Gene transfer: A review of methods and applications

Pathology (1998), 30, pp. 335-347

GENE TRANSFER: A REVIEW OF METHODS AND APPLICATIONS H. MILES PRINCE Department of Hematology, Division of Hematology and Medical Oncology, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia

Summary Gene transfer is a potentially powerful tool for the treatment of a wide variety of diseases. The transfer of these genes is achieved by utilizing a variety of vectors, including retroviral, adenoviral, adeno-associated virus (AAV) and a number of non-viral mechanisms. Numerous studies have successfully demonstrated transduction of genes into target cells with a variety of vectors, and have provided 'proof-in-principle' that gene transfer can result in prolonged in vivo expression of transduced genes, albeit at low quantities. Furthermore, gene marking studies in acute myeloblastic leukemia (AML), chronic myeloid leukemia (CML) and neuroblastoma have elegantly demonstrated that gene-marked tumor cells contribute to relapse following autologous transplantation. However none of the studies examining the therapeutic benefit of gene therapy has definitively demonstrated a clinically meaningful benefit. Nonetheless, the results of studies involving gene transfer for severe combined immunodeficiency (SCIO), chronic granulomatous disease (CGO), melanoma and lung cancer highlight the potential benefit of this strategy. This review will discuss mechanisms of achieving gene transfer into target cells. It will examine some of the pre-clinical and clinical results to date and will discuss some of the potential uses of gene transfer for therapeutic purposes. Key words: Gene transfer. Abbreviations: AAV, adeno-associated viruses; Ad, adenovirus; ADA, adenosine deaminase; AML, acute myeloblastic leukemia; APC, antigenpresenting cell; CGD, chronic granulomatou s disease; CML, chronic myeloid leukemia; CTI.., cytotoxic-T·lymphocyte; DNM, dominant negative mutation; GALV, gibbon ape leukemia viru s; HSC, hemopoietic stem cell; ITR, inverted terminal repeat; LTR, long terminal repeat; MDR, multi-drug resistance; MHC, major histocompatibility complex; MMLV, Moloney murine Jeukemia. virus; RCA, replication~competent adenovirus; RCR, replication-competent retrovirus; RRE, rev response element; SCIb, severe combined immunodeficiency; TAR, trans-activating response; TIL, tumor infil trating Iymphocytes; wt, wild-type.

Accepted 28 May 1998

INTRODUCTION The transfer of genetic material into a target cell has a wide range of potential applications in medicine. Consequently, there is an increasing need for clinicians and pathologists to have an understanding of its basic concepts, its potential applications and its shortfalls. This review will not attempt to cover the whole field of gene transfer, but will rather focus on three areas. Firstly, a description of the various vectors (vehicles to carry the gene of interest to the target

cell) with a particular emphasis on the safety issues and the advantages and disadvantages of these systems. The second section will focus on gene transfer into hematopoietic cells, which, largely because of their ease of access, have been the most widely investigated cells in gene transfer experiments. The results of gene marking studies and potential applications for gene therapy using hematopoietic cells, will also be discussed. The last section will explore the potential of gene transfer for cancer therapy. DEFINITIONS AND ETHICAL ISSUES Gene therapy can be defined as the transfer of genetic material into cells of the body in order to treat human disease. Somatic cell gene therapy involves the genetic manipulation of any cell in the body, except those involved in meiotic reproduction (germ line cells), namely spermatozoa and ova. Philosophically, somatic gene therapy does not differ greatly from current medical procedures, such as organ transplantation or the use of medical/surgical procedures for Phase I human studies, and has received general ethical acceptance!. At present, over 200 clinical protocols worldwide involving gene transfer into somatic cells, have been initiated or have received approval to begin I. Conversely, germ-line gene therapy has many scientific, moral and ethical issues which are yet to be resolved 1,2, and thus has not been as aggressively pursued to date. HISTORICAL PERSPECTIVE In 1928, Frederick Griffiths discovered that a transforming agent in a .virulent strain of Pneumococcus was able to confer virulence into a non-virulent strain3 . It was not until 1944 however that Avery et al. recognised that deoxyribonucleic acid (DNA) was the transforming agent required4 • Taken together, these discoveries hinted at the potential for using gene transfer as a therapeutic tool. The advent _of recombinant DNA technology 25 years ago, provided -the- tools which would be essential for effective gene therapy, ie; cloned genes and feasible gene transfer techniques. Despite substantial progress having been made since the first gene transduction into mammalian cells using the calcium phosphate method, the search for the perfect vehicle to carry genes into target cells has remained elusive. VECTORS FOR GENE TRANSFER The pe/fect vector Vectors are the vehicles used to carry the genetic material which is to be inserted into the target cell and they are

0031-3025/98/040335-13 © 1998 Royal College of Pathologists of Australasia

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TABLE 1 Comparison of the most commonly used vectors Retrovirus

Adenovirus

AAV

Herpesvirus

Plasmid-liposomal

Insert size

6-7kb

7.5 kb

2--4.5 kb

10-100 kb

Genome

RNA

dsDNA

ssDNA

dsDNA

Unlimited RNAlDNA

Site

Genome

Episome

Episome/genome

Episome

Episome

Efficiency

Low

Moderate

Variable

Moderate

Very low

Cell proliferation

Required

Not required

Not required

Not required

Not required

Expression

Permanent

Transient

Permanent/transient

Transient

Transient

Major advantages

Genomic expression

Relatively high transduction efficiency

Relatively high transduction efficiency +/-Genomic expression

Tropism

Safe

Inflammatory response Immune response

? Insertional mutagenesis Difficult to produce

Difficult to produce

Major problems

Insertional mutagenesis

broadly categorised into viral and non-viral. The perfect vector does not exist-and never will-as there are different vector requirements depending on the specific aims of the investigator (some approaches may require permanent gene expression [eg; treatment of a Factor IX deficiency], while others may require transient expression [eg; cytokine production]). However, in general terms the following attributes are considered desirable: safety to the patient and environment; non-inflammatory nature; non-immunogenicity; a large capacity for genetic material; efficient transduction; high/controlled expression of gene; target cell specificity; and the ability to be produced in high concentration and at a low cost (Table 1).

Retroviral vectors Retroviruses consist of a single-stranded ribonucleic acid (RNA) genome surrounded by a nucleocapsid core and glycoprotein envelopes,6. The retroviral genome is between 7 and 10 kb long and contains three conserved genes (gag, pal and env) required for its normal life cycle. The prominent features of the wild-type (wt) Moloney murine leukemia virus (MMLV) double-stranded provirus (genomic integration of viral DNA) are illustrated in Fig. 1. Retroviruses attach to the cell surface of host cells via the envelope surface protein? Contemporary amphotrophic (transduces cells across species) vectors, such as MMLV, specifically recognise the amphotrophic RAM-1 receptor8 • RAM-1 has been identified as an inducible sodium-

LTR~LTRI Fig. 1 Wild-type murine leukemia virus. The retrovirus genome encodes three genes: gag codes for the nucleocapsid protein and others required for the assembly of infectious viral particles at the plasma membrane; pol codes for reverse transcriptase (required for viral DNA synthesis), integrase (required for integration of viral double-stranded DNA into host DNA) and protease (cleaves viral polyproteins); env codes for a viral glycoprotein which recognises specific cell surface receptors and confers host range and tropism for the retroviral particle. The Ijt packaging sequence extends a few hundred base pairs into the gag gene; LTR; long-terminal repeats.

Cheaper Poor efficiency

dependent phosphate symporter expressed in a broad range of tissues 8 . Gibbon ape leukemia virus receptor-2 (GLVR2) has also been identified as a receptor for amphotrophic retroviruses9 . Following attachment, the virus enters the cell by receptor-mediated endocytosis5 • The virus sheds its outer envelope coat and then releases the nucleocapsid into the cell cytoplasm, at which point viral reverse transcriptase directs synthesis of a double-stranded DNA intermediate from the single-stranded RNA genomic template. The nucleoprotein complex then enters the cell nucleus, a process that requires cell division lO , and integrates into a random site in transcriptionally active chromatin domains7. Although the site of integration is random, the mechanism of integration is a highly ordered and precise process that is mediated by attachment sites in the long terminal repeats (LTRs) and in which viral integrase is essential. In this manner, genes located within the LTRs of a retroviral vector are stable and are faithfully transmitted without rearrangements. This integrated double-stranded viral DNA is termed the provirus. Following integration the proviral genome utilises the host cell machinery to synthesise RNA transcripts and translate viral proteins. Viral transcripts are either used to produce viral proteins or are packaged in newly assembled retroviral particles at the plasma membrane. Virions are released into the extracellular space by a budding process in which the host plasma membrane, embedded with envelope glycoproteins, is pinched off and becomes the outer viral envelopes. Retroviral vectors derived from the MMLV are currently the gene transfer vehicles of choice for the majority of approved clinical trials. The major advantage of retroviral vectors is that they have the potential to integrate into the genome of the target cell, and thus the gene is integrated for the lifespan of the cell and into the progeny of the genetically transduced parent cell. However, these viruses can only insert their genes into the genome of actively dividing cells. Efficiency of transduction is further limited by the ability of the virus to bind to receptors on the target cell surface. MMLV is part of the oncovirus sub-famili. Among oncoviruses, MMLV belongs to the group which can induce 1eukemias and lymphomas in mice after a long latent period5 . The induction of malignancy results from the insertion of wt viral DNA into the host genome,

GENE TRANSFER

337

A. _

B.

ILTRt-x~gag Upo{ Uenv ~LTRI

***** gag proteins ***** pol ****** env

Fig. 2 Schema for production of replication-incompetent retroviral vector particles.(A) plasmid containing a human gene, ie; geneX, is transduced into (B) the packaging cell line and the vector transcript is packaged by viral proteins (*****) provided by the packaging cell line and (C) released into packaging cell media supematant. Packaging sequence (1jI+) and 3' end of gag protein is left intact in the plasmid. The packaging sequence is removed in the packaging cell line (X). This supematant can be used to infect target cells and can be incorporated into the target cell genome. Adapted from Salmons and Gunzburg37 •

which subsequently functions as an oncogene or as a promoter to oncogenes with resultant clonal proliferation (ie; insertional mutagenesis). Two critical factors are needed to induce mutagenesis: wt viral DNA and a large quantity of this DNA. Consequently, central to the concept of producing safe retroviral vectors is the need to remove wt viral DNA and to make these retroviruses replication defective (unable to replicate in the host cell). If the viral vector cannot replicate, only small and controlled quantities of DNA will be targeted at the celL Other retroviruses related to the MMLV which are being investigated as gene transfer vectors, include gibbon ape leukemia virus (GALV) and human immunodeficiency virus (HIV)5,1l. The key to producing a replication-defective vector that is safe to the patient and the environment, is to remove the structural proteins from its genome. By removing these structural genes the virus cannot replicate after it has entered the cell. A virion that infects the target cell will therefore contain the proviral DNA containing the gene of interest with the structural protein genes removed. To produce the initial infecting virion, the genes for these structural proteins must be provided from elsewhere. Fortunately retroviruses do not require intact viral structural genes gag, pol, env in their genome in order to produce infectious viral particles. Indeed, these structural protein genes can be provided by co-infection with other retro-

viruses (so-called producer cells) (Fig. 2). As a result, the typical retroviral vectors are designed by cloning the foreign gene of interest into the coding region normally occupied by gag, pol and env in a MMLV backbone. Producer cell lines encode the structural viral genes deleted from replication-defective viral vectors. Transduction of a retroviral vector into producer cells leads to packaging of an infectious, replication-defective retroviral vector. Several generations of producer cell lines have been developed with safety features designed to minimise the creation of replication-competent retroviruses (RCR). Resultant replication-defective retroviral particles only produce one round of infection, and there is very low risk for virus spread in cells exposed to these replication-defective vectors during gene therapy. Another advantage is that the size of the gene inserted in wt retroviral vectors is limited and, by removing viral structural genes, foreign genes up to 8 kb in size can be inserted 12 • Fig. 2 is a schematic illustration of the packaging of a retroviral vector containing a human gene in a producer cell line to produce an infectious, replication-defective retroviral vector particle. There always remains however, a potential for homologous recombination (recombining two strands of distant DNA) of the retrovirus resulting in the development of RCR, thus increasing the potential of the target gene to act as an oncogene or as a promoter to oncogenes with resultant clonal proliferation (ie; insertional mutagenesis).

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Fig. 3 Structure of the AdS genome.Two regions of the viral genome (El and E3) have been used to make insertions or substitutions of DNA to generate helper independent recombinant viruses. Deletions of up to 3.2 kb can be made in E l. The other region of the AdS genome which has been extensively used for cloning foreign DNA is E3. Deletions of over 3 kb have been made in this region, permitting insertion of between 4 and 5 kb of foreign DNA, and generating recombinant viruses able to replicate in any normally permissive host. Combined deletions in El and E3 allow insertion of up to approximately 8 kb of foreign DNA. Adapted from 16 and 17.

Despite the evolution of producer cell lines to produce minimal, if any, RCR, potential exposure of human subjects to RCR remains a concern. To summarise the data concerning the safety of retroviral vectors: (1) there is no evidence that systems currently used for clinical trials produce RCR; (2) current RCR detection assays are extremely sensitive (to one RCRJ10 6 viral particles); (3) severe immunosuppression in the host, combined with an extremely high dose of infectious retroviral particles (2 X 10\ would be required for malignancy, as was the case in the monkeys that developed lymphoma 13; and (4) clinical gene therapy trials to date have shown that retroviral vectors can be safely used in human sUbjectsl.

Adenoviral vectors Several factors account for the growing interest in adenoviral vectors for gene transfer. Firstly, they can easily be rendered replication defective by deletion of critical regulatory genes. Second, they have a relatively high transduction efficiency into a wide variety of normal and tumor cells. Third, they transduce both dividing and non-dividing cells. Fourth, they can be produced relatively easily and at high titres (10 11 _10 12 infectious units per ml). Finally, they do not incorporate into the genome. This final fact can be both advantageous, as there is no risk for insertional mutagenesis and transient expression will result, and disadvantageous, if long-term expression (and passage of the proviral DNA into subsequent progeny) is required l4 . The major disadvantages of the adenovirus (Ad) as a vector include, firstly, its propensity for homologous recombination (particularly at high titres) with the consequent development of replication-competent adenovirus (ReA). However, one must also bear in mind that wt Ads are generally safe; millions of North American army recruits have been vaccinated by oral wt Ad, with rare respiratory side effects. Second, during transcription a number of adenoviral structural genes are transcribed along with the provirus. The expression of these genes makes the target cell immunogenic and prone to cell-mediated immune responses. In association with this, a humoral response against the adenoviral gene products is initiated, resulting in a brisk response on re-infection with rapid clearance of the vector. This propensity for an immune response (which can result in local tissue inflammation, as has been demonstrated in the cystic fibrosis clinical trials of in vivo gene transfer) is a potential limitation for Ad as an in vivo gene transfer vector 14. 15• Structurally, Ad particles are relatively stable. The virion is an icosahedral capsid about 70 nm in diameter, consisting exclusively of protein and DNA I6. The genome is a

linear double-stranded DNA molecule of about 36 kb. The most common isotypes are AdS and Ad2. Fig. 3 demonstrates a simplified map of the AdS genome 16•17 • There are two phases of the virus replication cycle: early, corresponding to events occurring before the onset of viral DNA replication; and late, corresponding to the period after initiation of DNA replication. Following virion production, adenoviral particles are released from the host celL Binding to the target cell requires ligation of the adenoviral fibre receptor to the target cell receptor. Two regions of the viral genome (El and E3) have been used to make insertions or substitutions of DNA to generate helper-independent recombinant viruses. El gene products are not required for viral replication in human 293 cells (a line which is transformed by AdS DNA and which contains and expresses the left end of the genome)1 8. Deletions of up to 3.2 kb can be made in El without compromising the ability of the virus to grow in 293 cells l9. The other region of the AdS genome which has been extensively used for cloning foreign DNA, is E3. Deletions of over 3 kb have been made in this region20, permitting insertion of between 4 and 5 kb of foreign DNA and generating recombinant viruses able to replicate in any normally permissive host. Combined deletions in El and E3 allow insertion of up to approximately 8 kb of foreign DNA into vectors which can be replicated in a helper-independent fashion in human 293 cells. Furthermore, although Ad DNA sequences can integrate into host cell chromosomes at low efficiency, there is no role for an integration mechanism in the life cycle of the virus, nor are any viral gene products known that might function in integration. This latter property of Ads is ~m important consideration in the context of gene therapy and marks a clear distinction between Ad vectors and vectors based on retroviruses or on parvoviruses (see below). Recently adenoviruses have been successfully produced by a helper virus (this helper virus provides all structural proteins for production). The wt Ad can thus have all the structural genes removed, allowing proviral DNA of up to 35 kb to be inserted. An additional advantage is that there is no production of structural proteins by the target cell, thus potentially reducing the normal immune response that is initiated when these structural proteins are recognised by the recipient's immune response 21 • Selective targeting, such as binding via folate receptors on the target cell surface22 or hybridizing the Ad to monoclonal antibodies,23.24 is also being investigated.

Adeno-associated virus (AA V) AAV belong to the family parvoviridae, are widely preva-

GENE TRANSFER

lent and infect over 90% of human adults. AAVs are unique among animal viruses in that they normally require coinfection with an unrelated helper virus (ie; Ad) for productive infection in cell culture. Without a helper virus, AAVs integrate into the host genome and remain as provirus. WtAAVs have a preferred site of integration into the human genome (Chromosome 19q13.4) and can exist as a latent infection for the life of the cell. Once incorporated into the genome, transcription is undertaken with the assistance of genes derived from the adenovirus. The AA V viral genome is transcribed in three overlapping regions producing seven primary transcripts. There are many attractive advantages of AA V as a gene transfer vector. WtAAV do not require target cell replication to infect the cell and are capable of infecting all human cell cultures tested so far. However, it appears that transduction into hematopoietic progenitors only occurs at high titers and is transient. WtAAVs have not been associated with any clinical symptoms in any host, and are not known to be tumorogenic. The sole sequence needed for AAV vector integration is the terminal 145-nucleotide inverted terminal repeat (ITR), making the cloning capacity of the AAV vector 4.7 kb. However, when the rep gene is deleted, AAV vectors integrate randomly. This raises the concern of potential insertional mutagenesis in the human genome. Another additional drawback is the viruses' propensity to undergo rearrangement during integration. Finally, viral stocks of 1 X 108_109 infectious units/ml are feasible but difficult to prepare, and are easily contaminated with wtAA V and helper adenovirus!5. Herpes simplex virus (HSV) The tropism of HSV to neural tissue is its major advantage and it can reliably infect neural cells both in culture and in vivo with both striatal and peripheral neurons successfully transduced. Most pre-clinical studies to date have utilized the HSV vector to transduce cells with the genes of neuronal growth factors. Two types of HSV derivative have been developed as transfer agents: the amplicon, a plasmid containing only the viral origin of replication and the necessary packaging information; and vectors, consisting of a nearly complete viral genome from replication-defective HSV strains. The primary problems with HSV-mediated gene transfer are: (1) the cytopathic nature of HSV; (2) the difficulty in maintaining long-term expression of inserted genes-introduced genes are generally shut down within weeks of infection; and (3) infection efficiency is relatively low compared to other viral systems 25-27 Electroporation Electroporation is the use of electrical currents to disrupt the cell membrane so as to allow the passage of 'naked' genetic material into the target cell. This method offers several advantages for gene transfer. It is applicable to a wide variety of cell types, including hematopoietic cells 28 and stromal elements29 • It is easy to perform and can be highly reproducible. It also allows the use of 'naked' DNA, eliminating the need for viral packaging systems. The major disadvantage of this method is the poor efficiency of transduction. Like adenoviral transduction, the DNA is not incorporated into the genome, and hence long-term ex-

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pression is not achieved and DNA is not transferred to the progeny of the cell transduced. Bead transduction This method of transduction combines the principle of physically producing breaks in the cellular membrane, as utilised in the scrape loading technique of Fechheimer et al. 30, with the use of beads, as described for the loading of macromolecules 3!. Several factors influence the efficiency of bead-mediated gene transfer, including the concentration of exogenous DNA, the timing of the addition of DNA, the size and condition of the beads and the buffers utilised. The efficiency, although relatively low, is equivalent to electroporation 32 • Gene gun or particle bombardment The gene gun, or DNA-coated particle bombardment, utilises heavy metal particles (either tungsten or gold, 1-5.um in diameter) that are accelerated to a velocity sufficient to penetrate target cells, and it continues to be an important method of plant cell transduction. Recently this technology has been applied to animal cells. These devices use a helium-driven plunger to pierce a Kapton disc placed in front of another disc on which the DNA-coated microprojectiles are fixed. When the gas is released, a shockwave three to four times the speed of sound is created and this launches microprojectiles against the target tissue. Again the efficiency is low and the DNA is not incorporated into the genome 33 . Liposomes This method utilises lipid chemistry to surround naked DNA plasmids with a liposomal coat which is subsequently endocytosed by the target cell. In theory, plasmidliposome complexes have many advantages as gene transfer vectors: they can be used to transfer expression cassettes of essentially unlimited size; they cannot replicate or recombine to form an infectious agent; and they may evoke fewer inflammatory or immune responses because they lack proteins. The disadvantage of these vectors is that they are inefficient, requiring thousands of plasmids be presented to the target cell in order to achieve successful gene transfer. The available data are not sufficient to determine whether repetitive administratIon of liposomes poses safety risks.

APPLlOATIONS OF GENE TRANSFER Over 200 clinical studies in gene transfer are currently in existence and have involved over 2000 patients worldwide. Approximately 25% of these studies utilise 'marker' genes to examine basic biological questions regarding the fate of transduced cells. A further 60% involve patients with cancer. The remaining studies examine the potential role of gene transfer· in single gene defects and acquired non-malignant disease (Taple 2). This review cannot cover all the possible applications of gene transfer and instead will focus on areas of hematopoietic gene transfer and potential gene therapy approaches for cancer.

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TABLE 2

Summary of clinical trials of gene transfer. Adapted from J

Disease

Number of studies (%)

Gene-marking studies (variety of diseases)

24

Cancer (by approach) Immunotherapy Pro-drug Chemoprotection Tumor suppressor gene Anti-sense

49

Inherited single gene disorders ADA deficiency /XJ-Antitrypsin deficiency Chronic granulomatous disease Cystic fibrosis Familial hypercholesteroiemia Gaucher's disease

29

10 4 4 2

18

<1 <1 <1

10

<1

3

Infectious diseases (HIV)

8

Acquired disorders (peripheral vascular disease, rhematoid arthritis, etc)

2

GENE TRANSFER INTO HEMATOPOIETIC PROGENITORS As bone marrow cells are easily accessible for in vitro manipulation, there has been considerable focus on the transfer of genes into hematopoietic cells. However, the challenge of developing a successful strategy which results in long-term gene expression in such cells is immense. To be effective, the target cells must be transduced with high efficiency, must survive the process and must engraft into the marrow niches or lymph nodes (and survive any immune or inflammatory response). The gene must be transcribed and the product expressed in sufficient quantities. Most studies to date have involved gene marking of the autograft which is infused following high-dose conditioning chemotherapy. These studies have been critical to our understanding of the fate of gene-marked cells once they are re-infused into the host and they have answered important biological questions relating to the influence on relapse of reinfused tumor cells. Other areas where gene transfer into hematopoietic cells is being investigated include the human immunodeficiency virus (mV) infection, the transduction of the multi-drug resistance (MDR) gene and the transduction of genes that are abnormal or absent from the hematopoietic stem cell or its progeny, ie; hemoglobinopathies, hemophilias, chronic granulomatous disease (CGD) and severe combined immunodeficiency (SCID). Although these latter diseases are exceedingly rare, the importance of these studies cannot be over emphasized as they provide 'proof-in-principle' that gene-marked cells can survive and be expressed for extended periods of time once re-introduced into the host. Hematopoietic stem cells (HSC) are, by definition, cells that are capable of long-term hematopoietic reconstitution and that possess the ability of self-renewal over the course of a lifetime34-36. In the steady state the majority of stem cells are dormant in the cell cycle. Indeed, the dormancy of these cells is the major hurdle to successful and efficient gene transfer. Most studies of gene transfer into hematopoietic cells have employed an ex vivo approach in which HSCs are harvested and the genes transduced while the cells are in culture. The genetically modified cells are subsequently

infused into autologous or syngeneic recipients who usually have received cytotoxic/immunosuppressive conditioning chemotherapy prior to adoptive transfer. The issue of chemotherapy is an important one. For hematopoietic cells to engraft, they must find an appropriate niche which has an appropriate stromal and cytokine milieu in which these cells can be sustained and can differentiate. The ability to engraft in a niche appears to be in part related to competition between other niches. In the allogeneic transplant setting, the ability to overcome that competition appears to be enhanced by prior conditioning chemotherapy, particularly if it is immunosuppressive. It remains unresolved if such prior therapy is necessary in the autologous setting, and clearly this issue is of importance in gene therapy approaches for non-malignant disease where conditioning chemotherapy is undesirable. The vast majority of studies of gene transfer into hematopoietic cells rely on recombinant retroviruses as the delivery system of choice, largely because they can integrate into the genome and have the potential to express their gene products permanently while being able to pass the gene onto their progeny37. As retroviral transduction into the most immature of these cells requires cellular division, one can predict that the most efficient transduction process will be one that has maximum retroviral exposure at the time of HSC division. Indeed, the optimal transduction protocol would be one that would actively stimulate HSCs into cycle. However, to date the exact mechanisms that trigger HSCs into division remain elusive34 . Indeed, none of the known hematopoietic growth factors or cytokines appears to have the ability to actually trigger a HSC into cycle. Consequently the various current clinical protocols attempt to stimulate proliferation and incubate the target cells with a cell-free retroviral supernatant, in the presence or absence of bone marrow stroma, with serum or serum-free media and with or without growth factors 38-42. Adenoviral vectors have been used to transfer genes into a variety of tissues, but the transgenes do not integrate into the host genome and they do not efficiently infect all hematopoietic cells4 3-45. Physical methods of gene transduction, such as electroporation, liposomal-mediated transfer or direct DNA injection, do not result in genomic integration. Beyond these difficulties in determining the conditions to optimise HSC division and consequently transduction, the ability to recognise and quantify successful transduction into the HSC is also problematic. No definitive assay to identify and measure the HSC exists36 . Consequently, to definitively demonstrate successful gene transduction into the most immature human HSCs, one must perform clinical trials to demonstrate that auto grafted human cells contain the gene and contribute to long-term marrow repopulation.

Gene marking studies Genetic marking of cells is a powerful approach for addressing biological questions in clinical trials of experimental cancer treatment46 . Such cell marking facilitates studies of the long-term distribution and survival of marked cells in vivo and assessment of their contribution to clinical outcome. In clinical trials involving only gene marking, cells to be marked are incubated ex vivo, with a replication-incompetent retrovirus bearing a reporter gene.

GENE TRANSFER

A commonly used reporter gene is neo, encoding bacteria] neomycin phosphotransferase which, when expressed, confers resistance to the neomycin analogue G41S 47 • The stable and unique integration pattern of proviral DNA in the genome of marked cells can provide a permanent marker for individual hematopoietic cells and their clonal descendants. The fates of marked hematopoietic stem cells, infused into autologous recipients following marrow-ablative cancer therapy, can thus be readily determined using sensitive genetic-based detection systems (eg; PCR) or by clonogenic assays for progenitors resistant to toxic concentrations of a41S. The first evidence for gene transfer into HSCs was provided by Brenner et al. in a clinical gene marking trial48 . Ex vivo transduction with the reporter gene neo was performed with 20% of bone marrow colonyforming units positive for proviral DNA at IS months. The marker genes continued to be detected and expressed for up to 3 years in the mature progeny of marrow precursor cells, including peripheral blood T- and B-cells and neutrophils. In peripheral blood cells, expression was five-fold and ten-fold lower and was variable between different lineages. The highest level of expression was seen in myeloid cells and the lowest level was seen in Blymphocytes. One critical aspect of these studies was that they were pelformed in the pediatric population, a population which consistently demonstrates higher transduction efficiencies than adults49. Importantly, in one case, leukemic progenitors expressed neo at 42 months, demonstrating not only successful gene transfer into a primitive malignant clone, but also that this clone (harbored within the autograft) contributed to relapse post-transplant48 ,5o. Deisseroth et al. have demonstrated similar marking of primitive malignant clones in patients undergoing autotransplant for CML and neuroblastoma51 •52 • Recent clinical applications in which genetic cell marking may provide new and important information, include the infusion of retroviral marked, autologous, tumorinfiltrating lymphocytes (TIL) into patients with advanced melanoma53- 55 and the infusion of retroviral marked bone marrow into patients with breast cance~6, myeloid leukemia50, myeloma57,58 and neuroblastoma52 undergoing autologous bone marrow transplantation. The salient findings in these important early trials were that: (a) retroviral gene transfer as currently practiced is safe; (b) gene-marked hematopoietic cells contribute to both early (weeks) and late (months) engraftment after high-dose chemotherapy (but note; the contribution to engraftment beyond this time is yet undetermined); (c) peripheral blood stem cells engraft at higher levels than bone marrow-derived cells; and (d) gene-marked cancer cells contribute to relapse in acute leukemia and CML and in neuroblastoma. As much energy and time is now being devoted to developing tumor-purging techniques, the resolution of this latter issue will define the role of ex vivo autograft purging for marrow-infiltrating malignancies and will help focus further protocols. Chronic granulomatous disease (CGD) As mentioned, although caD and SCID (see below) are exceedingly rare, the importance of these studies cannot be over emphasized as they provide 'proof-in-principle' that gene-marked cells can engraft in non-chemotherapy-exposed marrow, can survive and can be expressed for

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extended periods of time once re-introduced into the host. CaD is caused by mutations in the genes encoding gp91 phox , p22phoX, p47phox or p67 phox • Insertion of a functional copy of the involved gene into HSCs (even with low expression, ie; < 10% normal) should reconstitute burst oxidase activity in phagocytes and potentially provide lifelong cure. A Phase I gene therapy study in five patients with p47 phox has recently been conducted59 • Autologous CD34 + peripheral blood cells were transduced with a retroviral vector over a three day period and infused into a non-myeloablated patient. Oxidase-positive neutrophils were detected between three weeks and several months. However the inefficiency of current protocols was highlighted as only 0.04-0.05% of circulating neutrophils had oxidase activity. Severe combined immunodeficiency (SCID) Clinical gene transfer trials in adenosine deaminase (ADA) deficient patients were among the first undertaken. Peripheral blood lymphocytes were harvested and expanded in vitro, transduced using a retroviral vector, containing both the ADA gene and marker gene (neophosphotransferase), and subsequently infused monthly into patients. Follow-up has demonstrated detectable vector DNA, mRNA and functional marker DNA beyond three years from first treatment (and 545 days following last infusion). In addition, improvements in immune function have been demonstrated. Although ADA levels have been 25% of normal, these patients have, for obvious ethical reasons, remained on enzyme replacement therapy, and thus the true clinical impact has not been tested6o • Current studies are examining the effect of progressively withdrawing replacement therapy in such patients. Multi-drug resistance gene (MDR) The gene for MDR transcribes p-glycoprotein. This pglycoprotein pump exists on the cell sulface and actively 'pumps out' toxic substances (ie; plant-derived chemotherapeutic agents such as the anthracyclines, vinca alkaloids and taxanes) and is a major contributor to the resistance of tumor cells to chemotherapy. Investigators are examining the effect of transducing this gene (or alkylating agent resistance genes) into normal hematopoietic progenitors in an attempt to render them resistant to subsequent chemotherapy. This increased tolerance would allow for dose-intensification of chemotherapy. Such an approach may have an important role in protecting the marrow microenvironment (stromal and accessory cells) following high-dose therapy and transplantation 61 ,62. Coagulation factor deficiencies and hemoglobinopathies In patients with less than 1% factor VIII or IX activity, dramatic clinical improvements are seen if exogenous factor replacement increases plasma levels by only a few per cent. Hence, these diseases are conceptually attractive targets for gene therapy strategies, given that even an inefficient system may produce sufficient protein to have a clinically meaningful impact. However, issues such as large gene size, choice of appropriate target cell (liver, muscle, stroma, hematopoietic cells), efficiency of transduction, duration of expression, transcriptional regulation

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and post-transcriptional processing, have hampered development in this area63 -05 . Investigators examining other red cell disorders (ie; thalassemias), where transcriptional regulation is critical (excessive production of the gene may lead to further structural defects), have had to deal with similar issues. Human immunodeficiency virus (H/V)

A number of gene transfer approaches are being investigated as potential anti-HIV strategies and this area has been well reviewed elsewhere66-68. The vast majority of these protocols utilise viral vectors, and most early studies have focused on transducing mature lymphocytes. More recently the transfer of genes into the early hematopoietic progenitors (which would differentiate into myeloid, Band T-lineages) has been investigated. Briefly, the strategies used include: transducing the genes for dominant negative mutations (DNM) such as the rev DNM gene (these DNM genes produce proteins which compete for the binding sites on regulatory RNA); RNA decoys (the transduced genes produce RNA similar to trans-activating response (TAR) or rev response element (RRE) which compete for the binding of regulatory proteins such as Tat or Rev, respectively); anti sense RNA strands that bind and block transcription sites; ribozymes (catalytic antisense molecules) which bind and splice RNA; soluble CD4 genes to block HIV binding sites; and chimeric T-receptors formed by inserting the gp140 receptor gene. A particularly novel strategy is the use of intracellular 'intrabody' genes. This involves transducing CD4 T-Iymphocytes with the anti-gp120 gene, the anti-gp120 inactivates the gp120 receptor in these T-cells and thus blocks HIV virions entering the ce1l69 . GENE THERAPY FOR CANCER A failure of the host to mount an antigen-specific T-cellmediated immune response against malignant cells is critical for tumor proliferation. The mechanisms utilised by tumors to evade the immune response are numerous 70-75 and will only be briefly discussed here. A brief review of T-cell responses to foreign antigen serves as a background to understanding potential therapeutic strategies for gene transfer. After exiting the thymus, T-cells respond to antigens in an antigen-specific and antigen-nonspecific manner. The former requires the interaction of the T-cell with specialised (professional) antigen-presenting cells (APCs). The generation of an immune response by T-cells following the presentation of antigen by an APC, requires three distinct stages of cell to cell interaction, ie; adhesion, recognition and co-stimulation (Fig. 4). Cellular adhesion results following the interaction of APCs and T-cells via surface ligands and their receptors (adhesion molecules). The subsequent recognition stage involves the presentation of sufficient quantities of antigen by APCs in the context of the major histocompatibility complex (MHC). The MHC-T-cell receptor interaction initiates a number of complex signalling events via the CD3 subunits76 . This signalling (signal 1) is critical to T-cell activation but insufficient to complete antigen-specific T-cell activation. A further co-stimulatory signal (signal 2) is essential for T-cell activation. This second signal is initiated following

Pathology (1998), 30, November

the binding of co-stimulatory molecules to epitopes on the T-cell surface. Two of the most important co-stimulatory molecules are B7-1 (CD80) and B7-2 (CD86), which bind to their T-cell ligands, CD28 and CTLA4. Signal 2 ultimately results in initiation and enhancement of T-cell clonal expansion, lymphokine secretion and effector function. If signal 2 is not delivered, T-ceIls enter a state of anergy (long-term unresponsiveness to specific antigens)77. Tumors can evade the immune response at a variety of steps including absent or inadequate expression of adhesion molecules, absence of specific tumor-associated antigens (eg; MUC-l, MAGE 1, etc.), inadequate processing of antigen (ie; via TAP proteins), absent or limiting cell surface MHC, absent or limiting cell surface co-stimulatory molecules, production of immunosuppressive molecules by tumor (ie; TGF-f3, IL-lO), activation of T-suppressor cells after exposure to antigen and abnormal T-cell signal transduction following binding of ligand. Depending on the site of defect in relation to the three stage response, either no T-cell response will be elicited or tumor-specific anergy (tolerance) will result. The aim of immunological gene therapy for cancer hinges on overcoming or bypassing these evasive mechanisms to elicit an anti-tumor T-cell response. As mentioned, a cytotoxic-T-Iymphocyte (CTL) response is dependent on presentation of antigen in the context of MHC class I. Indeed, there is a large number (greater than 10,000) of different peptides presented by MHC class I molecules on any given cell. Given that to date there have only been about a dozen specific tumor-associated antigens identified, an immune anti-tumor strategy applicable to a large number of tumor probably requires a less specific approach, such as upregulating the mechanisms required to present tumor antigens or alternatively, increasing the ability of a CTL to recognise 'foreign' tumor antigens. One potential mechanism to achieve this is by the systemic delivery of cytokines, such as et-IF, TNFet, IL-2, IL-4 or IL-12, to upregulate adhesion, MHC and co-stimulatory molecules. However, the doses of cytokine required, when given systematically, to achieve sufficient concentrations in the local proximity of the target cell may be considerable and associated with intolerable side effects. Cytokine and co-stimulatory genes

An alternative strategy is to obtain high cytokine concentrations only locally; transduction of genes coding immunomodulatory molecules directly into tumor cells is one appealing way of achieving this. Such molecules include MHC I, MHC n, IL-2, IL-4, IL-12, GM-CSF,TAP 1 & 2 proteins, B7-1 (CD80) and B7-2 (CD86fo,71,78,79. This 'vaccination' approach usually requires that the target cells are transduced ex vivo with genes of one or more of these cellular proteins, and subsequently returned to the host. Viral vectors (retroviruses and Ads in particular) have proved useful in this regard. Several of these strategies have demonstrated the ability to generate systemic, tumorspecific immune responses in mice and humans. Indeed, a preliminary report suggests regression of melanoma in distant sites following repetitive injections into cutaneous melanomatous lesions utilising a GM-CSF-containing retroviral vector80•81 • One major limiting factor of various cytokine-gene clinical trials has been the difficulty in determining the appropriate dosing schedule of these vaccination approaches.

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Fig.4 Cell-<:ell interaction of tumor cell/APC with T-cell. (A) Adhesion molecules bind; (B) tumor-associated antigens processed; (C) tumor antigens processed and packaged (ie; TAP proteins); (D) antigen presented in context of MHC class I (or rarely, MHC class Il); (E) co-stimulatory molecules bind (ie; B7-1, B7-2); (F) cytokines released from antigen-presenting cell (ie; TGF-fl, IL-lO); (G) interaction (positive or negative) from surrounding cells; (H) T-cell signal transduction with subsequent cell activation.

Another factor that complicated earlier studies is that most patients had widely disseminated disease and had previously received intensive anti-cancer (immunosuppressive) therapy. Importantly, in a number of clinical studies to date where responses have been identified, definitive gene therapy-mediated anti-tumor responses have been difficult to demonstrate because no control vectors (ie; a 'dummy' vector containing an inactive gene) have been used. This latter issue is of importance because one could argue that some of the pathological findings may be due to an immune or inflammatory response induced by the vector itself (rather than by the gene it is carrying). The co-stimulatory molecule CD80 (B7-1) is a good example of the development of a candidate molecule from concept to clinical trials. As discussed, one critical component of the T-cell is the co-stimulatory molecule CD80 (B7-1) interaction with its T-cellligands, CD28 and CTLA4. Increasing the expression of CD80 on tumor cells is therefore one potential means of enhancing anti-tumor T-cell-mediated cytotoxicity. Indeed, most solid tumors, particularly those of epithelial origin, and most human leukemias, lack B7 expression. 75 ,82. To date, various vectors have been utilised to transduce the B7 -1 gene into a variety of murine cell lines including melanoma83 , sarcoma84 , transformed leukemia85 , mastocytoma86 and mammary adenocarcinoma87 • Injection of these B7-1-expressing tumor cell lines into immunocompetent mice can induce MHC-restricted anti-tumor immunity with consequent suppression of tumor growth 83- 86 , protection to subsequent inoculation with wt untransduced (B7-l -) tumor cells 83 ,85,86,88 and, perhaps more clinically relevant, regression of established tumors 83 ,85,86. In some studies the

insertion of the single B7 -1 gene into the target cell results in only a modest anti-tumor response, whereas the insertion of an additional gene results in a substantial synergistic response 89-92. Such combinations with B7-1 include the addition of the genes for yIp93,94, IL_1289-91 and IL_4 92 • Indeed, subsequent clinical trials with a B7-IL12 construct are now planned (AK Stewart, personal communication). Although these models indicate that manipulation of these immunomodulatory pathways can result in substantially enhanced tumor cytotoxicity, there are a number of potential obstacles to this approach. These include: tumor cell heterogeneity within a given tumor; selection pressure for the development of new genetic and structural tumor variants; a large tumor burden and rapid growth fraction which may exceed T-cell cytolytic response; insufficient antigen-specific precursor T-cells; T-cell immune suppression by prior chemotherapy; or downregulation of MHC by prior therapy (eg; prednisone).

Tumor suppressor genes A variety of tumors express abnormal tumor suppressor genes. Conceptually, if the wt (normal tumor suppressor activity) gene is replaced then tumor growth may be retarded. Indeed, Roth et al. recently demonstrated regression of non-small cell lung cancers in a small number of patients following transduction of their tumor with wt p53 gene95 . However the clinical relevance of these findings has been debated because of the lack of suitable control vectors. More importantly however, this strategy may have limited applicability because all tumor cells would need to be transduced with the wt (normal) gene.

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Nonetheless, this strategy may have a place in in vivo gene transfer studies where the tumor is easily accessible, small and/or relatively localised (ie; head and neck cancer, ovarian cancer).

Suicide genes Brain tumors, T-cells and plasma cells have been retrovirally transduced with the 'suicide gene' herpes simplex thymidine kinase (HSTK). This gene produces the inert enzyme HSTK in the target cell. However, when the target cell is exposed to the otherwise nontoxic ganciclovir, HSTK converts it to its toxic metabolite. Furthermore, this metabolite can escape through gap junctions resulting in the so-called 'bystander effect,96-98. Inhibitory proteins An example of this potential approach is in myeloma. In theory, inhibitors of known myeloma 'proliferation factors' could abrogate myeloma cell growth if delivered directly to the marrow microenvironment by genetically modified HSCs. Candidate genes currently thought to be potentially useful in this regard include iX-interferon, soluble interleukin-6 receptor, soluble gp130 (the non-ligand-binding component of the high-affinity IL-6 receptor) and an IL-6 receptor antagonist99- 102 •

REGULATORY ISSUES

There is no doubt that gene transfer has the potential to be a powerful tool for the treatment of a wide variety of diseases, and a great deal of energy (and money) has been devoted to exploring the area. In early studies the safety issues focused primarily on the risk of tumor induction by viral vectors. Although this remains of paramount importance, it is not prohibitive and a growing number of biotechnical companies are producing 'pharmaceutical-grade' vectors for investigators. Consequently the safety issues have focused on safe ex vivo manipulation of human cells. Indeed, as ex vivo cell manipUlation remains the cornerstone for many clinical trials in gene transfer, investigators will need to be prepared for the constraints that are being placed upon them by independent statutory bodies (such as the need for good manufacturing practice facilities to transduce these cells for clinical trials).

CONCLUSION

Numerous studies have demonstrated successful transduction into human target cells with a variety of vectors. Gene marking studies in AML, CML and neuroblastoma have elegantly demonstrated that gene-marked cells can be safely re-infused into patients, can survive and can express the marker genes for long periods of time. Furthermore, they have answered an important biological question, ie; that tumor cells in the autograft do contribute to relapse. However, none of the studies examining the therapeutic benefit of gene therapy has definitively demonstrated a clinically meaningful benefit. Nonetheless, the results of studies involving gene transfer for SCID, COD, melanoma and lung cancer highlight the potential benefit of this strategy. Along the way much has been learnt about gene structure, transcription and control, while the area of can-

cer immunotherapy is expanding exponentially. If gene therapy is to succeed, it will require improvements in vector design, transduction strategies, administration protocols and, importantly, the design of clinical trials. Address for correspondence: H. M. P., Division of Hematology and Medical Oncology, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett St, Melbourne, Vic 3000, Australia.

References

1. Orkin SH, Motulsky AG. NIH Proceedings-Dec 95 Report and recommendations of the panel to assess the NIH investment in research on gene therapy, 1995 2. Wivel NA, Walters 1. Germ-line gene modification and disease prevention: some medical and ethical perspectives. Science 1993; 262: 533-7. 3. Raven PH, Iohnson JB. Biology 2nd edn. St Louis: Times Mirrorf Mosby College Publishing, 1989; The genetic materiaL 4. Avery OT, Macleod CM, McCarty M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J Exp Med 1944; 79: 137-58. 5. Watson ID, Hopkins NH, Roberts JW, Steitz lA, Weiner AM. Molecular Biology of the Gene 4th ed. The Benjamin/Cummings Publishing Company Inc, 1988; The extraordinary diversity of eukaryotic viruses. 6. Temin HM. The Retroviradae New York: Plenum Press, 1992; Origin and general nature of retroviruses. 7. Coffin IM. Virology New York: Raven Press Ltd. 1990; Retroviradae and their replication. 8. Kavanaugh MP, Miller DG, Zhang W, Law W, Kozak SL, Kabat D. et al. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci USA, 1994; 91: 7071-5. 9. Van Zeul M, lohann SV, Closs E, Cunningham J, Eddy R, Shows TB. et al. A human amphotrophic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family. Proc Natl Acad Sci USA 1994; 91: 1168-72. 10. Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell BioI 1990; 4239-42. 11. Porter CD, Collins MKL. Tailor CS, Parkar MH, Cosset F, Weiss RA. et al. Comparison of efficiency of infection of human gene therapy target cells via four different retroviral receptors. Hum Gene Ther 1996; 7: 913-9. 12. Gelinas C, Temin HM. Nondefective spleen necrosis virus-derived vectors define the upper size limit for packaging reticuloendotheliosis viruses. Proc Natl Acad Sci USA 1986; 83: 9211-5. 13. Donahue RE, Kessler SW, Bodine DM, McDonagh K, Dunbar CE, Goodman S. et al. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med 1992; 176: 1125-35. 14. Trapnell BC, Gorziglia M. Gene therapy using adenoviral vectors. Curr Opin Biotechnol 1994; 5: 617-25. 15. Kremer El, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfcr. Br Med Bull 1995; 51: 31-44. 16. Ginsberg HS. The Adenoviruses New York: Plenum, 1984. 17. Chroboczek J, Bieber F, Jacrot B. The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2. Virology 1992; 186: 280-5. 18. Graham PL, Smiley I, Russell wc. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 1977; 36: 59-72. 19. Bett AI, Prevec L. Graham FL. Packaging capacity and stability of human adenovirus type 5 vectors. J Virol 1993; 67: 5911-21. 20. Bett AJ, Haddara W, Prevec L, Graham FL. An efficient and flexible system for construction of adenovirus vectors with insertions and deletions in early regions 1 and 3. Proc Natl Acad Sci USA 1994; 91: 8802-6. 21. Parks RI, Chen L, Anton M, Sankar U. Rudnicki M, Graham FL. A helper-dependent adenovirus vector system: removal of the helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci USA 1996; 93: 13565-70. 22. Michael SI, Douglas IT, Miller CR, Krasnykh V. Hong JS, Engler lA. et al. Strategies to accomplish targeted gene delivery implying

GENE TRANSFER

23. 24.

25.

26. 27. 28. 29. 30.

31. 32.

33. 34. 35. 36. 37. 38.

39.

40. 41.

42.

43. 44. 45. 46.

47. 48.

tropism-modified recombinant adenoviral vectors. Cancer Gene Ther 1995; 321. Wickham TJ, Lee GM, Titus JA, Sconocchia G, Bakas T, Kovesdi I. et al. Tatgeted adenovirus-mediated gene delivery to T cells via CD3. J Virol 1997; 71: 7663-9. Wickham TJ, Catrion ME, Kovesdi I. Tatgeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs. Gene Ther 1995; 2: 750-6. Neve RL, GeHer AJ. A defective herpes simplex virus vector system for gene delivery into the brain: compatison with alternative gene delivery systems and usefulness for gene therapy. Clin Neurosd 1996; 3: 262-7. Turner SL, Jenkins FJ. The roles of herpes simplex virus in neuroscience. J Neurovirol 1997; 3: 110-25. Latchman DS. Herpes simplex virus vector for gene therapy. Mol Biotechnol 1994; 2: 179-95. Toneguzzo F, Keating A. Stable expression of selectable genes introduced into human hematopoietic stem cells by electric field mediated DNA transfer. Proc Natl Acad Sd USA 1986; 83: 3496-9. Wu D, Keating A. Engraftment of donor-derived bone marrow stromal cells. Exp Hematol 1991; 19: 485. Fechheimer M, Boylan JF, Patker S, Sisken JE, Patel GL, Zimmer SG. Transfection of mammalian cells with plasmid DNA by scrape loading and sonicating loading. Proc Natl Acad Sci USA 1987; 84: 8463-7. MacNeil PL, Watder E. Glass beads load macromolecules into living cells. J Cell Sd 1987; 88: 669-78. Mathews KE, Mills GB, Horsfall W, Hack N, Skorecki K, Keating A. Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells. Exp Hematol1993; 21: 697-702. Mathews KE, Keating A. Gene therapy with physical methods of gene transfer. Transfusion Sci 1996; 17: 29-34. Ratajczak MZ, Gewirtz AM. The biology of hematopoietic stem cells. Semin Oncol 1995; 22: 210-7. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood 1993; 81: 2844-53. OrIic D, Bodine DM. What defines a pluripotent hematopoietic stem cell (PHSC): will the real PHSC please stand up! Blood 1994; 84: 3991-4. Salmons B, Gupta C. Tatgeting retroviral vectors for gene therapy. Hum Gene Ther 1993; 4: 129-41. Carter RF, Abrams-Ogg ACG, Dick JE, Kruth SA, Valli, V, KamelReid S. et al. Autologous transplantation of canine long-term matrow culture cells genetically matked by retroviral vectors. Blood 1992; 79: 356-64. Sekhat M, Kotani H, Doren S, Agatwal R, McGatrity GJ, Dunbat CE. Retroviral transduction of CD34-enriched hematopoietic progenitor cells under serum-free conditions. Hum Gene Ther 1996; 7: 33-8. Hatzfeld A, Batatd P, Panterne B, Taieb F, Hatzfeld J. Increased stable retroviral gene transfer in eatly hematopoietic progenitors released from quiescence. Hum Gene Ther 1996; 7: 207-13. Dube ID, Kruth S, Abrams-Ogg A, Kamel-Reid S, Lutzko C, Nanji S. et al. Preclinical assessment of human hematopoietic cell transduction in long-term marrow cultures. Hum Gene Ther 1997; 7: 2089-100. Stewart AK, Prince HM, Cappe D, Chu P, Lutzko C, Sutherland DR. et at. In vitro maintenance and retroviral transduction of human myeloma cells in long-term marrow cultures. Cancer Gene Ther 1997; 4: 148-56. Mitani K, Graham FL, Caskey CT. Transduction of human bone marrow by adenoviral vector. Hum Gene Ther 1994; 5: 941-8. Neering SJ, Hatdy SF, Minamoto D, Spratt SK, Jordan CT. Transduction of primitive human hernatopoietic cells with recombinant adenovirus vectors. Blood 1996; 88: 1147-55. Prince HM, Dessureault S, Gallinger S, Graham F, Sutherland DR, Stewart AK. Adenoviral mediated B7 gene transduction of malignant human plasma cells. Exp Hematol 1998; 26: 27-36. Miller A, D., Skotzko MJ, Rhoades K, Belldegrun AS, Tso CL, Kaboo R. et at. Simultaneous use of two retroviral vectors in human gene matking trials: feasibility and potential applications. Hum Gene Ther 1992; 3: 619-24. Dick JE, Kamel-Reid S, Murdoch B, Doedens M. Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays. Blood 1991; 78: 624-34. Brenner MK, Rill DR, Holladay MS, Heslop HE, Moen RC,

49. 50.

SI.

52. 53.

54. 55.

56.

57. 58. 59.

60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73.

345

Buschle M. et at. Gene matking to determine whether autologous marrow infusion restores long-term haematopoiesis in cancer patients. Lancet 1993; 342: 1134-7. Dilloo D, Rill DR, Grossmann ME, Leimig T, Brenner M. Gene matking and gene therapy for transplantation medicine. J Hematother 1996; 5: 553-5. Brenner MK, Rill DR, Moen RC, Krance RA, Mirro Jr J, Anderson WF. et al. Gene matking to trace origin of relapse after autologous bone matrow transplantation for acute myelogenous leukemia. Lancet 1993; 341: 85-6. Deisserotb AB, Zu Z, Claxton D, Hanania EG, Fu S, Ellerson D. et al. Genetic marking show that Ph + cells present in autologous transplants of chronic myelogenous leukemia (CML) contribnte to relapse after autologous bone marrow in CML. Blood 1994; 83: 3068-76. Brenner MK, Rill DR, Moen RC, Krance RA, Heslop HE, Mirodr J, et al. Gene matking and autologous bone marrow transplant. Annals New York Academy oj Sciences 1994; 1: 204-14. Rosenberg SA, Aebersold P, Conetta K, Kasid A, Morgan RA, Moen R, et al. Gene transfer into humans-immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. New Engl Med 1990; 323: 601. Platsoucas CD, Freedman RS. Tumour-infiltrating lymphocytes in gene therapy. Cancer Bull 1993; 45: 118-24. Merrouche Y, Negrier S, Bain C, Combatet V, Mercatello A, Coronel B. et al. Clinical application of retroviral gene transfer in oncology: results of a French study with tumor-infiltrating lymphocytes transduced with the gene of resistance to neomycin. J Clin Oncol 1995; 13: 410-8. Dunbat CE, CottIer-Fox M, O'Shaughnessy JA, Doren S, Carter C, Berenson R. et al. Retrovirally matked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation. Blood 1995; 85: 304857. Stewart AK, Prince HM, Dube ID. Background and update to a Phase I trial of stem cell gene matking in mUltiple myeloma. Transfusion Sd 1995; 17: 175-84. Munshi NC, Ding LM, Kornbluth J, Naugler S, Saylors R, lyer R. et al. Gene therapy strategies for the treatment of multiple myeloma. Blood 1994; 84 (Suppl 1): 172a Malech H, Maples PB, Whiting-Theobald N, Linton GF, Sekhsatia S, Vowells SJ et al. Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease. Proc Natl Acad Sci USA 1997; 94: 12133-8. Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M. et al. T lymphocyte-directed gene therapy for ADA(-) SCID: Initial trial results after 4 yeats. Science 1995; 270: 475-·80. Koc ON. Transfer of drug resistance benes into hematopoietic progenitors to improve chemotherapy tolerance. Semin Oncol 1997; 23 (SuppJ 1): 46-65. Bertino JR, 'Turning the tables'-normal marrow resistant to chemotherapy. J NaIL Cancer Inst 1990; 82: 1234. Thompson AR. Status of gene transfer for haemophilia A and B. Thrombosis Haemostasis 1991; 66: 1I9~22. Brownlee GG. Prospects for gene therapy of haemophilia A and B. Br Med Bull 1995; 51: 91-105. Connelly S, Kaleko M. Gene therapy for hemophilia A. Thrombosis Haemostasis 1997; 78: 31-6. Dropulic B, Jeang K. Gene therapy for human immunodeficiency virus infection: genetic antiviral strategies and tatgets for intervention. Hum Gene Ther 1994; 5: 927-39. Buchschacher GLJ. Moleculat tatgets of gene transfer therapy for HlV infection. J Am Med Assoc 1993; 269: 2880-6. Lever AM. Gene therapy for HlV infection. Br Med Bull 1995; 51: 149-66. Chen S, Bagley J, Marasco WA. Intracellular antibodies as a new class of therapeutic molecules for gene therapy. Hum Gene Ther 1994; 5: 595-601. Zier KS, Gansbacher B. The impact of gene therapy on T cell function in cancer. Hum Gene Ther 1995; 6: 1259-64. Patdoll D. Immunotherapy with cytokine gene-transduced tumor cells: the next wave in gene therapy for cancer. Curr Opin Oncol 1992; 4: 1124. Urban nJ, Schreiber H. Tumor antigens. Annu Rev Immunol 1992; 10: 617-44. Boon T, Coulie P, Matchand M, Weynants P, Wolfel T, Brichatd V. Important Advances in Oncology. Philadelphia: Lippincott, 1994; Chapter 4, Genes coding for tumor rejection antigens: perspectives for specific immunotherapy.

346

Pathology (1998), 30, November

MILES PRINCE

74. Boon T, Cerottini J, Van den Eynde B, van der Bruggen P, Van Pel A. Tumor antigens recognized by T lymphocytes. Annu Rev Immunol 1994; 12: 337-65. 75. Chen L, Linsley PS, Hellstrom KE. Costimulation of T cells for tumor immunity. Immunol Today 1993; 14: 483-6. 76. Janeway CAJ. The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu Rev Immunol 1992; 10: 645-74. 77. Jenkins MK, Pardoll DM, Mizuguchi J, Quill H, Schwartz RH. T cell responsiveness in vivo and in vitro fine specificity of induction and molecular characterization of the nnresponsive state. Immunol Rev 1987; 95: 113-35. 78. Salvadori S, Gansbacher B, Wemick I, Tirelli S, Zier K. B7-1 amplifies the response to interleukin-2-secreting tumor vaccines in vivo, but fails to induce a response by naive cells in vitro. Hum Gene Ther 1995; 6: 1299-306. 79. Tepper RI, Mule JJ. Experimental and clinical studies of cytokine gene-modified tumor cells. Hum Gene Ther 1994; 5: 153--64. 80. Ellem KA, O'Rourke MG, Johnson GR, Parry G, Misko IS, Schmidt CW. et af. A case report: immune responses and clinical eourse of the first human use of granulocyte/macrophage-colony-stimulatingfactor-transduced autologous melanoma cells for immunotherapy. Cancer Immunol Immunother 1997; 44: 10-20. 81. Dranoff G, Soiffer R, Lynch T, Mihm M, Jung K, Kolesar K. et al. A phase I study of vaccination with autologous, irradiated melanoma cell engineered to secrete hnman granulocyte-macrophage colony stimulating factor. Hum Gene Ther 1997; 8: 111-23. 82. Guinan EC, Gribben JG, Boussiotis VA, Freeman GJ, Nadler LM. Pivotal role of the B7: CD28 pathway in transplantation tolerance and tunior immunity. Blood 1994; 84: 3261-82. 83. Chen L, Ashe S, Brady WA, Hellstrom I, Hellstrom KE, Ledbetter JA. et al. CostimuJation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell 1992; 71: 1093-102. 84. Basker S, Ostrand-Rosenberg S, Nabavi N, Nadler LM, Freeman GJ, Glimcher LH. Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class IT molecules. Proc Natl Acad Sci USA 1993; 90: 5687-90. 85. Matulonis UA, Dosiou C, Lamont C, Freeman GJ, Mauch P, Nadler LM. et af. Role of B7-1 in mediating an immune response to myeloid leukemia cells. Blood 1995; 85: 2507-15. 86. Chen L, McGowen P, Ashe S, Johnston J, Li Y, Hellstrom I. et al. Tumor immunogenicity determines the effects of B7 co stimulation on T cell-mediated tumor immunity. J Exp Med 1994; 179: 523-32. 87. Cayeux S, Beck C, Aicher A, Dorken B, Blankenstein T. Tumor cells cotransfected with iriterleukin-7 and B7.! genes induce CD25 and CD28 on tumor-infiltrating T lymphocytes and are strong vaceines. Eur J Immunol 1995; 25: 2325-31. 88. Hellstrom KE, Hellstrom I, Linsley PS, Chen L. On the role of costimulation in tunior immunity. Ann NY Acad Sci 1993; 690: 225-30. 89. Putzer BM, Hitt M, Muller WJ, Emtage p, Gauldie J, Graham FL. Interleukin 12 and B7-1 co stimulatory molecule expressed by an adenovirus vcctor act synergistically to facilitate tumor regression. Proc Natl Acad Sci USA 1997; 94: 10889-94. 90. Ciuderc B, Zitvogel L, Tahara H, Gilles F, Lotze MT, Robbins PD. Improvement of murine gene therapy using IL-12 by tumour cell stably expressing co stimulatory molecules of the TNF family. Gene Ther Meet Cold Spring Harbor 1996; 69--4F69. 91. Ksander BR, Geer D, Podack ER, Chen PW. Tumor cells transfeeted with B7-1 + IL 12 cDNA induce lasting protective immunity. Cancer Gene Ther 1995; 2: 317-32. 92. Cayeux S, Beck C, Dorken B, Blankenstein T. Coexpression of interleukin-4 and B7.! in murine tumor cells leads to improved tumor rejection and vaceine effect compared to single gene transfectants and a classical adjuvant. Hum Gene Ther 1996; 7: 525-9. 93. Katsanis E, Bausero M, Panoskaltsis-Mortari A, Dancisak BB, XU Z, Orchard PJ. et af. Irradiation of singly and doubly transduced murine neuroblastoma cells expressing B7-1 and producing interferon-gamma reduces their capacity to induce systemic immunity. Cancer Gene Ther 1996; 3: 75-82. 94. Albertini MR, Emler CA, Schell K, Tans KJ, King DM, Sheehy MJ. Dual expression ofhumau leukocyte antigen molecules and the B7-1 co stimulatory molecule (CD80) on human melanoma cells after particle-mediated gene transfer. Cancer Gene Ther 1996; 3: 192201. 95. Roth JA, Nguyen D, Lawrence DD, Kemp BL, Carrasco CH, Ferson

DZ. et af. Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat Med 1996; 2: 985-91. 96. Dilber MS, Abedi MR, Bjorkstrand B, Christenssen B, Gahrton G, Xanthopoulos KG. et al. Suicide gene therapy for plasma cell tumours. Blood 1996; 88: 2192-200. 97. Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM. In vivo geue transfer with retroviral vector producer cells for treatment of experimental brain tumours. Science 1992; 256: 1550-

2.

98. Ram Z, Culver KW, Oshiro EM, Viola JJ, DeVroom HL, Otto E. et al. Therapy of malignant brain tumors by intramural implantation of retroviral vector-producing cells. Nat Med 1997; 3: 1354-61. 99. Dunbar CE, Nienhuis AW. Multiple myeloma: new approaches to therapy. J Am Med Assoc 1993; 269: 2412--6. 100. Sporeno E, Savino R, Ciapponi L, Paouessa G, Cabibbo A, Lahm A. et al. Human Interleukin-6 receptor super-antagonists with high potency and wide spectrum on multiple myeloma cells. Blood 1996; 87: 4510-9. 101. Saggio I, Ciapponi L, Savino R, Ciliberto G, Perrieaudet M. Adenovirus mediated gene transfer of an IL6 antagouist. Gene Ther Meet Cold Spring Harbor 1996; 270. 102. Klein B, Xue-Guang Z, Zhao-Yang L, Bataille R. Interleukin-6 in human multiple myeloma. Blood 1995; 85: 863-72.

APPENDIX: Glossary of Terms Adeno-associated virus

AAV

Adenovirus

Ad Capable of transducing cells across species (cf; xenotrophic which is species specific).

Amphotrophic vector

Antigen-presenting cells

APCs

Chronic granulomatous disease CGD Cytotoxic T lymphocytes

CTL

The transfer of a gene of interest into a target cell for the purpose of producing a therapeutic effect. Transfer of genetic information into cell. (Transduction usually implies the use of a 'conduit' vector whereas transfection does not. However, the terms are often used interchangeably).

Gene therapy

Gene transduction

The transfer of a gene of interest into a target cell.

Gene transfer Hematopoietic stem cell

HSC

Insertion of a foreign gene into the host genome which subsequently functions as an oncogene or as a promoter to oncogenes with resultant clonal proliferation.

Insertional mutagenesis

Inverted terminal repeat

ITR

Interleukin

IL

(aka: reporter gene) Gene that can be used to easily trace the fate of successfully transduced target cells (ie; the gene for neomycin resistance).

Marker gene

Major histocompatibility complex

MHC

Moloney murine leukemia virus

MMLV

Neomycin resistance gene

neo

Producer cells Provirus

Common retroviral vector. A commonly used marker gene to trace the fate of successfully transduced target cells. Cells which provide structural protein genes for virion. Genomically integrated viral DNA.

GENE TRANSFER

Replication-competent adenovirus

RCA

Replication-competent retrovirus

RCR

Replication-defective adenovirus

RDA

Adenovirus that is capable of infecting a target cell and remains capable of further replication. Retrovirus that is capable of infecting a target cell and remains capable of further replication. Adenovirus that is capable of infecting a target cell but incapable of further replication.

347

Replication-defective retrovirus RDR

Retrovirus that is capable of infecting a target cell but incapable of further replication.

Reporter gene

See marker gene.

Severe combined immunodeficiency

SCID

T -cell receptor

TCR

Vector Wild-type

wt

Vehicle for transferring gene of interest into cell. Naturally occurring type (ie; gene, virus).