore increase the tolerance of the patient toward traditional doses of chemotherapeutic agents and might even allow the use of vastly increased doses to permit more effective tumor treatment. Recent results with mice expressing the mouse MDR-1 gene in stem cells have shown that they tolerate a substantially increased concentration of some chemotherapeutic agents (A. Nienhuis, personal communication). Similarly, the use of murine bone-marrow stem cells expressing a methothrexate-resistant mutant form of the dihydrofolate reductase gene can lead to improved survival after reimplantation into mice being treated with anti-proliferative drugs (Corey et al., 1990). The liver

The liver is an organ central to most metabolic processes in mammalian organisms, and it therefore constitutes one of the most attractive potential target organs for gene therapy. One of the major problems with retroviral infection of hepatocytes is a problem common to other slowly replicating or non-dividing cells, i.e. retroviruscs cannot infect cclls in the absence of DNA replication and cell turnover. The adult mammalian liver consists almost entirely of differentiated and relatively quiescent hepatocytes which are not efficiently infected with retroviral vectors. Fortunately, methods have become available to infect these cells with retroviral vectors. Primary cultures of hepatocytes undergo partial dedifferentiation and resume more active cellular proliferation transiently, making them susceptible to rctroviral infection (Wolff et al., 1987). Hepatocytes can therefore be readily infected in vitro and subsequently grafted back into recipient animals by infusion of the cells directly into the portal vein or into the spleen, from where they migrate with reasonable efficiency to the liver (Ponder et al., 1991). The ability of the grafted genetically modified hepatocytes to survive and to provide new liver functions is enhanced by inducing a transient state

of gcneral hepatocyte regeneration through partial hepatectomy or toxin-induced damage to the liver (Wilson et al., 1988). Because of the central metabolic role played by the liver, many hepatic diseases have become attractive targets for a gene-therapy approach, including familial hypercholesterolemia resulting from deficiency of low-density lipoprotein (LDL) receptors (Miyanohara et al., 1988), coagulation defects such as those caused by absence of clotting factors VIII and IX (Armentano et al., 1990), and phenylketonuria resulting from phenylalanine-hydroxylasedeficiency (Peng et al., 1988). In the case of the LDL-receptor-deficiency model, in vivo studies with a relevant animal model have been made possible by the existence of the Watanabe hypercholesterolemic rabbit, in which absence of LDL-receptor function leads to premature atherosclerosis and coronary-artery disease. precisely as it does in the human counterpart (Watanabe, 1980). Hepatocyles from Watanabe rabbits have been cultured and infected with a retrovirus expressing the LDL receptor from the 8-actin promoter. After infection, genetically modified cells were injected into the spleen of the animals from where they migrated to the liver, restoring LDL-receptor function and leading apparently to a stable reduction in total serum cholesterol levels (Chowdhury et al., 1991). Other investigators have used genetically modified hepatocytes on solid matrices to form orgdnoids for implantation into the liver (Miyanohara et al., 1992a) or have even introduced retroviral and herpes-simplex-virus(HSV)type-1-derived vectors directly into the liver for in vivo gene transfer (Miyanohara et al.. 1992b; Fig. 3 and discussion on HSV-derived vectors).

Muscle A relatively new and as yet incompletely characterized area of interest is the use of muscle as a target organ, not only for the potential therapy of intrinsic muscle disease but also for the correction of non-muscle disorders through the production in muscle tissue of humoral factors. Skeletal muscle has been shown to be susceptible to direct in vivo gene modification after direct injection of plasmid DNA , albeit at low efficiency (Wolff et al., 1990). Naked DNA injected directly into skeletal muscle can be expressed for very long periods of time in vivo, even as long as a year, although there is no evidence that the injected DNA becomes integrated into the genome of the recipient cells. This approach has been used in dystrophin-deficient mdx mice to express the normal dystrophin gcne that is also defective in humans with Duchenne’s muscular dystrophy (Acsadi et al., 1991). However, the efficiency of gene expression after direct delivery of this sort seems to be very low, and no reports have appeared on the efficacy of direct gene delivery for an apparent disease phenotype in vivo. Nevertheless, even low levels of gene expression in muscle may be sufficient for vaccination purposes. A potentially more efficient method of gene transfer into differentiated muscle cells (myotubes) has recently taken advantage of the fact that undifferentiated myoblasts found normally as satellite cells in skeletal muscle can be cultured and genetically modified in vitro by infection with retroviral vectors. Such cells have the property of fusing with existing myofibers after grafting into skeletal muscle, thereby, in principle, providing new genetic functions to muscle (Barr and Leiden, 1991; Dhawan et al., 1991; Fig.4). It has also been possible to show gene expression and circulating levels of the coagulation protein factor IX after grafting genetically modified primary myoblasts into mice (Roman et al., 1992). It seems very likely

21 8 A

vector to include the use of housekeeping promoters have recently led to more prolonged and stable gene expression in vivo (Scharfmann et al., 1991).

neurotrophic

oducl boiite

Epithelial, endothelial and smooth muscle cells

Transgene product transfer to neurons

Infection

B retrovirus

& cell fusion

change 01 phenotype or secretion 01 new gene product

Infection

Cell fusion

Fig. 4. Indirect and direct genetic modifications of neurons and skeletal muscle cells. (A) Disorders of the central nervous system can be approached by bringing genetically modified non-neuronal cells such as fibroblasts or glia cells into close proximity to the target neurons. The latter then take up gene products or metabolites sythesized by the graft by paracrine mechanisms or by metabolic cooperation. Another potential approach makes use of vectors derived from transducing neurotrophic or other viruses which are able to infect neuronal cells. (B) Myoblasts, the satellite cells of skeletal muscle cells (myotubes), are cultured and infected with retroviral vectors. Modified myoblasts can then be grafted into skeletal muscle, where they fuse with endogenous myotubes and either alter the phenotype of these cells to correct an intrinsic muscle disease or release a gene product into the cirulation to modify the disease phenotype of distant cells.

that this system will become of great use for a number of different kinds of genetic disease. The skin

The skin represents an attractive target organ for therapeutic gene manipulation since it is obviously readily accessible and since major skin-cell types such as fibroblasts and keratinocytes are usually easily infectable with retroviral vectors. Genetic disorders characterized by the absence of circulating products are in principle easily approached by grafting genetically modified fibroblasts or keratinocytes into recipient animal skin. Skin fibroblasts have been grown in vitro on a collagen matrix after infection with vectors expressing the human clotting factor IX, and after implantation into mice, circulating factor IX produced by the graft was readily detectable (St Louis and Verma, 3988). While early cxperirncnts showed only transient expression of factor IX, due partly to an immune response to the heterospecific gene product and also to transcriptional shut-down of gene expression from the provirus (Palmer et al., 1991), modifications of the retroviral

An alternative approach for the introduction of new humoral factors is made possible through the use of vascular endothelial cells or smooth muscle cells that normally make intimate contact with the circulation. In the case of vascular endothelial cells, efficient gene transfer has been accomplished by introduction of retroviral vectors directly into a segment of a blood vessel that had been mechanically clamped for short periods of time (Nabel et al., 1989). While it has been possible to show a very high efficiency of transfer of reporter genes such as lacZ , it remains to be demonstrated that sufficient numbers of cells can be transduced by this approach to provide physiologically useful amounts of a humoral factor. Since these problems are likely to be soluble technical ones, this approach for providing new circulating gene products may be useful. In a similar fashion, smooth muscle cells of vascular origin have also been modified genetically by retroviral infection and implanted into the vascular system of the rat (Lynch et al., 1992). In this study, the transferred human ADA gene was expressed at about the same level as the endogenous rat ADA gene for at least six months. Recently, the gene for the cystic-fibrosis transmembraneconductance regulator (CFTR) protein has been identified (Riordan et al., 1989) and shown to be expressed in epithelial cell types. Almost certainly, airway epithelial cells of a still vaguely defined type are the targets for genetic correction in patients with cystic fibrosis, which is the most common lethal recessive genetic disease in the Caucasian population, with an incidence of one affected person in every 2000 births. Cystic fibrosis is caused by two defective copies of the CFTR gene, which encodes a chloride channel required for electrolyte homeostasis (Collins, 1992). There have been several approaches for both in vitro and in vivo transfer of functional copies of the CFTR gene into epithelial cells (Rich et al., 1990, Rosenfeld et al., 1992). A particularly promising approach involves the use of adenovirus-derived vectors for gene transfer (see Fig. 3 and below). The central nervous system

There are probably a large number of genetic and other disorders of the central and peripheral nervous systems that may be amenable to correction at the genetic level. Unfortunately, the straightforward application of retroviral gene transfer to many of these disorders is difficult since the target cells of many of these diseases, the neurons, are almost all terminally differentiated and non-replicating and therefore not susceptible to infection with retroviral vectors. Nevertheless, some central-nervous-system disorders represent very attractive and possibly even ideal models for a form of gene therapy that involves transfer of the gene for a trophic factor or a neurotransmitter into an irrelevant donor cell such as an autologous fibroblast followed by grafting of the genetically modified cells into the central nervous system into a site that provides a locally high concentration of the therapeutic gene product (Gage et al., 1987; Fig. 4). In this way, it is possible in principle to avoid the problems of drug delivery to many regions of the brain that are not affected by the disorder and whose function may be disturbed by doses of an agent that may be therapeutic to other regions of the brain.

219 Disorders that may be amenable to this kind of gene therapy include the degenerative disorders Parkinson’s disease and Alzheimer’s disease, among others. Despite the fact that the immediate causes of the neuronal degeneration in these disordcrs are not understood, it has become possible to devise strategies to replace the function of the cells or to prevent their further degeneration. In the case of Parkinson’s disease, the partial success of treatment by systemic delivery of the neurotransmitter precurser 3,4-dihydroxyphenylalanine,and the promising results obtaincd through grafts of normal fetal tissue to the affected areas in Parkinson’s-disease patients (Lindvall et al., 1989), supports the notion that long-lived grafts of cells stably producing 3,4-dihydroxyphenylalanine or 3,4-dihydroxyphenylethylaminemay be an effective method of cxpressing this vital neurotransmitter in patients with Parkinson’s disease (Wolff et al., 1989). The potential therapeutic role of exogenous trophic factors such as nerve growth factor to degenerating neurons may suggest a potential genetic approach to the treatment of Alzheimer’s disease (Hefti and Weiner, 1988). It is obvious that a different approach must be taken for disorders involving more global dysfunction of neurons (Rosenberg et al., 1988). In that regard, other methods of efficient gene transfer must still be developed which probably involve viruses capable of infecting neurons (Fig. 4 and discussion on HSV-derivcd vectors). Cancer cells

Our understanding of the deranged cell-proliferation characteristic of tumors has increased markedly since the discovery of virus-transduced oncogenes. Despite that fact, while treatment of some kinds of human cancer has improved greatly, the explosion of information about oncogenes and their role in tumor development has not yet been matched by major new forms of thcrapy. We now know that the transformation of a normal cell into a tumor cell is usually a multistep process, involving independent but interactive mutations in a variety of genes important for signal-transduction and cell-cycle regulation. The number oT such mutational steps probably varies between different tumors and tissues. Many tumor-related genes have been identified, including dominantly acting oncogenes that represent mutated forms of normal genes involved in cellular growth-regulatory and cell-cycle runctions (Bishop, 1991). When mutated, these genes drive constitutive and unregulated cell proliferation leading to tumor development. Another class of genes are those represented by the tumor-suppressor genes, whose normal function in cells is to act as growth and replication brakes (Weinberg, 1991). It is through the loss of function of both alleles of these tumor-suppressor or growth-suppressor genes that the regulation of cell replication is so deranged that cells become tumorigenic. The classical example of this kind of gene is the retinoblastoma gene whose homozygous loss of function leads to retinal tumors and later in life to a variety of other cancers including osteosarcoma. A more recently discovered tumor-suppressor gene, pS3, has been found to be mutated and/or lost in a very high percentage of human cancers and is therefore thought to play a central role in growth regulation of human cells (Hollstein et al., 1991). It is now recognized that most or all human cancers are associated with multiple mutations in oncogenes and tumorsuppressor genes, and that it is the balance of function of these multiple genes and cooperation between their gene products that dcterrnines the final phenotype of the cell. The existence of these multiple genetic defects has prompted many investi-

gators to assume that the genetic correction of any one of the mutant oncogenes and tumor-suppressor genes may not have a major and predictable effect on the growth properties of cancer cells. This conclusion seems not to be correct. A number of studies have now shown that restoration of the function of a single tumor-suppressor gene can cause suppression of the tumorigenic properties of many kinds of tumor cells. The introduction of a normal retinoblastoma gene into retinoblastoma cells has been found to suppress the neoplastic properties of these cells (Huang et al., 1988). Similarly, the normal allele for the tumor-suppressor gene p53 has been introduced into cells deficient in wild-type p S 3 expression, as in some T-cell leukemias, colon carcinoma cells, breast cancer cells and others, and the tumorigenic properties of the cells have been suppressed (Baker et al., 1990; Cheng et al., 2992; T. Friedmann, unpublished results). Due to the presumed existence of multiple mutations in these cells, it has been somewhat surprising that restoration of the function of a single tumor-suppressor gene can produce such marked phenotypic changes. The interpretation of these results is that pS3, and possibly other tumor-suppressor genes, play very central and pivotal roles in events of the cell cycle and in cell replication, and are major determinants of the tumorigenic or non-tumorigenic properties of mammalian cells. The extension of these kinds of studies to a demonstration of clinically useful tumor suppression in vivo will be difficult, given the presumed need in such a study to infect a very large percentage of the existing tumor cells while either avoiding infections of non-tumorigenic cells, or employing tumor suppressors which do not interfere with the functions of non-tumorigenic cells (Fig. 5). No doubt strategies to target tumor cells specifically and selectively with viruses or other pharmacological agents will be developed. Such strategies promise to be useful, not only for the selective delivery and expression of tumor-suppressor genes to tumor cells in order to growth-arrest those cells, but may allow the introduction of sequences in an antisense orientation to sequences transcribed from dominant oncogenes, in order to bind and eliminate the oncogene transcripts (Sklar et al., 1991; Calabretta, 1991) or the transfer of potentially cytotoxic genes like the HSV thymidine-kinase gene to the site of the tumor. Other procedures may aim at the selective lysis of glia-cell-derived tumors by introducing cytotoxic viruses which are capable of establishing a latent and non-destructive state in the healthy neuronal tissue surrounding the glia-cell tumor (Martuza et a]., 1991). Recently, in still another approach to attack tumor cells in vivo, researchers have shown that exprcssion and release of cytokines by genetically modified tumor cells abrogates tumorigenicity and induces protective immunity in mice against tumors which are not normally attacked by the immune system (Gansbacher et al., 1990). Other studies have demonstrated a reduced tumorigenicity of tumor cells genetically modified to express and secrete ?-interferon (Esumi et al., 1991). Along these lines, several human clinical studies are now in progress (see below). If and how these approaches can be used efficiently in the genetic approach toward cancer treatment remains to be determined. A number of investigators are proposing clinical studies involving the introduction of foreign cytokine genes in tumor cells derived from a patient and the subsequent implantation of the genetically modified cells as a vaccine to the patient to induce a host immune response against tumor cells anywhere in the body. In still another use of the host immune system, cells that accumulate in some tumors, probably as part of an immune attack on the tumor (tumor-infiltrating lymphocytes, TI L) are being modified to express potentially

220 A

responding T cells

@@a Harvest of rumor cells

0

@@

Lethal irradiation of tumor cells, iransplaniafion

0 0 Tumor

In vitro-Infection of tumor cells with retrovirus vectors bansducing cytokine or other immuno-regulatory genes

Expression of cytokines or other immuno-regulators

immune response to cytokine-expressing tumor cells, original tumor and metastases

B Initial epigeneiic or genehc damage (demethylation; dvomosome loss etc.)

Somatic cells

Inmrporation and expression of tumor suppressor gene

Secondary generic modifications (i.e.. loss or mutation of one allele of tumor suppressor X )

Pre-malignant state 1

Activation of proto-oncogene(s)

Pre-malignant state 2

Vector-mediatedtransfer of rumor suppressor gene

Loss of heterozwositv .. . of tumor suppressor X, divation of merastasisrelated genes

Reversion of tumorigenic phenotype

Nan-tumorigenic cells m a i n unaffected

@ Full malignancy metastasis

Pre-malignant

state 3

Fig. 5. Models of potential genetic approaches to cancer treatment. (A) Tumor cells are isolated from a tumor and infected in vitvo with retroviral vectors transducing cytokine or other immuno-regulatory genes. Implantation into the patient or the lethally irradiated cells coexpressing thc immune-rcsponsc-stimulating factors and putative tumor cell antigens may elicit a specific immune response mediated through cytotoxic T cells and directed against the tumor cell. (B) The transrormation of a normal somatic cell into a fully malignant and metastatic tumor cell is a multistep process. The number and order of steps arc largely unknown and vary with the tumor type. Vectors transferring the tumorsuppressor gene (T) can be retroviral or othcr vcctors which bind specifically to the tumor cells and its parent tissue.

antitumor functions such as the tumor necrosis factor. Those cells are being returned to the patient and the hope is that they will again accumulate in the tumor and provide concentration of potent anti-tumor gene products. Other viral vector systems

In the past several years, retrovirus-derived vectors have been successfully employed for the transfer and expression of many types of genes into a wide variety of different tissues: however, there are problems with retroviral vectors intrinsic to their biology and life cycle that make their use in some gene-therapy applications difficult. Their capacity for foreign DNA is limited to less than 10 kb, they can be produced in only low to moderate amounts, they may exhibit a tendency to be unstable and to shut-down of gene expression especially in vivo, and they are no1 capable of infecting non-dividing cells. For these reasons, a number of investigators have been developing alternative vectors from several different parent viruses including adenoviruses, adeno-associated viruses and HSV. Both adenoviruses and HSV-1 contain comparatively large DNA genomes of 36 kb and 150 kb, respectively. In contrast to retroviruses, these viruses usually do not integrate into the

host-cell genome, although integration of parts of the HSV-1 genome into the infected-cell genome has been reported (Roemer et al., 1992). Many common serotypes of adenoviruses have been found to be human pathogens gcncrally associated with upper airway infections. lntact adenoviruses have been extensively used for vaccination purposes (Top et al., 1971), providing extensive information on the safety of this system and making them attractive as potential parent viruses for the production of vectors for human application. Onc major advantage or adenovirus vectors is that thcy can be produced in very high titers, up to 10" infectious unitsiml medium, titers that can be augmented still further by physical concentration up to l o ' * infectious units/ml, or even higher. Adenovirus vectors made replication-defective by removal of the El region of the viral genome (Fig. 3A) are capable of transducing foreign DNA of about the same size as retroviruses (Berkner, 1988). They have been used recently for efficient gene transfer into airway epithelial cells (Rosenfeld et al., 1991) and into several other cell types including mouse hepatocytes in vivo, where they expressed the gene for the ornithine transcarbamoylase and corrected the phenotype of ornithine-transcarbamoylase-deficient mice (Stratford-Perricaudet el al., 1990). Adenovirus vectors may be particularly promising for the efficient transfer of the CFTR gene, because

221 the airway epithelium, the natural target for most adenoviral infections in humans, represents also the target organ principally effected in this disorder. While airway epithelial cells can be infected by retroviruses in v i m (Stanley et al., 1991), vastly increased titers of retrovirus vectors would be necessary to achieve reasonably efficient gene transfer in vivn. However, it should be remembered that the ubiquity of adenoviruses and their presence in the upper airway may pose a source of potential problems, since genetic recombination between a therapeutic vector and naturally occurring adenoviruses in airway cells may result in the rescue of replication-competent vectors with altered pathogenic properties. This event might lead to spreading infection and an immune response to the transgene product. It is also clear that there has been little study of the potential cellular damage caused by persistent or repeated infection with replication-defective adenovirus vectors. HSV-1 has recently gained much attention as sources of useful vectors, especially because of its ability to infect neurons and to become latent in some neural cells (Breakeficld and DeLuca, 1991). Like adenoviruses, HSV-1 can be grown to very high titers, is ubiquitous in most human populations, and is able to infect non-dividing fully differentiated cells like neurons, hepatocytes and others refractory to retrovirus infection. Of the 150-kb HSV-1 genome, at least 30 kb are known to be dispensable (Longnecker et al., 1988). The capacity for foreign DNA o f these vectors therefore is potentially much greater than that of any other vector system currently available and many different dispensable sequences in the HSV genome have been used to insert foreign DNA (Fig. 2B). A number of investigators have used HSV-1-derived vectors to express reporter genes such as Escherichiu coli 8-galactosidase (lucZ) gene in mouse and rat neurons in vivo (Dobson et al.. 1990; Chiocca et al., 1990), or foreign genes in mouse liver, either by direct injection into the liver or by delivery of vector via the portal vein (Miyanohara et al., 1992b). However, it seems likely that many of the defective HSV-1 vectors, in particular replication-defective vectors deficient for the essential viral immediate-early gene 3 (DeLuca et al., 1985), continue to be cytopathic because of the persistent overexpression of the other immediate early genes in the vectors (Johnson et al., 1992). In addition, gene expression from heterologous promoters in the context of the entire genome of the replicationdefective HSV-1 seems to be regulated in complicated patterns defined by the viral genome by mechanisms that are as yet poorly understood (Panning and Smiley, 1989; Roemer et al., 1991). The regulatory sequences of the latency associated transcripts which are known to be the only transcripts expressed during viral latency, are therefore of particular interest for the regulation of transgenes in HSV-1 vectors (Dobson et al., 1989). To avoid problems and constraints posed by continued cxpression of toxic gene products of the remaining intermediate-early genes and the complex regulation of transgenes with foreign promoters in full-length HSV-1 vectors, packagable HSV-I-derived plasmid vectors have also been used in a number of studies (Kwong and Frenkel, 1984; Geller and Breakefield, 1988). However, to be most useful, plasmid vectors must exhibit a very high ratio of vector/helper virus to minimize problems intrinsic to helper viruses. Another vector system specially designed for high expression and correct processing of transgene products makes use of recombinant vaccinia viruses (Moss and Flexner, 1987). Vaccinia-virusderived systems may prove particularly useful for overexpression of genes products which are secreted into the circulation. Table 1 summarizes the properties of the principal

current viral vectors with the highest potentials as gene-transfer vehicles in human gene therapy.

Human clinical studies The concepts and methods of efficient gene transfer, in particular those involving retroviral vectors, have progressed to the point where a number of investigators have proposed and received permission from the appropriate regulatory bodies to pursue studies with human patients. These studies have included both non-therapeutic cell-marking studies aimed simply to determine the Pdte of genetically marked cells after grafting into patients as well as studies with a more specific therapeutic goal. The first human study involved the introduction by retroviral transduction of the neo' gene into tumor-TIL, which accumulate in some solid tumors (Rosenberg et al., 1990). The cells were obtained by biopsy of the tumors, infected in vitro with a nee'-transferring retrovirus and grown to very large volumes in the laboratory before being reintroduced into the patients. When the tumors were rebiopsied, cells carrying and expressing the neo' marker were detectable in the tumor, indicating that at least some of the TIL cells are able to find their way back to the tumor of origin and express the foreign gene for many months (Kasid et al., 1990). This kind of experiment does not truly define the efficiency of tumor targeting by TIL cells, but it does lend some credence to the notion that these cells may serve as delivery vehicles for antitumor reagents. An extension of these studies is currently in progress in which the human tumor-necrosisfactor gene is being introduced into TIL cells in an attempt to deliver this potentially tumor-cytotoxic agent specifically to the site of a solid tumor through this form of indirect gene therapy. One of the major metabolic deficiencies in many tumor cells apparently is the failure to elicit a cytotoxic T-cell immune response aimed against the tumor cells by stimulating immune cells to express one or a number of classes of cytokines in response to the presence of cell-surface tumor-specific markers. A potential genetic approach to overcome this deficiency is to use tumor cells as surrogate sources of the cytokines in the context of the tumor antigens and thereby elicit and immune response. Several human studies are now in progress in which retroviral vectors are being used to introduce cytokine genes directly into cells derived from a tumor. Not only do those cells themselves become incapable of forming tumors, but in animal studies are able to immunize animals against subsequent challenge with unmodified tumor cells (Gansbacher et al., 1990; Esumi et al., 1991; Fearon et al., 1990; Golumbeck et al., 1991). They apparently function as a tumor vaccine. In human studies now being carried out, the interleukin-2 gene and other lymphokine genes are being introduced into tumor cells which are then irradiated to prevent replication and returned to the patient. If extrapolations from the animal studies are relevant, it seems possible that this form of immunotherapy will become useful in elucidating an immune response that will prevent the growth of unseen and inaccessible tumor metastases (Fig. 5). One of the major problems complicating and impeding successful bone-marrow-transplantation approaches for the treatment of leukemic malignancies is the uncertainty regarding the origin of cells responsible for relapse of patients after bone-marrow transplantation. It is not certain whether the cells responsible for the relapse have their origin in the transplanted bone marrow that had been inadequately purged of

222 Table 1. Principal viral vectors of potential application for human gene therapy. Parent virus

Potential targct cells

Advantages

Disadvautages

Retrovirus

Fibroblasts, endothelial cells. myoblasts, smooth muscle cells, hepatocytes, hematopoietic cells and stem cells

Nonpathogenic Integration into host cell genome Relatively simple design Biology well understood

Relatively low virus titers Limited capacity for foreign DNA (< 10 kb) Inefficient in vivo infection Do not infcct non-dividing cells Transgene expression may not be prolonged

Adcnovirus

Hcpatocytes, airwaj epithelial cclls, lymphoid, hematopoietic and myeloid cells

Nonpathogenic rcplication-defective mutants are available Tlumans are natural hosts High virus titers High efficiency of irz vivo infections Infects cell types that are largcly refractory to retrovirus infection Biology well understood Capacity of 7.5 kb foreign DNA or morc

Virus does not integrate into host cell genome Vector design more complicated than for retroviruses May recombine with naturally occurring adenoviruses

Adeno-associatcd virus

Hematopoietic cells, fibroblasts, epithelial cells

Nonpathogenic and noncytotoxic Humans are natural hosts Prcfcrred site-specific integration a t human chromosome 19 Abilily to establish a latent state Relatively simple design

Relatively low virus titcrs Limited capacity for foreign DNA (4kb) Infection cfficiency low, depending on ccll type Requires adenovirus as helper Many aspccts of biology not well understood

HSV

Non-dividing cells such as differentiated neurons and hepatocytes

Replication-defective mutanls and packagable plasmid are availablc High virus titers Broad host-cell range High efficiency of infection Infects cells refractory io retrovirus infection Biology well understood Capacity of 30 kb foreign DNA

Plasmids are packaged with low efficiency Plasmids recombine with helper virus Keplication-defective viruses are still cytotoxic Viral genome is more difficult to manipulate Gene regulation is complcx

tumor cells, or rather whether the cells arise from portions of the patient's bone marrow that had been incompletely ablated by chemotherapy or irradiation. A number of clinical studies now are aimed to study that question. In principle, the purged bone marrow is being modified genetically to express a nontherapeutic marker gene such as neo'. If cells derived from relapsed leukemia patients contain the neo' gene, it can be concluded that at least these kinds of relapse are caused by inadequate purging in vitro of the material returned to the patient in the form of the transplant. The use of lymphocytes as carrier cells has also been incorporated into genetic approaches to the treatment of severe combined immunodeficiency disease (SCID) resulting from a deficiency in the enzyme ADA. T lymphocytes, isolated and cultured from patients with ADA deficiency, are being infected with a retroviral vector expressing the human ADA gene, and after amplification in vitro to very large cell numbers, the cells are being transfused into patients. Since these cells have relatively long lifetimes in vivo, and since they are also expected to have a growth advantage over ADA-deficient cells, it has been hoped that they would provide a means of restoring T-cell function to patients in a reasonably stable form. There have been several patients treated over the past year or so by this approach, and it seems likely that encouraging initial clinical reports describing their response will be appearing shortly. At least two patients have been described

and attending school. In addition. they have been reported to have steadily increasing levels of adenosine deaminase in their circulation, levels approaching normal levels. Furthermore, it has also been reported that immunological reconstitution may have taken place, as evidenced by the appearance, for the first time in these children, of blood isohemagglutinins. An additional therapeutic clinical protocol has been proposed and is underway relating to potential genetic therapy of hypercholesterolemia secondary to absence of LDL-receptor function. Studies with the Watanabe-hypercholesterolemicrabbit model have suggested that it is possible lo return genetically modified and corrected hepatocytes to Watanabe rabbits by direct infusion into the portal vein or into the spleen and that such cells express sufficient LDL-receptor function to lead to prolonged and stable reductions in serum cholesterol levels in these animals (Chowdhury et al., 1991). Based o n these studies, human experiments are soon to be underway in which patients with homozygous familial cholesterolemia will first have a partial hepatectomy, followed by in vitro infection of their hepatocytcs with retroviral vectors expressing the human LDL-receptor gene and reimplantation of the genctically corrected cells.

to have done well clinically, to be essentially free of infection

concepts and developments of tools for gene transfer and

Future strategies and developments Although there have been very impressive advances in the

223 complementation of genetic defects in humans, there arc a number of remaining difficulties and technical problems that must be solved before techniques of gene therapy bccome readily available and applicable to a large number of human diseases. In many cases, the tissue or organ affected by a genetic disease may not be readily accessible to in v i m manipulation by the kinds of approaches discussed above. This makes the development of improved concepts and methods for targeting of genetic information to predetermined sites in vivo particularly important. Disorders which may require targeting procedures might include systemic and body-wide degenerative diseases and atherosclerosis, metastasizing cancers and diseases resulting from global neuronal dysfunction. Unfortunately, there has been little progress in the development of efficient vector-targeting methods in vivo; however, several studies have begun to suggest powerful approaches to this problem. One approach takes advantage of the concepts of receptor-mediated endocytotic uptake into hepatocytes of DNA bound to asialoglycoproteins for which specific rcceptors exist on liver cells (Eisenberg et al., 1991). This approach has led to the partial phenotypic correction of inherited analbuminemia in rats (Wu et al., 1991) and hypercholesterolemia in Watanabe rabbits. Other researchers have reported that the CD4 surface glycoprotein on T4 lymphocytes, the cell-surface receptor for the human immunodeficiency virus (HIV), can be incorporated into particles of retroviral vectors (Young et al., 1990). These vectors may therefore have the potential to deliver therapeutically useful substances specifically to HIV-infected cells as a potentially powerful approach to AIDS therapy. Given today’s standards of techniques and stratcgics for altering the genetic content of subpopulations of cells, one has to conclude that considerable progress has been made toward the genetic correction of disorders either exclusively or partly causcd by genetic defects. Yet the permanent correction of a phenotype based on a genetic defect in humans by means of gene transfer and expression has so far not been achieved. However, as described above, several studies of human gene therapy are currently being performcd. These and other experiments will certainly provide information in the near future about whether our current technical skills are sufficient to permit long-term correction of genetic disorders in human patients. Whatever the conclusions of these studies may be, we are optimistic that further progress in manipulating the human genome will eventually lead to means of overcoming many human maladies. We argue that this application of powerful genetic techniques toward the amelioration of human sufrcring is important and justifiable, when carried out according to the noblest and most rigorous principles of medicine. As with any human undertaking, technical or not, there will inevitably be errors and missteps along the way, but thesc must not in themsclves determine the appropriateness of the approach. By carrying out the human applications openly, in full view and with thorough scrutiny ofall our society’s formal or informal review bodies, we will be able to achieve a major victory in the battle against human disease.

Wc thank our colleagues at the Center for Molecular Genelics for the helpful comments on the manuscript. This work was supported by Public Health Scrvice grant HD 20034 (T. F.), HL47119 and CA 58317 from the National Institutes of IIealth. K. R was supported by the German Rcsearch Foundalion (DFG).

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