Gene transfer and antisense nucleic acid techniques

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Gene Transfer and Antisense Nucleic Acid Techniques N. Miller and R.G. Vile Attempts to suppress a harmful genetic trait by antisense means, OY to restore a normal phenotype by gene transfer, attract much publicity. This is especially the case where clinical trials incorporating such methodologies have been initiated, such as antisense oligonucleotide therapies for some types of leukaemia, an tisense gene-transfer therapy for a form of lung cancer, and gene-transfer therapies for adenosine deaminase deficiency, severe combined immunodefciency disease, and various forms of cancer including brain tumours and melanoma. However, translation of laboratoy success into treatment OY control of disease is unlikely to be straightforward. Here, Nick Miller and Richard Vile summarize the rationale, problems and potential of such techniques as applied to parasitic disease. Research aimed at developing new antiparasite chemotherapies has conventionally focused on the hope of identifying as a target a biochemical pathway that is unique to the parasite; this requires not only an immense amount of groundwork to identify the pathway, but also a degree of serendipity (as there is no guarantee that exploitable biochemical differences between host and parasite actually exist). In addition, there is no defence against the development of drug resistance by parasite populations. A significant advance would be the development of strategies that can bypass the secondary, phenotypic characteristics of metabolism and strike directly at the genotype or at the immediate consequences of transcription, where differences between host and parasite are easily identified and probably impossible for the parasite to disguise. Although antisense strategies have this potential, the realistic extent of their applicability to the treatment or prevention of parasitic disease is debatable. In-depth reviews of antisense techniques have already been publishedi-4; this article is intended to summarize such approaches and their possible application to parasitic disease. In addition, it may be possible to alter the host’s susceptibility to parasites or the parasites’ susceptibility to therapeutic or immunological agents by genetic alteration of host or parasite, respectively, and this article also considers the potential of gene-transfer strategies as antimagsparasite therapies or prophylactics. Antisense theory Therapeutic approaches generally rely on antisense nucleotide sequences being synthesized and delivered to the target cell. Binding of antisense to sense sequences may then occur at one or other of various

Nick Miller and Richard Vile are at the Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, UK WC2 3PX.

stages in the pathway between DNA and protein; sufficiently stable binding can result in blocking the protein expression of a particular gene. Theoretically, the natural process of recognition of complementary nucleotide sequences can be used in this way to disrupt expression of any gene for which the sequence is known. Where a protein is essential to the survival of a parasite, its antisense-mediated ablation would represent a potential therapeutic approach to the disease caused by that organism. But, however great the theoretical attraction of antisense therapeutics, they must first fulfil the same practical criteria required of all antiparasite chemotherapies; that is, they must encompass: (1) a target, ie. a structure or biochemical characteristic that is limited to the parasite; (2) a therapeutic agent, ie. an agent that can recognize the target and that has a specific, or at least localized, toxic or biostatic effect where recognition occurs; and (3) a delivery system that promotes the maximum contact between target and therapeutic agent. The criteria of a target and a therapeutic agent are exquisitely fulfilled by sense and antisense sequences, respectively, but an adequate in vivo delivery system remains a fundamental problem in the practical application of antisense therapeutics. Antisense DNA Antisense DNA strategies in the context of parasitic disease have been reviewed recentlys. These techniques involve the use of short sequences of synthetic DNA that are compatible with naturally occurring sense sequences in the pathogenic cell; RNA, especially mRNA, is the usual target for such antisense oligodeoxynucleotide (ODN) sequences. Binding of antisense ODNs to target RNA occurs via Watson-Crick base pairing, and can inhibit translation by incompletely understood means that may include steric hindrance of ribosomes or increased susceptibility of the RNA:DNA duplex to cleavage by RNase He. The ubiquitous presence of nucleases in vivo requires that all realistic therapeutic strategies involving antisense ODNs employ DNA analogues such as the methylphosphonates, which combine the same binding specificity with increased nuclease resistance. The use of such analogues generally precludes cleavage by RNase H (which does not usually recognize duplexes formed with DNA analogues), but allows the incorporation of residues that confer various advantageous characteristics on the ODN. For instance, the addition of poly-L-lysine to the 3’ end of the oligonucleotide can improve its stability and uptake by eukaryotic cells7. Other modifications have resulted in the class of ‘active’ analoguess such as those that possess crosslinking agents (resulting in 0

1994,ElsewrSmnce Ltd

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more-permanent binding) or catalytic moieties that actually cleave the RNA target. The latter group of analogues represents an extremely promising avenue, as just one catalytic antisense strand could, in theory, destroy several RNA target molecules. The application of antisense ODNs to parasite RNA has been most actively explored in systems of mammalian cells parasitized by virusess. In cells infected with herpes simplex virus, addition of antisense methylphosphonates complementary to viral RNA inhibited viral but not cellular protein synthesis, resulting in an up to 98% decrease in virus titres9. Similarly, in human cells infected with hepatitis B virus, in vitro, application of antisense sequences resulted in up to 96% inhibition of expression of the hepatitis B surface antigen10 and there has been some success with blocking human immunodeficiency virus (HIV)11 and influenza virus.12 replication, in vitro, by means of antisense ODNs targeted against viral RNA. However, such data concern cells grown in culture rather than animal models, and therefore may be of limited relevance in viva. The miniexon sequence. There have also been attempts to apply these strategies to some of the parasitic protozoa. It is now well established that most or all mRNAsl3 from Leishmaniu and Trypanosoma have at their 5’ end a 39 nucleotide sequence14 that is identical in all members of a given species. This led to suggestions that ODNs targeted against this ‘miniexon’ sequence might inhibit the expression of virtually the entire genetic complement of the parasite, and indeed such antisense sequences not only suppress translation of trypanosomal mRNAraJsJ6 in vitro, but also can kill T. brucei*7. In the latter study, the antisense sequences were coupled. to an intercalating agent (an acridine derivative), which had the dual property of promoting stable binding between the ODN and the target sequence and of promoting uptake of the oligonucleotides by the parasites. ODNs that did not carry an intercalating agent were ineffective. The presence of such ODNs in the medium resulted in the death of T. brucei in about 40 h. However, to prevent rapid degradation of the ODNs, this experiment was performed in serum-free medium, in which trypanosomes do not divide’8. Also, the trypanocidal effect was observed only where the ODN concentration was 80 ~J,Mor morel7 and it may not be possible to achieve this concentration in vertebrate tissues without nonspecific side-effects. For miniexon-complementary antisense sequences to have any therapeutic potential, the ODNs would either have to be imbued with nuclease resistance by modifications of the sort already discussed, or be protected from enzymes (eg. by encapsulation in liposomes; see below). It remains to be seen if the antisense sequence can have a deleterious effect on a healthy, dividing trypanosome population, ie. in the presence of serum. In any case, the substantial hurdle of adequate delivery would still remain. Targeting translation initiation. There have also been attempts to apply antisense approaches to Plasmodium. One report19 showed that ODNs targeted against the translation initiation site or a coding region of the dihydrofol.ate reductase-thymidine synthase gene (DHFR-TS) of P. fulcipurum could inhibit translation of the DHFR-TS mRNA, in vitro. However, significant translation *arrest was achieved only at

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high concentrations of ODN, ie. 45->200~~. More encouraging data 20 show that nuclease-resistant antisense DNA analogues are effective in sub-micromolar concentrations against the growth of Plasmodium, in vitro. Human erythrocytes grown in culture and infected with P. falcipurum were used; the antisense sequences were targeted either to the DHFR-TS gene or to the ~195 gene (which encodes the precursor common to three major merozoite surface antigens). All such sequences had antimalarial properties as measured by the death of parasites in vitro, and by the decreased percentage of infected erythrocytes in culture. Again, there remains the problem of delivering the ODNs to a sufficient number of infected host cells, in zko, to reverse falciparum malaria; this issue is fundamental to any realistic application of antisense therapies. Topical application. Antisense strategies have not yet been directed against living Leishmuniu cells, but these organisms may, in some cases, be more amenable to such techniques than Plasmodium or African trypanosomes. Antisense should be particularly applicable to cases of cutaneous leishmaniasis where the lesion is quite discrete, since the restriction of the majority of the parasite population to a confined and relatively accessible area would minimize the problem of delivery of antisense constructs. Topical application of formulations containing conventional antiprotozoals has been shown21 to be beneficial in such cases; since some DNA analogues are thought to be capable of dermal penetration22, antisense agents might also be suitable for external application. More significantly, a case of human visceral leishmaniasis has been cured by treatment with amphotericin B encapsulated in liposomes23, indicating that this approach can deliver the antibiotic to a sufficient number of parasites to reverse the disease. Liposome-mediated delivery is useful in the leishmaniases [and possibly in other infections of the reticuloendothelial system (RES)24] as the parasitized cell (the macrophage) preferentially takes up such vesicles; encouraging results have also been obtained with amphotericin B liposomes in a mouse model of T. cruzi Y strain infection25. Encapsulation of antisense sequences or therapeutic genes (see below) rather than antibiotics would avoid any problems of emergence of drug resistance and of toxicity to uninfected macrophages. Therefore, antisense therapeutics may be of genuine clinical use for the leishmaniases. Another parasite that could offer fewer problems than most in terms of efficient delivery of the antisense agent is Giurdiu lumbliu, in view of its extracellular habit and its restriction to the upper small intestine; if a solution of ODNs resistant to gut conditions could be applied to this area, perhaps antisense sequences could be delivered to every single parasitic cell. Other gut-living parasites might be susceptible to this approach for similar reasons. The effect of binding of ODNs to target sequences is likely to be transitory, as an active gene will be continually producing transcripts, and there will only be a finite population of ODNs of finite lifetime delivered to the target cell. Where RNA is the target, it is necessary that the protein which it encodes is so crucial for the survival of the parasite that its ablation or downregulation is rapidly lethal (such considerations

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Double helix

ODN

5

5

Homopyrimidine

a

!

3

IE

G

T:AT and C:GC

A G 3

A G A

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G A G

iomopurine !

! A

4:AT and G:GC

G A G A 5

G

T

A

T

A

G G T i

c-4

A

d

IC

T

IA

Mixed ODN

JA

G

G

A

A

G

G

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A G

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5

Fig. 1. Mechanisms of triple helix firmotion. An extended pine sequence (A or G) in the duplex may be specifically bound by complementary pine or pyrimidine oligodeoxynucleotides(ODNs), firming in general A:AT/G:GC or T:AT/C:GC triplets, respectively. Note that pyrimidine ODNs bind parallel to the target sequence, whereos purine ODNs are ontiporal/el with respect to the target This might allow the design of purin+yrimidine ODNs that con switch between different strands of the duplex to bind to different homopurine runs’? as shown.

may not be a problem in the exceptional case of the trypanosomatids, where, as has been discussed, the presence of a common sequence in all transcripts seems to allow a single type of antisense ODN to interfere with synthesis of all proteins). Transcripts of genes that are responsible for parasite metabolism or drug resistance are obvious candidate targets. More interesting possibilities are those gene products responsible for the pathogens’ ability to evade the immune system, where modest downregulation (rather than complete ablation, which is difficult to achieve) of the appropriate protein might be sufficient, in some cases, to tip the balance in favour of the host’s defences.

Triple helix formation. It is also possible to suppress gene expression by direct targeting of the DNA duplex by designing ODNs to bind specifically to certain regions of the duplex, thus forming a triple helix4. Stable formation of triplex structures in essential promoter or coding regions of a gene could, theoretically, block expression of that gene. Indeed, in cell-free systems, a triple helix in a eukaryotic promoter can block the binding of endonucleases and of a transcription factor specific to that region27. Triple helices can be formed in one of two ways, both of which at present require the target region of the DNA duplex to contain a homopurine strand, and both of which involve binding via Hoogsteen rather than Watson-Crick hydrogen bonding. In the first case, a homopyrimidine ODN can bind to the duplex by specific recognition of the duplex base pairs AT and GC, giving T:AT and C:GC triplets28; for the latter triplet to form, the cytosine must be dissociated, thus cytosine-containing ODNs only form triplexes at acid pH. Homopyrimidine ODNs form triplexes by lying in the major groove of DNA parallel to the homopurine tract of the duplex. Alternatively, a homopurine ODN may also form a triple helix by specific recognition of base pairs, in this instance usually forming the triplets G:GC and A:AT29; such triplexes form independent of pHs0, as cytosine residues are not found on the ODN. Homopurine ODNs also lie in the major groove of DNA, but are antiparallel with respect to the homopurine tract of the duplex (see Fig. 1). The great attraction of the triple helix approach is that theoretically only one copy of the antisense sequence per copy of the targeted gene need reach the pathogenic cell to shut off production of the encoded protein. This approach also avoids natural compensatory mechanisms (such as an increase in transcription rate or a decrease in mRNA degradation rate) which are inherent problems where RNA is the target. The application of triple helix strategies in viva has been hampered by a number of factors: mainly the lack of stability of the triple helix under physiological conditions; and the requirement for an extended homopurine sequence in the target region of the duplex. However, these restrictions may be surmountable. For instance, the incorporation of chemical analogues in the homopyrimidine ODN sequence can obviate the requirement for low pH31. Also, incubation of T cells with an oligonucleotide designed to form a triple helix at the promoter region of the interleukin 2 (IL-2) Ra gene was found to suppress transcription of IL2Ra32, thus demonstrating the possibility of the triple helix approach in living cells. Another interesting study showed triplex formation, albeit only in an intramolecular context, by an ODN that consisted of alternate homopurine and homopyrimidine sequences 33. This greatly extends the theoretical range of possible duplex targets for triple helix formation by potentially obviating the need for an extended homopurine sequence in the target DNA. No-one has yet attempted to apply triple helix technology to parasite-host systems. While the problem of delivery of the ODN to each parasite remains, the advantage of the triple helix approach lies in the greatly reduced number of antisense molecules needed to reach each parasite. A potential drawback is the necessity for the antisense sequence to reach the

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nucleus; thus, where a parasite is intracellular, the antisense sequence woald have to cross host cell membranes, parasite cell membranes and parasite nuclear membranes in order to reach its target. Also, triplex formation would need to mediate its effect prior to division of the -target cell (as the third strand would not be duplicated along with the target DNA), and it should be emphasized that the extent of accessibility to ODNs of DNA in chromatin is not clear. In summary, there are still substantial technical obstacles to overcome before triple helix strategies reach the clinic. Antisense RNA Where the appropriate antisense RNA sequences can be generated in z&o, they can in theory be applied to the cells directly, like DNA analogues. However, since naked RNA would be rapidly degraded, and since nuclease-resistant analogues are not readily available, the use of antisense RNA in this way is strictly limited to delivery systems that somehow protect the RNA (eg. by encapsulation of the RNA into liposomes). Another approach is to integrate into the pathogenic genome a sequence of DNA that is the strand complementary to the sense sequence encoding the protein that is to be ablated, but in the reverse orientation, and under the control of a strong promoter element. Constitutive transcription of this antisense gene gives rise to antisense RNA sequences that may (where the two RNA species end up in the same cellular compartments) bin.d to the wild-type sense RNA sequences, thus hindering translation of a specific protein. While the insertion of the foreign DNA, in vitro, may be accomplished by any of the conventional methods of gene transfer, the application of this methodology, in vivo, is likely to rely upon vectors such as engineered viruses or targeted liposomes (see below). For instance, viral vectors have been used to target viral RNA; an adenovirus-associated vector was used to integrate into mammalian cells a stretch of DNA encoding antisense RNA complementary to the long terminally repeated (LTR) sequence present on the ends of all HIV-l transcripts. These cells subsequently showed marked inhibition of production of infectious HIV particle+. Transfer of exogenous $;enes to parasite cells Gene-transfer strategies involve the insertion of a functional gene into the target cell with consequent alteration of the phenotype of the cell. Either parasite or host cells could be targeted for genetic alteration. In the parasite, the exogenous gene would be chosen to have a lethal effect; in the host, the gene could be designed either to have a toxic effect (on infected host cells) or a protective effect (on uninfected cells). Such protection could be achi.eved either passively, by preventing recognition or attachment by the parasite, or actively, by enabling host cells to produce a factor toxic to the parasite. There are few studies describing the genetic transformation of parasitic protozoa, due to the absence (until recently) of suitable transforming vectors. Such vectors are now available and have been shown to allow gene transfer to some protozoans in culture; clearly the next step is to develop vectors that allow

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gene transfer in vivo, ie. in an infected host, as this is a prerequisite for the therapeutic application of genetic modification of parasites. However, much useful groundwork is being done using the in vitro protocols now available. Transient expression of foreign genes such as bacterial chloramphenicol acetyltransferase has been achieved in Leptomonas seymouri35, Leishmania spp36 and Toxoplasma gondii37. Such studies provide experimental systems that allow the identification of promoter elements that are constitutively active, ie. that continually facilitate transcription of any appropriately positioned adjacent gene, in parasite cells but not in host cells. This kind of deletional analysis has begun for Leishmania38. Specific promoter elements seem likely to exist for the parasitic protozoa in view of the genetic oddities of many of the members of this group (eg. higher eukaryotic polyadenylation signals do not seem to be functional in Leishmania39). Genes regulated by parasite-specific sequences would be expressed only in parasite cells and not in host cells; this would allow the therapeutic application of genes encoding toxic products. Such ‘suicide genes’ include the herpes simplex virus thymidine kinase (TK) gene and the bacterial cytosine deaminase gene. These encode enzymes that can convert non-toxic prodrugs to lethal products (eg. TK initiates the conversion of the prodrug ganciclovir to an agent which disrupts DNA synthesisJO). Thus, treatment of an infected host with vectors that deliver the TK gene driven by a parasite-specific promoter, so that TK is expressed only in parasite cells, followed by systemic treatment of the host with ganciclovir, would result in selective killing of the parasite cells. (This approach effectively targets cancer cells while avoiding damage to normal bystander cell+.) No-one has yet attempted to express suicide genes in parasitic protozoa. Where the product of the therapeutic gene is rapidly or irreversibly toxic, transient expression will be adequate to kill the parasite. In many cases, it may be that optimal therapy requires stable expression. Such long-term expression of exogenous DNA has been demonstrated for some parasites (eg. Leptomonas‘Q, Leishmaniu38,43 and trypanosomes44-46), in vitro. Transformation of T. brucei was achieved by the use of DNA vectors containing trypanosomal sequences: in one case, the neomycin-resistance gene (neon) was flanked by 5’ and 3’ elements of the I$ tubulin gene clustel”‘5; in another, by 5’ and 3’ elements of the calmodulin locust; and in the third approach, neoR was driven by a known trypanosomal promoter element (the procyclic acidic repetitive protein promoter) and flanked by non-coding elements of the trypanosome genome4. Such approaches allowed stable integration of the minigene into the trypanoby homologous recombination. somal genome (Homologous recombination involves the integration of DNA into the genome at specific points where the genome has sequences in common with the vector DNA, and has been irrefutably demonstrated in L. enrietti47.) That parasitic protozoans are readily transformed by a mechanism involving homologous recombination is exciting for treatment of disease. In one case4, stable transformation of T. brucei was achieved using a minigene flanked by non-coding sequences of the

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parasite genome, so that homologous recombination would result in integration at a site that did not involve disrupting normal genes. But for antiparasite approaches, the disruption of essential genes would be desirable. Therefore, a vector could be designed in which the therapeutic gene was flanked by sequences compatible with, for example, a gene involved in drug resistance. Such a vector could confer susceptibility to one drug while suppressing resistance to another drug; these drugs would then be expected to act synergistically in the treatment of an infected host. More subtle interactions between the effects of knocking out a wild-type gene and inserting an exogenous gene can be imagined. The application of gene-transfer techniques for the relief of disease is still distant. The major barrier is again the impossibility, with present technology, of delivering a gene to every parasite in an infected host. This problem might be overcome by the use of viral vectors. Retroviruses are well established as vectors for the transfer and expression of DNA in mammalian cells48, and other types of virus are starting to be used; for safety reasons, strains of virus are employed that can infect cells but cannot replicate (replication incompetent). The application of similar techniques for the transfer of antisense genes to parasites depends on the development of similar vectors capable of infecting protozoan cells, preferably exclusively. Possible candidate vectors have been identified for Leishmania@,sQ, Giardiasl, Trichomonas52, Entamoebas3, Eimerius4 and Babesias5 and could be developed into important therapeutic agents in the future. Viruses that show specificity for parasite cells rather than for cells of the vertebrate host are of especial interest (eg. T. vaginalis virus cannot infect other trichofor monads and G. Zamblia virus is non-infective other protozoa and for mammalian cell line@). This specificity could eventually allow administration of a replication competent virus carrying a therapeutic gene (eg. a suicide gene or an antisense gene producing antisense transcripts complementary to parasite RNA). Such a virus could in theory infect every parasitic protozoan in a parasitized vertebrate host, resolving the problem of delivery of the antisense sequence to most or all of the target cells; the restriction of the virus infection to protozoan rather than mammalian cells would need to be absolutely established prior to taking this route. (The therapeutic gene could be constructed so that it is under the control of parasite-specific regulatory elements, as described earlier; even if a virus carrying such a gene infected mammalian cells, the gene should not be active and so host cells would not be harmed. However, this should be regarded as a desirable additional precaution, not as a substitute for using a virus to which mammalian cells are demonstrably refractory.) A single administration of a replication-competent protozoan virus-derived therapeutic vector might not deliver the gene to every parasite, as the host immune system would probably clear virus relatively rapidly; the strategy might therefore be most effective in localized rather than disseminated infections, or in conjunction with transient immunosuppression of the host. Experiments using animal models are urgently needed in this area.

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Another way of delivering genes is by the use of liposomes57,58. Liposomes are artificially produced vesicles consisting of a phospholipid bilayer enclosing an aqueous compartment, and may be manufactured so that they carry agents such as antibiotics or DNA. The primary drawback of liposome-mediated delivery, in viva, is the preferential uptake of liposomes by macrophages of the RES (although this can be modulated to a certain extent by altering the make-up of the liposome membranesv) and the limited extent of liposome extravasation. However, these characteristics can be turned to advantage, for instance where macrophages are the target cells; hence the great promise, mentioned earlier, of liposome-mediated therapies in the leishmaniases. One strategy for increasing the uptake of liposomes by a particular tissue involves the incorporation into the lipid membrane of antibodies or other molecules to confer specificity for the target tissueGO. Thus, binding of liposomes to the hepatocyte membrane can be promoted by inclusion of lactosylceramide into the liposome membrane58 as the terminal galactose of the glycolipid is bound by a hepatocyte lectin. Such systems might allow the targeting of the liver stage of Plasmodium. Binding of the liposome to the target cell is only the first step; frequently, such binding merely results in internalization and degradation of the liposome and its cargo via receptor-mediated endocytosis. Effective delivery of the cargo to the cytoplasm of the infected cell requires fusion of the liposome membrane with the target cell membrane. This phenomenon can be promoted by the inclusion into the liposome membrane of viral envelope proteins; the resultant vesicles are known as proteoliposomes. The viral envelope proteins not only confer the tropism displayed by the virus from which they were derived, but also promote fusion of proteoliposome and cell membranes, thus permitting delivery to the cytoplasm of undegraded vesicle contents. Study of the viruses of the protozoa may reveal proteins that can be used to construct proteoliposomes capable of targeting extracellular parasites. Where liposomes are intended to mediate the therapy of intracellular parasites, they must be targeted against the host cell type for which the parasite is tropic. Recently it has been shown61 that intravenous administration of untargeted liposome-DNA complexes in mice results in the exogenous DNA being expressed primarily in heart and lung, with the DNA in one case being localized only in the heart. Therefore, even the use of untargeted liposomes could be of value in delivery of therapeutic agents to T. cruzi when intracellular in cardiac muscle. Even with specific targeting, there will still be significant uptake of liposomes by the RES; it is important therefore that the agent carried by the liposome has an effect limited to the parasite or, via a bystander effect, to the infected host cell. Such approaches would therefore require the use either of antisense nucleotides specific to parasite sequences or of therapeutic genes under the control of parasite-specific elements, so that although the liposomes would adhere to both healthy and infected cells, toxic effects would be limited to infected cells. The application of gene-transfer technology in human therapies rightly proceeds with caution. This is

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largely due to theoretical safety considerations (eg. random integration of DNA into the genome could result in malignant transformation by insertional mutagenesis). However,, where the delivery system can be accurately targeted to the parasite, the possibilities of such harmful side effects for the host are much decreased. A thorough working of the (presently embryonic) field of protozoan virus research might turn up efficient vectors for the specific delivery of functional genes to eukaryotic parasites. For instance, protozoan retroviruses might exist that will allow the stable integration of exogenous genes into the parasite genome; the possibility of discovering a retrovirus that possesses, or can be engineered to possess, a parasite-specific tropism should be sufficient reason for pushing substantial human and financial resources into this research area. Conclusion Antisense strategies have been heralded as a new class of therapeutic agent. There is no doubt that antisense techniques can h,ave dramatic effects in vitro, and in this context their potential as research tools for the study of parasites in culture has only just begun. Their application to parasite disease in vim is likely to be limited to that set of infections where the natural history of the disease mitigates the fundamental problem of delivery of the therapeutic agent to most or all of the pathogenic cells in an infected host. In these cases, however, antisense techniques offer real hope as treatments that may suffer neither from toxicity to the host nor from the eventual development of resistance by the parasite. Similarly, most theralpies based on gene transfer will require delivery of the gene to nearly every parasite in a given infection. This might be attainable in some cases by continuous or protracted treatment with gene delivery vectors such as proteoliposomes or replication incompetent virus. Modification of protozoan viruses to produce an infective agent carrying a therapeutic gene could be an ideal solution since administration of a small inoculum might result in the delivery of the gene to each parasite in a vertebrate host. Large-scale production of such viral DNA could result in ,a product cheap enough to supply to Third World countries. Acknowledgements We would like to thank Sina 9orudi for animated discussions, and Peter Billingsley and Simon Croft for commenting on initial drafts of this article. References 1 Crooke, S.T. (1993) FASEB j’. 7,53%539 2 H&ne, C. and Touhn~, J.J. (1990) Biochim. Biophys. Acta 1049, 99-125 3 Agrawal, S. (1992) Trends Biotechnol. 10,152-158 4 Maher, L J. (1992) BioEssuys 14,807-815 5 Sartorius, C. and Franklin, R.M. (1991) Parasitology Today 7, 90-93 6 Murray, J.A.H. and Crockett, N. (1992) in Antisense RNA and DNA (Murrav, T.A.H., ed.), DD l-49, Wilev-Liss 7 Degok, G. e’t aI. (1942) k’kisense RfiA and DNA (Murray, J.A.H., ed.), pp 255-265, Wiley-Liss 8 Toulm6, J.J. (1992) in A&sense RNA and DNA (Murray, J.A.H., ed.), pp 175-194, Wiley-Liss 9 Kulka, M. et al. (1989) Proc. Nutl Acud. Sci. USA 86,6868-6872 10 Goodarzi, G. et al. (1990) J. Gen. Viral. 71,3021-3025 11 Lisziewicz, J. ef al. (1993) Proc. Nutl Acud. Sci. USA 90,

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