WO2000009731A1 - Transgenic parasites as gene therapy agents - Google Patents

Abstract

This invention relates to a transgenic eukaryotic vector organism comprising a heterologous coding sequence which encodes (a) a therapeutic polypeptide, or (b) a polypeptide capable of generating a therapeutic agent, which vector organism is capable of living non-pathogenically within a mammalian or avian host organism, and of expressing the coding sequence as a therapeutic polypeptide.

Description

TRANSGENIC PARASITES AS GENE THERAPY AGENTS

FIELD OF THE INVENTION

The invention relates to organisms which can generate therapeutic agents within mammalian or avian hosts. These may be polypeptides, including proteins and glycoproteins, or non-polypeptide molecules. The invention further relates to uses of such organisms for therapy, including preventive therapy.

BACKGROUND TO THE INVENTION

The invention relies upon the use of organisms that, in nature, live in close association with a host, such as symbiotes, commensals or parasites, and which act as vectors of genetic information and thereby synthesis e and release one or more therapeutic agents whilst resident within a mammalian or avian host.

Many methods of delivering therapeutic agents already exist. Some of these are based on direct administration. Such methods include local injection of material formulated to provide sustained release, use of medical apparatus designed to provide sustained or programmed infusion from an external source via an in-dwelling intravascular or subcutaneous cannula. However, these methods typically demand frequent expert supervision and only give an effective concentration of therapeutic agent in blood or tissue for a limited period, especially when the active principle is an autocoid, cytokine or other labile cell product.

Other methods rely on the delivery to the host of nucleic acids that encode a therapeutic polypeptide and are expressed in vivo.

For example, direct application or injection of naked or encapsulated DNA can effect transfection locally but inconsistently. Viruses (e.g. retroviruses, adenoviruses and adeno-associated viruses) are therefore prominent as prospective vectors for introduction of genes into man. A characteristic of viral vectors is irredeemable modification of the genome, a process whose long-term consequences cannot be anticipated but may not be inconsequential. For instance, T-cell lymphomas have occurred in primates following use of a gene therapy based upon a retrovirus that was formerly thought to cause such pathology only in rodents. Although there are projects to develop therapies for a range of diseases use of viral vectors may ultimately be limited to tumour therapy for this reason.

A further deficiency of viral vectors is the limited quantity of heterologous DNA that can be incorporated. This may preclude use of human promoters and other endogenous regulators that may be needed for expression and secretion.

Also, viral vectors have a tendency to induce immune reactions and, by becoming incorporated into the genome, may activate latent genetic diseases.

Still other methods involve delivery of eukaryotic cells, typically mammalian host cells or other cells, modified so as to effect sustained secretion of a therapeutic agent, to the host. Generally, this approach suffers from the drawback that heterologous cells provoke an immune response. Two methods are available for avoiding this immunological rejection of infused eukaryocytes: (i) use of leukocytes freshly collected from individual recipients; and (ii) use of cultured stem cells. The first of these approaches, collection of peripheral blood leukocytes for re-infusion after genetic manipulation, has been shown to be effective in man. Being customised to individual patients, this method is not amenable to widespread use and fails to circumvent non-immunological mechanisms that eliminate infused leukocytes that sequester at inappropriate loci (c.f. dissolution of trophoblast fragments which shower into the maternal circulation in late pregnancy). Modified stem cells provide an alternative to peripheral blood leukocytes, but have the defects of not being amenable to elimination and lacking episomal stability if clonal expansion occurs. Alternatively, eukaryocytes can be placed at an immunologically privileged micro-environment (e.g. within semi-permeable containers or gels). These procedures, however, are not established as being suitable for general application.

Also, there is considerable concern that viral vectors and cultured stem cells might compromise recipients by inadvertent introduction of human pathogens (e.g. of passaged viruses or prions) or activation of genetic diseases (for instance by activation of oncogenes). Furthermore, there is concern over the possibility of transmutation of viral vectors into novel pathogenic organisms.

Therefore, it is clear that all the presently available methods of delivering polypeptide therapeutic agents to hosts suffer from serious drawbacks, either because their therapeutic effectiveness is limited, because they involve a risk of pathogenic side effects, or because they are difficult to apply generally. The present invention seeks to overcome these difficulties.

SUMMARY OF THE INVENTION

The invention provides a new alternative means of delivering therapeutic polypeptides and therapeutic products generated in vivo by the action of polypeptides to hosts. It takes advantage of:

(a) the capacity of certain parasites, symbionts and commensals to reside in their definitive host for prolonged periods unaffected by either immunological or non- immunological defence mechanism; and

(b) the ability of parasites in unusual circumstances to coexist with the host as a commensal.

Expression of a genetic construct encoding a suitable therapeutic polypeptide, or a polypeptide capable of generating in vivo a therapeutic product, in vector organisms which combine characteristics (a) and (b) converts an ordinarily parasitic organism into a commensal or symbiont which produces the therapeutic agent, for example a protein or glycoprotein, or a non-polypeptide therapeutic product generated by the encoded polypeptide, in vivo. Thus the present invention provides such transgenic vector organisms as a means of delivering therapeutic agents which are polypeptides, including proteins and glycoproteins, or agents generated in vivo by the action of polypeptides.

The present invention seeks to overcome the problems listed above, and the vector organisms of the invention do not suffer significantly if at all, from the above- mentioned deficiencies of conventional methods. In particular, no known human pathogens are associated with the vector organisms of the invention, perhaps because their complex life-cycles include replication within non-mammalian species. This distinguishes them from both viruses and eukaryotic cells as means of delivering therapeutic agents.

Further, as the vector organisms of the invention are parasitic in nature, they do not give rise to an immune reaction in the host. This is a considerable advantage over the use of viral vectors.

Yet further, the vector organisms of the invention can deliver large amounts of nucleic acids, unlike viral vectors.

Also, the vector organisms of the invention are intrinsically stable, in the sense that they can reliably deliver therapeutic proteins over a long period of time. This is due to the fact that they can survive for long periods of time (up to 30 years) in their hosts. This stability provides an advantage over all the conventional techniques mentioned above, especially the use of stem cells, (which behave unpredictably if clonal multiplication occurs) and the direct delivery of proteins (where it is difficult to ensure a constant rate of delivery).

Accordingly, the invention provides:

A transgenic eukaryotic vector organism comprising a heterologous coding sequence which encodes (a) a therapeutic polypeptide, or (b) a polypeptide capable of generating a therapeutic agent, which vector organism is capable of living non- pathogenically within a mammalian or avian host organism, and of expressing the coding sequence as a therapeutic polypeptide.

A population of vector organisms as defined above wherein all the individuals are of one sex.

A pharmaceutical composition comprising a vector organism of the invention or a population of such organisms and a pharmaceutically acceptable carrier. A vector organism or population of the invention for use in a method of treatment of the human or animal body; or a population of vector organisms.

Use of a vector organism or population of the invention in the manufacture of a medicament for the treatment of the human or animal body. A method of treatment of the human or animal body which comprises administering to a subject in need thereof an effective amount of vector organisms according to the invention, a population of such organisms or a pharmaceutical composition of the invention. A cell harbouring such a construct.

A process of producing a vector organism or a population of vector organisms of the invention, which process comprises (a) transforming or transfecting a vector organism with a nucleic acid construct of the invention; (b) propagating the transformed vector organism; and (c) recovering a transformed vector organism or population thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Depiction of the life cycles of three representative schistosomes

DETAILED DESCRIPTION OF THE INVENTION

The vector organisms of the invention may be taxonomically diverse but all show certain common characteristics. Firstly, they are eukaryotic, though they may be either unicellular or multicellular. Secondly, in nature, they live in close association with a host, preferably a mammalian or avian host; and preferably they live within the host's body for at least a portion of their life-cycle. Thus, they are typically symbionts, commensals or parasites, for example endosymbionts or endoparasites. Thirdly, when used according to the invention, the vector organisms do not cause significant pathological effects and preferably do not provoke a significant immune response. Preferably no immune response at all is provoked.

Helminths (worms belonging to several metazoan phyla), for example parasitic helminths, are preferred.

One particularly preferred group of vector organisms are trematodes, particularly schistosomes; for example Schistosoma mansoni, Schistosoma haematobium and Schistosoma japonicum, especially Schistosoma mansoni. Another preferred group of organisms are parasitic nematodes, especially Onocera volvus and Ascaris.

Members of the family Schistosomatidae are trematode worms that are parasitic in the blood vascular system of vertebrates. Schistosomes are dioecious and the female is enveloped within the body folds of the male, hence the descriptive term "schist-o- some", which means "fissured body". There are twelve genera of Schistosoma, seven having birds and five having mammals as definitive hosts. For Schistosoma mansoni, individual life-span within the portal vasculature of man is of the order of 10 to 15 years and survival for up to 30 years has been documented. Such longevity is indicative of morbidity (disease), rather than mortality, as the consequence of parasitization and is consistent with the absence of any local host response to adult worms, other than an inconsequential loss of the erythrocytes upon which the parasites feed. In schistosomiasis, pathology is exclusively due to lodgement of eggs in tissues such as liver, intestinal mucosa, bladder and ureter where cellular immune reactions to egg antigens evoke inflammatory reactions that facilitate emission of eggs from the host via urine or faeces. Thus, the vector organisms of the invention are non-pathogenic, and do not provoke an immune reaction, except when they produce eggs.

The Schistosome life cycle can be summarised as follows. On being shed from the host into water, the egg hatches as a ciliated larval stage (miracidium) that must encounter and penetrate the epidermis of a particular species of gastropod snail (Biomphalaria for S. mansoni). Once within the snail, the miracidium loses those structures associated with a motile existence and residual germinal cells grow and divide repeatedly prior to encapsulation by somatic cells to form embryonic clusters. These clusters of undifferentiated cells are the precursors of vermiform larvae (sporocysts) which migrate to the digestive gland of the snail where they may undergo further cycles of multiplication to form daughter sporocysts. This phase of asexual replication ensures regular release of large numbers of free-swimming larvae (cercariae) whose instinctive behaviour, including phototropism, favours contact when a definitive host comes in contact with water. Extensive replication both in the definitive host (bird or mammal) and in the intermediate (snail) host reflects the difficulty of ensuring that cercariae will enter a definitive host in numbers sufficient to ensure pairing within the portal vasculature.

On entry into the epidermis of a definitive host, cercariae shed their tails and transform into vermiform schistosomula within 48 hours and subsequently migrate to the dermis where they will enter a venule within the next 24 hours. The schistosomula become entrapped within the pulmonary vasculature and remain at that location for several days during which they enlarge and elongate to about 10 mm. At this stage, the larvae traverse the pulmonary capillaries and enter the systemic circulation. Those which enter the portal circulation remain in enterohepatic veins by a combination of motility and the use of suckers. Those schistosomula that enter other sectors of the vasculature traverse capillary beds but return to the systemic circulation. Transfer from lung to liver is highly efficient (50 to 70%) and there is an overall probability that 30 to 40% of cercariae which enter the epidermis will achieve location and pairing in portal vessels. Pairing refers to the association of male and female schistosomes in the portal vessels so that sexual reproduction can occur.

The immunological response of the mammalian host to Schistosomes, which only occurs following egg production, includes both antibody-mediated and cellular immunity. Antibodies are secreted in response to cercarial proteins released into epidermal tissue during metamorphosis into schistosomula and such antibodies also develop in response to proteins released into blood during oviposition, whereas cellular immunity results from entrapment of eggs within capillary beds. Cellular immunity favours granuloma formation and tissue damage to facilitate release of eggs from the host. Secretion of antibodies following egg production provides protective immunity whereby migration of cercariae across the epidermis and dermis is hampered by antibody-dependent chemoattraction of cytotoxic leukocytes. This hampers subsequent infection by new populations of parasites.

Nevertheless, substantial numbers of cercariae avoid immunological recognition by incorporating host DNA during metamorphosis and expressing this DNA to generate host proteins which are incorporated into the specialised integument of the schistosomulum. In consequence, there is a reduction rather than an abolition of transdermal migration of schistosomula. This expression of host proteins allows adult schistosomes to remain unaffected either by circulating antibodies directed against proteins in cercariae or schistosomula, or by cellular immunity directed towards egg antigens, proteins that are not exposed on the surface of adults, especially adult males. Hence, once within the vascular system, schistosomula are impervious to immunological rejection and do not induce activation of leukocytes, platelets or circulating proteins. This capacity to evade immunological and non-immunological surveillance is retained by mature adults.

Therefore, schistosomes have the ability to take up and express host DNA, as well as the ability to evade the host's immune system. These characteristics are exploited in the present invention by providing heterologous DNA that the Schistosomes express as polypeptides. These may be released into the host's circulation. Alternatively, they may act in vivo to generate other products, generally non-polypeptide products which are themselves released. Thus the invention provides vector organisms capable of delivering therapeutic polypeptides, or of delivering therapeutic products generated by encoded polypeptides, without raising an immune response. Therapeutic polypeptides may act in any way, for example by converting biologically active materials into less toxic or inert metabolic products.

Parasitic nematodes of the invention, such as Onocera volvus have similar characteristics, both in terms of their life-cycles and their advantages as vectors for heterologous DNA.

When vector organisms of the invention are administered to hosts such as humans, they are attenuated, in the sense that they are incapable, or substantially incapable, of giving rise to the pathological effects that may result from natural infection.

For example, attenuation may be achieved by classical techniques of serial passage and selection of decreasingly pathogenic strains, by transfer to a different host species, by chemical treatment or it may be effected by manipulation of the genome of the organism, for instance by deletion of a gene required for pathogenicity. Alternatively, the vector organism may be of a species, or strain which even in the wild type is non-pathogenic to the host. In this respect, one method of attenuation that is particularly preferred is discussed below.

Substantial numbers of male schistosomes (up to 100 to 500, or more) can survive in laboratory mice for at least eighteen months with no females present. Despite such a large burden of adult males, equivalent to several hundred thousand in an adult human, host mice were indistinguishable from normal animals and did not exhibit acute or chronic pathology. Since death of the circa 200 million humans afflicted by

Schistosoma mansoni is rare, infection by unisexual populations is unlikely to have serious adverse side effects. By contrast, 50 to 100 paired S. mansoni (i.e. a mixed-sex population) kills all host mice within three months. Further, such male-only populations do not induce any protective immunity in mice, in contrast to the situation when mice are infected with mixed-species populations.

Following natural infection with S. mansoni, both cell-mediated (delayed type hypersensitivity) and antibody-mediated cytotoxicity can be detected at the onset of oviposition. These defence mechanisms neither impair survival of the adults, nor influence physiological activities (e.g. feeding, formation and release of eggs).

Established adults are unaffected because their immunological identity has been obscured by inclusion of host antigens (e.g. histocompatibility antigens within their integument). Therefore, some product of female Schistosomes determines this form of host immunity, since male-only populations of cercariae do not induce protective immunity in mice.

Debilitating consequences of entry and maturation of schistosomes are solely attributable to endogenous inflammatory (immune) reactions, which result from embolisation of eggs within the microvasculature of host tissue, particularly in liver and intestine. Males of S. mansoni reside in the host in a manner indistinguishable from paired organisms but, lacking the ability to produce eggs cannot elicit a pathological response.

Unisexual populations of Schistosomes do not exhibit the pathology of mixed sex populations (K.S. Warren and E.O. Domingo "Schistosoma mansoni: Stage specificity of granuloma formation around eggs after exposure to irradiated cercariae, unisexual infections, or dead worms" Experimental Parasitology 27:60-66 (1970)) and although some pathological changes can be detected (CA. Baki and J.A. Grimaud "Unisexual murine schistosomiasis: portal hepatitis in subacute infections" Experientia 41 : 1423-1426 (1985)), it has been possible to infect mice with unisexual populations without detectable effects (e.g. colonisation of mice by male worm populations (> 500) did not result in overt acute or chronic (1.5 years) pathology.

Therefore, male-only populations of Schistosomes neither give rise to pathological effects nor provoke on immune reaction in the host. Accordingly, for the purposes of the invention, it is preferred to use Schistosomes of one sex only, preferably male Schistosomes. A further advantage in this respect is that, as male-only populations do not provoke protective immunity, it is possible to introduce successive doses of Schistosomes, for example to increase the delivery of therapeutic protein, to deliver a different therapeutic protein, or to replace a population that has died or been eliminated by chemotherapy. In populations of the invention, at least some of the vector organisms are organisms of the invention, i.e. transgenic vector organisms. Preferably, a high proportion for example at least 50%, at least 60%, at least 70%, at least 80%), at least 90%>, at least 95% or at least 99% of the vector organisms are organisms of the invention. More preferably, all of the vector organisms are organisms of the invention.

Some parasitic nematodes, including Onocera volvus, are also dioecious so that the sexes can be separated for use as single-sex populations. Others may be used as mixed-sex populations, especially if the host to which they are delivered is not their normal host and they are unable to reproduce, and yet the host may respond to constituents or secretory products of the vector and thus experience diminished pathology (c.f New Scientist, page 4, 7 August 1999).

Vector organisms having this effect can desirably be used in some embodiments of the invention.

It is known that pairs of Schistosoma mansoni may survive in humans for up to thirty years, although a more usual life span is of the order of a decade. As survival of unisexual populations in mice is comparable with that of paired organisms and exceeds 1 year, it is reasonable to expect that the survival of unisexual populations of organisms in man will be not dissimilar to that of bisexual populations. Therefore, unisexual populations will survive for long enough to release polypeptides of the invention for therapeutically effective periods.

The vector organisms of the invention are transgenic in that they include one or more heterologous nucleic acid sequences, preferably DNA. The heterologous nucleic acid encodes a polypeptide of therapeutic benefit to the host. The vector organism expresses this nucleic acid and therefore generates the polypeptide of interest. In one preferred embodiment, the polypeptide is then secreted by the vector organism into the host's tissues, where it has a therapeutic effect. In another preferred embodiment, the polypeptide is not released but acts inside the vector organism to generate a further product which has a therapeutic effect. This can be either a further polypeptide product, or a non-polypeptide product, e.g. a small molecule with therapeutic effects. For example, if the encoded polypeptide is an enzyme, the further product may be the product at the enzyme's action in its substrate.

The nucleic acid may be introduced into the vector organism by any means known in the art. For example, the vector organisms may be transformed or transfected by any suitable method, such as the methods described by Sambrook et al (Molecular cloning: A Laboratory Manual; 1989) or by Ausubel et al (Current Protocols in Molecular Biology, Wiley Interscience). For example, nucleic acid constructs comprising nucleic acid sequences according to the invention may be packaged into infectious viral particles, such as retroviral particles. The constructs may also be introduced, for example, by electroporation, lipofection, scrape loading, calcium phosphate precipitation, lipofection, biolistic methods or by contacting naked nucleic acid constructs with the vector organisms.

Of these methods biolistics or particle bombardment is preferred. (See Davis et al; Transient expression of DNA and RNA in parasitic helminths by using particle bombardment, (1999) PNAS 96, 8687-8692). A further preferred technique is transformation by means of Micromechanical piercing structures (Hashmi et al; Biotechniques 19:766-770), November 1995).

Electroporation is also preferred (see: Sukharev et al Electrically-induced DNA transfer into cells. Electrotransfection in vivo. In "Gene Therapeutics" Ed. J.A. Wolff, Birkhauser 1994 pp 210-232). ^• Another preferred transformation technique is introduction of exogenous DNA into the germline of vector organisms by microinjection. This is described by Fire, A. in respect of transformation of the free-living nematode Caenorhabditis elegans. (The EMBO Journal 1986, 5:2673-2680).

Transformation or transfection may be effected at any stage in the vector organism's life-cycle. One preferred stage is the sporocyst, which is formed following entry of a miracidium into a snail host. The sporocyst is capable of asexual replication and thereby facilitates a naturally occurring form of clonal expansion. For this reason, and because sporocysts can be cultured in vitro, this larval stage is particularly suitable for transformation. Another preferred stage is the adult. At this stage, a preferred transformation technique is the introduction of the transformation construct into the germ cells, for example by microinjection.

Also, isolated cells from the vector organism may be transformed or transfected by any of the methods described above, and cultured to regenerate transgenic vector organisms.

The transformation construct may be of any type known in the art, and may comprise DNA or RNA, as appropriate. For example, the construct may be in linear or circular form. Plasmids are one preferred type of transformation construct, as this is the form in which Schistosomes and other similar organisms are thought to maintain the host DNA that they take up naturally and express as the proteins that preclude the host immune response. In C. elegans, for example, injected DNA becomes assembled into large arrays by recombination; and the extrachromosomal compartmentalisation of highly reactive, newly injected DNA leads to the production of plasmids as heritable transgenic structures C.C. Mello, J.M. Kramer, D. Stinchcomb and V. Ambros "Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences". The EMBO Journal 10:3959-3970 (1991).

Plasmids similar to, or derived from, those that are naturally developed by the vector organisms, or related organisms are particularly preferred.

Plasmid constructs derived from S. japonicum that incorporate reporter genes for readily monitored expression products (e.g. β-galactosidase, catecholamine acetyl transferase, glutathione S-transferase) are available commercially.

Those of skill in the art will be able to prepare suitable vectors comprising nucleic acid sequences encoding therapeutic polypeptides starting with widely available vectors which will be modified by genetic engineering techniques known in the art, such as those described by Sambrook et al (Molecular cloning: a Laboratory Manual; 1989). A transformation construct according to the invention typically comprises one or more origins of replication so that the construct can be replicated in the vector organism of the invention, and preferably in other cells as well, such as bacterial cells or yeast cells (this enables constructs to be replicated and manipulated, for example in E. coli, by standard techniques of molecular biology). A construct also typically comprises at least the following elements, usually in a 5' to 3' arrangement: a promoter for directing expression of the nucleic acid sequence and optionally a regulator of the promoter, a transcription start site, a translational start codon, and a nucleic acid sequence encoding polypeptide whose expression and delivery to the host organism is therapeutically desirable.

The construct may also contain one or more selectable marker genes, for example one or more antibiotic resistance genes, for example a gene that confers resistance to G418 (geneticin), an ampicillin resistance gene, or a neomycin resistance gene. Of these, neomycin resistance genes are particularly preferred, as they allow simultaneous evaluation of transformation efficiency and demonstration of heterologous gene expression. Alternatively or additionally, screenable or scorable marker genes may be included. These enable identification of transformants without killing non- transformants. Examples include green fluorescent protein (GFP) and luciferase. Such marker genes allow identification of transformants, both transformed vector organisms and transformed microorganisms useful in replication and manipulation of the construct. Optionally, the construct may also comprise an enhancer for the promoter. The construct may also comprise a polyadenylation signal, typically 3' to the nucleic acid encoding the functional polypeptide. The construct may also comprise a trans criptional terminator 3' to the sequence encoding the therapeutic polypeptide. The construct may also comprise one or more introns or other non-coding sequences 3' to the sequence encoding the therapeutic polypeptide. The intron or introns may be from the vector organism or from the organism from which the nucleic acid encoding the therapeutic polypeptide is derived, or from another source.

In a typical construct, the nucleic acid sequence encoding the therapeutic polypeptide is operably linked to a promoter capable of expressing the sequence.

"Operably linked" refers to a juxtaposition wherein the promoter and the nucleic acid sequence encoding the therapeutic polypeptide or protein are in a relationship permitting the coding sequence to be expressed under the control of the promoter. Thus, there may be elements such as 5' non-coding sequence between the promoter and coding sequence. Such sequences can be included in the construct if they enhance or do not impair the correct control of the coding sequence by the promoter.

Any suitable promoter that is capable of directing expression of the nucleic acid encoding the therapeutic polypeptide or protein may be included in the construct. For example, the promoter may be a bacterial, eukaryotic or viral promoter. The promoter may be constitutive or inducible.

Promoters derived from the vector organisms of the invention, or from closely related organisms such as nematode or trematode worms, are preferred. For example, promoters derived from Schistosome genes are preferred, such as the cathepsin G promoter. An example of a promoter from a related species is the 16kDa promoter from the nematode, C. elegans. Further preferred promoters and other regulatory elements derived from schistosomes are identified in the Examples. Similarly, promoters from other sources that function outside their normal environment may be used. For example, viral promoters, such as the SV40 promoter may be used.

Alternatively, expression of polypeptides of the invention may be achieved without the inclusion of a promoter in the construct, as it is possible to transform the cells of some vector organisms of the invention by inserting the nucleic acid encoding the therapeutic polypeptide downstream of a pre-existing promoter. Such expression of genes be achieved in C. elegans without promoters as illustrated by use of lacZ gene (see e.g. J.M. Young and I. A. Hope "Molecular markers of differentiation in Caenorhabditis elegans obtained by promoter trapping" Developmental Dynamics 196:124-132 (1993)).

Constructs that rely on this approach, and do not include a promoter, will typically comprise a dominant selectable marker gene that enables identification of transformants. Desirably, in one embodiment, the vector organisms of the invention secrete the expressed therapeutic polypeptide.

Accordingly, preferred constructs of the invention comprise a nucleic acid sequence that encodes a signal sequence that ensures this. In these constructs, the signal sequences will be operably linked to the promoter, if present, and to the sequence encoding the therapeutic polypeptide such that, on expression of the therapeutic polypeptide, the signal sequence is expressed, typically attached to the therapeutic polypeptide at its C- or N- terminus. Thus, the nucleic acid encoding the signal sequence is typically located immediately 3' or immediately 5' to that encoding the therapeutic polypeptide and therefore the therapeutic polypeptide is typically expressed as a fusion protein including the signal sequence.

Any suitable signal sequence may be used, as long as it is capable of directing or facilitating secretion of the therapeutic polypeptide. Signal sequences derived from the vector organisms of the invention or from related organisms, are preferred.

Thus, Schistosome signal sequences, such as the cathepsin G signal sequence, are preferred. Other signal sequences can readily be isolated by those of skill in the art from Schistosome genes whose products are known to be secreted. There are many other putative schistosome excretory-secretory proteins released in vitro (Murrell, K. E. et al., Exp. Parasitol., 36, 136 (1974); Rotmans, J. P. et al, Exp. Parasitol., 52, 171 (1981). Another way to facilitate secretion may be to express the polypeptides of the invention as fusion proteins. These will typically be encoded by a single DNA sequence under the control of a single promoter and comprising, in-frame, sequences encoding (i) the polypeptide of the invention and (ii) all or part of a polypeptide that is naturally secreted from the vector organism to be transformed or from another organism closely related enough for this part of the fusion protein to be secreted from the transformed vector organism.

Constructs of the invention may also comprise nucleic acid sequences whose expression allows the efficiency of transformation to be monitored. These function in an analogous way to selectable markers. An example of a gene which allows transformation efficiency to be monitored is the human placental alkaline phosphatase gene, whose product is, conveniently, secreted and can be assayed by simple techniques known to those of skill in the art. Another preferred gene for this purpose is the neomycin resistance gene (neo). Further examples of genes which are used as reporters of recombinant expression include luciferase and green fluorescent protein (GFP). Other suitable genes for monitoring transformation efficiency are known to those of skill in the art.

The constructs of the invention comprise one or more nucleic acid sequences that encode therapeutic polypeptides, for example therapeutic proteins or glycoproteins; or polypeptides that are capable of generating a further product which is itself therapeutic. In principle, any therapeutic polypeptide may be thus encoded for expression in vivo and delivery to a mammalian or avian host.

In particular, the in situ generation and release of therapeutic polypeptides or therapeutic products generated by encoded polypeptides by vector organisms of the invention allows the use of molecular species whose inherent chemical instability limits conventional modes of delivery. Plasma levels of therapeutic agent can therefore be maintained over prolonged periods (months rather than days). Secretion can be enhanced by injection of larger numbers of therapeutic organisms or by inclusion of multiple gene copies or other methods for increased expression.

Therapeutic agents, for the purposes of the invention, include polypeptides that contribute to curing, ameliorating or preventing any disease or other pathological condition of the human or animal body. Preferred therapeutic agents include autocoid hormones; neurosecretory agents, neurotransmitters and other naturally occurring molecules used for intercellular communication, and non-natural analogues or derivatives thereof that may be generated biosynthetically; enzymes; and mammalian or avian enzyme inhibitors. Further preferred therapeutic polypeptides are as identified in the Examples.

Two particularly preferred therapeutic polypeptides are growth hormone, particularly human growth hormone, and growth hormone-releasing hormone, preferably human growth homone-releasing hormone, which may be administered, according to the invention, to subjects deficient in the polypeptide or glycoprotein, such as children with retarded growth due to an inability to secrete growth hormone, or adults with reduced (<35OIU/litre) levels of insulin growth factor 1. In both instances, it is known that regular systemic administration of growth hormone preparations ameliorates these deficiencies and leads to measurable clinical benefit (Parra et al (1979) Metabolism 28:858-857; Rudman D. et al: (1990) N. Engl. J. Med 323: 1-6).

The construct may encode the therapeutic agent in its active form or a precursor thereof. The polypeptide therapeutic agent may be subjected to further processing, such as cleavage of signal sequences and other precursor moieties, glycosylation .and other modifying reactions performed either by the vector organism of the invention prior to secretion, or by the mammalian/avian host following secretion.

As far as polypeptides that are not themselves directly therapeutic but rather generate therapeutic agents indirectly are concerned, any suitable polypeptide may be expressed in order to generate a product that is a therapeutic agent. In many cases, such agents will be non-polypeptide molecules but they may be polypeptides. Examples of non-polypeptide therapeutic agents that can be generated in this way include prostaglandins, e.g. PGE2 (see Example 6), which are generated by cyclooxygenase enzymes.

Therapeutic polypeptides released by the vector organisms may act in any way, for example by converting biologically active materials into less toxic or inert metabolic products. Examples of metabolic elimination that can be achieved in this way include phenylalanine elimination (see Example 5). Phenylalanine which can be metabolised by phenylalanine ammonia lyase (EC 4.3.1.5)

In order to prepare the vector organisms of the invention for transformation with nucleic acid constructs of the invention, it may be necessary to grow them in culture. Also, it will typically be necessary to propagate or maintain the vector organisms of the invention in culture after transformation. Such culture of the organisms may be carried out by any means known in the art, and vector organisms of the invention, or populations comprising such organisms are then recovered by any suitable means known in the art. For example, any suitable stage in the vector organism's life cycle may be cultured and in this way, S. mansoni, for example, can be maintained in laboratory culture with high efficiency.

Eggs of S. mansoni can be isolated from the homogenised livers of infected mice by filtration and sedimentation in saline. Resuspension in water induces emergence of free-swimming miracidia, which shed their cilia and transform into sporocysts upon exposure to tissue culture media. Sporocysts and their progeny remain viable for several days. Sporocysts are convenient for transfection studies, since they can be maintained in vivo (see e.g. L.M. Cohen and L.K. Eveland (1989) J. Parasit. 74:963- 969); or in vitro (see e.g. C.J. Bayne et al (1994) J. Parasit. 80:29-35 (1994); Ivanchenko, M.G., Lerner, J.P., McCormick, R.S., Toumadje, A., Allen, B., Fischer, K., Hedstrom, O., Helmrich, A., Barnes, D.W., Bayne, C.J. (1999) continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni. Proc. Nat. Acad. Sci. USA 96:4965-4970.

Once transfected, they can proliferate whilst exposed to a selective pressure (e.g. in the presence of an amino glycoside antibiotic such as G418 (geneticin)) in order to ensure that only cells which have taken up and expressed the marker gene proliferate. Transfected sporocysts experience further clonal amplification on being introduced into the intermediate host gastropod (Biomphalaria spp.), so that large numbers of transfected organisms may be produced. Within a month, snails can be induced to shed cercariae (several thousand cercariae per snail per day) which, following application to the skin, enter the epidermis and then metamorphose into Schistosomula. Adult worms become established in the enteric vasculature within 4-5 weeks, with a survival rate of approximately 40%. Worm numbers can be estimated by measurement of concentrations of circulating anodic antigen (CAA: see below) by ELISA and the concentration of secretory product of the transfection construct in sequential plasma samples by ELISA or calorimetry.

In preferred culture techniques, single-sex, preferably male-only, populations of vector organisms will be obtained and/or maintained. For example, by adding individual male miracidia to single host snails, populations of sporocysts can be produced that are exclusively male. Transfer of sporocysts from individual to individual by direct injection ensures maintenance of a male population in the intermediate host snail. This procedure generates adequate numbers of cercariae for research purposes with yields of 10³-10⁴ cercariae per snail per day.

By infection of mice with shed cercariae from each individual snail, the gender of such populations can be ascertained by inspection of the morpho logical characteristics of adult worms following sacrifice of the mouse after approximately 30 days. Populations of snails that shed cercariae of male genotype can be pooled to provide cercariae for intravenous injection into host mammals or birds, in order to effect therapy. For larger scale culture, in vitro replication of schistosomulae is preferred.

There is no evidence that male S. mansoni can transform into females; however, to guarantee an absence of females, a cloned DNA sequence which binds only to DNA from female S. mansoni (detection limit 50 female cells) will provide quality control for large scale production of symbiont schistosome vectors. Schistosomulae can be cultured, even on a large scale, by standard culture techniques known in the art.

Preferably, vector organisms of the invention are cultured under a selection pressure that ensures that only, or mainly, transfected organisms survive and propagate. This makes use of the selectable markers incorporated in the constructs of the invention, as described above. In particular, it is preferred that a dominant selectable marker such as the neo gene (neomycin phosphotransferase) be used to allow simultaneous evaluation of transformation efficacy as well as demonstration of heterologous gene expression. Using this device, sporocyst cells are grown under selective pressure (e.g. in the presence of an amino glycosidic antibiotic such as G418) to ensure that only those cells which have taken up and expressed the marker gene are amplified.

In this respect, the vector organisms of the invention are advantageous as means of delivery of DNA to human and/or animal hosts when compared to conventional methods of delivery. Owing to the possibility of contamination by human pathogens, introduction of heterologous DNA into mammalian cells requires specialised facilities for containment, especially when scaled-up, in order to avoid contamination by pathogens. By contrast, clonal expansion of the schistosomes and other vector organisms of the invention is technically simple and even expansion to large scale in vitro culture can be achieved using conventional culture conditions. No known human pathogen is associated with S. mansoni, so that pathogen-free production of symbiont schistosome vector organisms is straightforward.

The vector organisms of the invention may be administered to suitable mammalian or avian hosts by any means known in the art, alone or in combination with any suitable additional ingredients. The invention therefore provides pharmaceutical compositions comprising organisms of the invention and a pharmaceutically acceptable diluent or carrier therefor. Suitable compositions include those formulated for oral, percutaneous, topical, intravenous or parenteral administration. Carriers and diluents include aqueous media such as water, typically sterile water, for injection. The compositions may contain conventional accessory ingredients such as buffers and isotonic agents not detrimental to the viability of the vector organisms or their ability to express the therapeutic agent.

Administration of the vector organisms or compositions comprising them may be by any suitable route. Preferred routes of administration include topical or percutaneous application of cercariae and intravenous injection of schistosomula. For laboratory studies in small animals, percutaneous application is preferred since this method is as efficient as intravenous injection and technically more convenient. For repeat administration this is also the preferred route. Topical application of cercariae can be expected to have a high level of efficiency that is relatively consistent between subjects (i.e. 30 to 45% maturation as adults). This contrasts with the highly variable uptake and expression following insufflation of conventional viral vectors into the airways. The simplicity of topical application of the symbiont schistosome vector to the skin will allow paramedical staff to effect the therapeutic procedure, which is advantageous. Alternatively, vector organisms or pharmaceutical compositions of the invention may be administered orally, parenterally, intramuscularly or intradermally. The invention may be used in therapeutic treatment of any mammalian or avian species, preferably man and domesticated livestock, such as cows, sheep, poultry and

Pigs- Further, constructs and transformed vector organisms of the invention may be evaluated using a variety of host organisms. S. mansoni affects a range of mammalian species which can be presumed to account for stability of the life cycle: thus, an abundance of definitive hosts favours suppression rather than expression of sub-species formation. The capacity of S. mansoni to propagate naturally in animals such at the mouse is especially convenient, since symbiont schistosome vectors can be tested in animals which serve as disease models in circumstances that are closely comparable with clinical situations. This is useful not only for research purposes but also for quality control of batches of cercariae of symbiont schistosomes since S. mansoni will also mature in either rhesus monkeys or baboons, these primates can be employed to simulate clinical performance of symbiont schistosome vectors prior to evaluation in man. Unlike the vector organisms of the invention, conventional leukocyte vectors can be evaluated in vivo only by introduction into humans, so that the vector organisms of the invention provide a further advantage over known methods.

The vector organisms of the invention may be administered in any suitable amount, and many different factors will contribute to the choice of dose size, including the route of administration, the size of the recipient, the level at which the vector organism expresses the therapeutic polypeptide, the nature of the polypeptide, and the amount of that polypeptide that is desired to deliver to the host.

A typical dose for an adult human may comprise from 10² to 10⁶ vector organisms of the invention, for example 10² to 10³, from 10³ to 10⁴ or 10⁴ to 10⁵ vector organisms, preferably male-only vector organisms. Estimates have been made of worm burdens in man that correspond to those of the mouse (A.W. Cheever (1969): Transactions of the Royal Society of Tropical Medicine Hygiene 63, No.6). From these estimates, 1 worm pair per mouse corresponds to 3500 worm pairs per adult (70 kg) human, of which 1000 reside in the liver. There is close correlation between mouse and man (K.S. Warren 1964 Nature 201,899).

It is known that up to 100 to 500 male schistosomes can survive in a single mouse without causing adverse effects. Therefore, based on the above estimate that 1 worm pair per mouse corresponds to 3500 worm pairs per human, doses in the region of 100,000 to 1,000,000 vector organisms of the invention are preferred, with doses of from 300,000 to 500,000 being particularly preferred.

Dosage schedules will vary according to, for example, the route of administration, and the species, size and condition of the recipient. However, single doses and multiple doses and multiple doses spread over periods of days, weeks, months or years are envisaged. Such multiple doses may be administered following the death of a previous population of vector organisms, or in order to supplement a previously administered population.

The number of vector organisms present in a host can be determined by assaying the host tissues, or bodily fluids for any suitable product of the vector organism. This allows dosages to be monitored, with a view to deciding whether further doses are required.

Amongst materials secreted by S. mansoni are proteoglycans, such as circulating anodic antigen (CAA). CAA appears in the plasma of the host following maturation of schistosomula and secretion is sustained throughout life. An ELISA which allows measurement of CAA is available, this provides a non-invasive method to estimate worm burden. Thus, numbers of symbiont schistosome vectors can be estimated using blood or urine samples whose CAA content can be monitored regularly and accurately using a standard laboratory assay. No comparable simple method is available for monitoring numbers of viral vectors or modified leukocytes, so the vector organisms of the invention are advantageous over conventional methods of delivering nucleic acids to mammalian/avian hosts.

Therefore, the present invention provides methods of treating human or avian subjects, which methods comprise administering to the subjects an effective amount of vector organisms of the invention, or of a pharmaceutical composition containing them. The vector organisms then express, and preferably secrete, the therapeutic polypeptide encoded by the heterologous DNA construct that they contain, and the polypeptide exerts its therapeutic effect.

In a preferred embodiment of the invention, the vector organisms of the invention will be eliminated from the host after a suitable time, for example after several weeks, months or years, thus terminating the treatment. This may be done by any means known in the art, such as chemotherapy. For example, when the vector organism of the invention is a schistosome, schistosomicidal drugs may be used in the case that a schistosome vector organism is employed. An advantage of using Schistosoma mansoni as a vehicle is that the organism may be completely eliminated by the use of chemotherapy. The preferred drugs for human therapy are Praziquantel (Bayer) and Oxamniquine (Pfizer) which may be used as single doses, or multiple doses, preferably single doses. Thus, a single oral dose of either Praziquantel or of Oxamniquine can eliminate symbiont schistosome vectors. Dead organisms are phagocytosed by blood leukocytes without significant side effects. Elimination of the symbiont schistosome vector can be confirmed conveniently by measurement of CAA in plasma.

In mice, it has been shown that either compound is effective in eliminating mixed cell populations of Schistosoma mansoni, but, unexpectedly, oxamniquine eliminates unisexual populations, whilst praziquantel has proved to be ineffectual. The differential effect of these two drugs in bisexual and unisexual infections has not previously been disclosed. Thus, any schistomicide may be employed, with Praziquantel and Oxamniquine being preferred. Oxamniquine is particularly preferred when male-only schistosome populations are employed as it eliminates male-only schistosome populations. Other drugs that effectively eliminate male-only populations of vector organisms are also preferred.

The following Examples illustrate the invention.

EXAMPLES

EXAMPLE 1: Interaction of male S. mansoni with a mammalian host

(i) Male-only populations of schistosomes cause no significant pathology, as is documented below.

Individual snails of Biomphalaria sp. are exposed to a single miracidium of S. mansoni, thereby ensuring that all miracidia produced by that snail will be of a single sex, identifiable by infection and sacrifice of individual mice. In this way, a substantial number of snails shedding only male cercariae provide a reliable and constant source of male cercariae. These clones are expanded by transplanting fragments of infected snail digestive glands into further snails.

(ii) Exposure of mice to between 50 and 100 cercariae of both sexes kills all recipients within 100 days, whereas it has been observed that exposure to 500 male cercariae does not influence survival over 1.5 years.

On this basis, individual mice are exposed to male-only cercariae (50, 100,

200, 400, 800) by the percutaneous route or, following transformation into schistosomula, by intravenous injection.

(iii) Blood samples are used to monitor worm burden by measurement of CAA, using an ELISA assay.

(iv) The effects of infection with the vector organisms of the invention are investigated.

At regular intervals over a prolonged period (500 days), individuals from each group are sacrificed, using portal, pulmonary and whole body perfusion to assess distribution of parasites. Individual worms are sexed and categorised by dimensions and weight. Liver pathology is assessed by measurement of SGOT in blood samples and systemic stress is monitored by ACTH determination in blood samples and by recording thymic weight. Liver, intestine and lung are fixed for histological examination. Cercariae that have been radiolabelled with Selenium are used to provide quantitative data of the migration from skin to lung and enteric vasculature by sampling animals at shorter (1-3 day) intervals over a 21 day period. Comparisons are made with groups of animals either exposed to 100 cercariae of mixed sex or not exposed to S. mansoni. (v) Chemotherapy

The capacity of doses of Praziquantel and Oxamniquine to eliminate mixed- sex populations are confirmed for single-sex populations.

In this way, it is established that male cercariae develop and survive as adults in a manner closely comparable with that of paired males of S. mansoni, but without inducing tissue pathology.

EXAMPLE 2: Transformation of S. mansoni using a reporter gene demonstrates the possibility of heterologous gene expression in S. mansoni

(i) Transformation Constructs A heterologous reporter gene is stably transformed into S. mansoni cells, and its expression is measured. Initially, a plasmid carrying a suitable promoter and reporter gene is constructed. A number of suitable promoters have been described in the literature, such as the C. elegans 16kDa heat shock promoter, the β-action promoter, the CMV promoter or the SV40 promoter, all of which have been shown to function in heterologous species. Alternatively, appropriate S. mansoni promoters will become available as sequence data from the S. mansoni genome sequencing project is deposited into the Genbank or EMBL (European molecular biology laboratory) databases. One of these could be incorporated into the construct using standard techniques. The reporter gene is preferably one for which a specific, quantitative assay exists (e.g. the β-galactidase gene); alternatively, the construct would express resistance to a compound such as G418, an amino-glycoside antibiotic. The neo gene (encoding an amino-glycoside phosphotransferase resistance marker) would also be suitable; transformants can be selected on the basis of resistance to neomyciyo. For the neo gene, appropriate vectors are available commercially (e.g. pcDNA, Invitrogen, Holland) and require little modification.

(ii) Transformation Protocols

Transformation protocols which have been used successfully with the nematode C. elegans are appropriate for use with S. mansoni, using assays of the reporter genes (as described above) to determine the efficiency of transformation.

(i) A suitable stage of the S. mansoni life cycle (e.g. sporocyst) is cultured as described by Yoshino and Laursen (1) and transformed by electroporation. Such an approach has been used to achieve stable transformation of Caenorhabditis elegans. By using sporocysts, successfully transformed cells are coned and amplified before allowing the life cycle to continue.

(ii) Alternatively, adult worms are removed from mice and heterologous genetic material injected by use of a micromechanical piercing array as described by Hashmi et al. (2) or by direct injection, as described by Mello et al. (3) Such worms will be returned (by intravenous injection into the enterohepatic hepatic circulation of normal mice. Eggs are collected from the faeces of recipient mice and used for infection of individual snails. Cercariae are harvested, with male cercariae being selected. Male populations are introduced into normal mice to establish that the gene product is expressed and exported into host blood.

This procedure can be repeated using a nucleic acid sequence encoding a therapeutic polypeptide, in order to generate transgenic vector organism of the invention.

REFERENCES (Examples 1 and 2)

(1) Yoshino, T.P. and Laursen, J.R. Production of S. mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic cells. (1995) J. Parasitol., 81 :714-722.

(2) Hashmi, S., Ling, P., Hashmi, G., Gaugler, R., and Trimmer, W. Genetic transformation of nematodes using arrays of michromechanical piercing structures. (1995) BioTechniques, 19:766-770.

(3) Mello, C.C., Kramer, J.M., Stinchcombe, D., and Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. (1991) EMBO Journal 10, 3959-3970.

EXAMPLE 3: Administration of transformed S. mansoni to a host (mouse)

Demonstration that a heterologous protein is secreted from transfected worms into the plasma of mice suffices to illustrate the potential of the vector. A series of iterative experiments are undertaken to define conditions which achieve plasma concentrations that are adequate for therapeutic objectives. Procedures that may influence steady state plasma concentration (worm number, utilisation of multiple gene copies, modification of the control of construct secretion etc), are investigated.

Example 4: Construction of a transformation vector and transformation of S. mansoni

(i) Vector construction

The vector pGL-3 is prepared as follows. The promoter and 5' UTR region of the S. mansoni SL RNA gene (-450 to +38; Genbank accession No. M34074) and the 3' UTR and polyadenulation signal of the S. mansoni enolase gene (225 bp; Genbank No. U33177; nucleotides 1006-1230) are used to flank the cDNA sequences encoding human thrombopoietin (Genbank accession No. L36052). Alternatively, another cDNA (or genomic DNA) sequence could be used, for example one encoding an enzyme, e.g. or an enzyme such as phenylalanine hydroxylase serine protease or phenylalanine ammonia lyase (Sarkissian et al (1999) Proc. Natl. Acad. Sci. USA 96:2339-2344) or antigen capable of raising an immune response to a pathogen. Examples of an encoded antigen could be the HIV gp 120 or gp 160 protein (Vinner et al Vaccine 17:2166-2175). DNA sequences encoding hormones, e.g. interleukins, interferons and cytokines may also be used.

Alternatively, other promoters, 5' and 3' UTR and polyadenylation sequences could be selected, based on the requirement to produce more transcripts of enhanced stability which would be expected to increase the amount of protein translated. Such variant sequences could be selected from those found in GenBank and it will be apparent to those skilled in the art that it will be necessary to test empirically various combinations of these sequences and that the optimal sequences may vary according to the cDNA being transcribed. Suitable S. mansoni promoters, 5' and 3' UTRs and polyadenylation sequences could be selected from those of the S. mansoni glutathione S-transferase gene (Serra et al., (1997) Eur. J. Biochem 248: 113-119), S. mansoni actin genes (Oliveira and Kemp, (1995) Mol. Biochem. Parasitol. 75: 113-117) S. mansoni elastase gene (Pierrot et al (1995) Mo. Biochem. Parasitol. 75:113-117, S. mansoni hsp70 gene (Levy-Holtzman and Schechter, (1995) Biochim. Biochem. Biophys. Acta. 1263:96-98) or S. mansoni phosphagen kinase gene (Mol. Biochem. Parasitol. 75: 119-122).

Other alternatives could include the use of promoters regulated according to the life cycle of S. mansoni of the presence of an external ligand, e.g. an antibiotic such as tetracycline. Furthermore, it may be possible to improve translation efficiency by changing the codon usage (bias) of the protein to be translated from that of human to S. mansoni (Milhon and Tracy, (1995) Exp. Parasitol. 80:353-356)

It will also be apparent that many different cDNAs could be cloned into a suitable DNA vector such that a whole range of proteins could be produced by S. mansoni. The cDNA could be introduced into the vector such that it produces a fusion protein either with a S. mansoni protein that is known to be secreted by the adult, such as circulating cathodic antigen (CAA) (Abdeen, H.H., Attallah, A.F., Mansour, M.M., Harrison, R.A. (1999) Molecular cloning and characterization of the polypeptide backbone of Schistosoma mansoni circulating cathodic antigen. Mol. Biochem. Parisitol. 101 : 149-159], serine protease or amido peptidase (Cocude et al (1999) J. Parasitol. 118:389-396) or another protein or fragment of protein. This is expected to enhance secretion of the polypeptide by S. mansoni into the host. Another way to enhance secretion is to link the nucleic acid of the therapeutic protein to a nucleic acid sequence encoding a signal peptide derived from S. mansoni. The expressed signal peptide will then direct secretion.

(ii) Transformation by particle bombardment

Adult S. mansoni are removed from 45-day infected Syrian golden hamsters by perfusion and sorted according to sex (Gupta et al (1987) J. Parasitol. 73:481-486). The adult male S. mansoni are placed in RPMI 1640 culture medium containing an additional 2 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, 10% heat inactivated FBS, 100 units penicillin and 100 ug/ml of streptomycin for 15 min to 24 hrs before bombardment (for further information see Gregoire et al., (1987) J. Immunol. 139:3792-3801).

The plasmid DNA (5-7 μg) is precipitated onto 1-1.25 mg of 1.6 μm of gold particles providing sufficient material for one bombardment. Further experimental details are described by the manufacturer (Bio-Rad) and by Sanford et al., (1993) Methods Enzymol. 217:438-509). Male adult S. mansoni worms are placed in a petri dish along with culture medium and the medium is then removed. The animals are then bombarded with the DNA covered gold particles with a chamber vacuum of 15 inHg and a target distance of 3 cm, 1500-2,000 psi of pressure is used for acceleration.

(iii) Culture of schistosomes after bombardment

Medium 199, supplemented with calf serum (10%) is added to the Petri dish (Mercer, J.G., Chappell, L.H. (1985) Parisitology 90:339-349) and the animals are left to recover at 37°C in 5% CO₂ for 1-12 hours before injection into the ileocolic vein of a host mouse (Basch et al, J. Parisitol. 67: 191-195). Blood can be sampled at suitable (e.g. daily) intervals and assayed for TPO by enzyme-linked immunosorbent assay (Wolber et al (1999) Blood 94:97-105). Alternatively, the medium can be assayed for thrombopoietin, or other product of transfection expression after 8-24 hrs of incubation. Alternatively or additionally, worms may be homogenised and RNA for TPO measured by reverse transcriptase/competitive polymerase chain reaction (Wolber et al (1999) Blood 94:97-105). Culture medium may also be assayed for depletion of a substrate as when transfected phenylalanine ammonia lysate is used to convert phenylalanine into trans-cinnamic acid , which is readily assayed by chromatographic and mass spectra properties (Nutley et al (1994) Food Chem. Toxicol. 877-886). Example 5: Generation of a non-polypeptide therapeutic product (PGE2) by adult male schistosomes

In mammals, cyclooxygehase or prostaglandin G/H synthase (PGHS) catalyses the rate limiting step in the conversion of arachidonic acid to bioactive prostaglandins and to thromboxane. Two isoforms of cyclooxygenase have been cloned (Cox-1 and Cox-2). In many tissues, Cox-1 is expressed constitutively whereas Cox-2 is rapidly induced in response to a wide range of biological and inflammatory mediators. The disparate function of the two isoforms of cyclooxygenase has lead to selective inhibitors of Cox-2 being proposed as novel non-steroidal anti-inflammatory drugs (NSAIDS). It is anticipated that selective inhibitors of Cox-2 will have less side effects than established non-selective NSAIDS, since Cox-1 will retain biological activity and will be available for beneficial effects, such as vasodilatation, cytoprotection and limitation of lymphokine secretion during inflammatory and immunological reactions. These effects of Cox-1 products imply therapeutic potential for an in situ generator of PGE2. It has been well documented that the Cox-1 gene product can be more easily expressed and to higher levels than the Cox-2 gene product, a property which may relate to the unstable nature of Cox-2 mRNA. In adult S. mansoni, arachidonic acid (C-20:4) is the predominant unsaturated fatty acid, being present at concentrations which exceed those of host plasma (Fripp et al (1976), Comparative Biochemistry and Physiology). Comparative Biochemistry and Physiology 53B:505-507). By using (HC)-arachidonic acid, it has been established that adult S. mansoni metabolise arachidonic acid into prostaglandins, but do not generate PGE2 (Rumjanek, F.D. (1987) Biochemistry and Physiology in "The Biology of Schistosomes" eds. D. Rollinson & A.J.G. Simpson. Academic Press, London pi 70). Hence any PGE2-formation that can be detected in worms transfected to express Cox-1 can be attributed exclusively to expression of the construct.

Human Cox-1 cDNA is cloned into a mammalian expression vector in a similar manner to that described in Example 4. The vector additionally comprises containing the neomycin resistance gene behind the CMV promoter. Expression vectors of choice include pcDNAl and pcDNA3 (Invitrogen, California). The coding sequence of Cox-1 is transferred into a S. mansoni expression vector downstream of a suitable promoter with the S mansoni enolase 3' UTR, added onto the Cox-1 coding region, (see Davis et al (1999) Proc. Natl. Sci. USA 96:8687-8692). As in Example 4, adult S. mansoni are removed from 45-day infected Syrian

Golden Hamsters. Females are separated from males and discarded (Gupta and Basch (1987) J. Parasitol. 73:481-486). Adult male S. mansoni are placed in RPMI 1640 culture medium, containing supplementary 2mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, 10% heat-inactivated FBS, 100 units penicillin and 100 ug/ml streptomycin for 15 min. to 24 hours before bombardment with gold particles.

Plasmid DNA (5-7 ug) is precipitated onto 1.0 - 1.25 mg of 1.6 um of gold particles to provide sufficient material for one bombardment as detailed by the manufacturer (Bio-Rad) and by Sanford et al (1993) Methods Enzymol 217:438-509. Approximately 30 adult male S. mansoni are placed in a Petri dish and washed using culture medium. Adult males are then bombarded with DNA-coated gold particles, using a chamber vacuum of 15-in, a target distance of 3cm and 1500-2000 psi particle acceleration pressure.

Incubation medium is replaced using medium 199, supplemented with foetal calf serum, and to which arachidonic acid (750μM) has been added to a final concentration of 30μM. Samples of culture medium are collected at intervals of 2,4,8 and 24 hours. PGE2 levels in samples are determined using an EIA Kit according to manufacturers instructions (RPN222, Amersham).

Example 6: Secretion of macrophage migration inhibitory factor (MIF), a polypeptide product, by adult S. mansoni

Recognised initially as a lymphokine that affects macrophages, MTF is now known to play a critical role in the activation of mammalian lymphocytes. During physiological responses to inflammation, infection or stress, secretion of glucocorticsteroids is increased. However, the suppression of lymphocyte activation by glucocorticosteroids is limited by concurrent release of MTF, which augments secretion of tumor necrosis factor alpha, LL-1, LL-6 and EL-8 and permits development of a primary immune response (Bacher, M., Metz, C.N., Calandra, T., Mayer, K., Chesney, J., Lohoff, M., Gemsa, D., Donnelly, T., Bucala, R. (1996)]. An essential regulatory role for macrophage migration inhibitory factor in T-cell activation. Proc. Natl. Acad. Sci. USA 93:7849-7854]. Additionally, MJF is co-secreted with insulin when islet cells of the pancreas respond to glucose. By promoting the action of insulin, MTF counters the metabolic derangement that accompanies chronic inflammation. Thus, although MJJ may intensify responses to bacterial endotoxin, endogenous generation of MTF may also have effects which are therapeutic in subjects with impaired cellular immunity and/or chronic inflammation. As parasitic nematodes secrete a MJF homologue that shares (42%) structural identity with human MTF and that can modify the immunological response of the host (Pastrana, D.N., Raghavan, Ν., FitzGerald, P., Eisinger, S.W., Metz, C, Bucala, R., Schleimer, R.P., Bickel, C, Scott, A.L. Filarial nematode parasites secrete a homologue of the human cytokine macrophage migration inhibitory factor. (1988) Infect. Immunol. 66:5955-5963), nematodes are appropriate vehicles for expression and secretion of human MJJ⁷.

In a similar manner to that described in Example 4, a construct to express MIF comprises a promoter and 5' UTR region of the S. mansoni SL RΝA gene (-450 to +38; Genbank accession No. M34074) and the 3' UTR and polyadenylation signal of the S. mansoni enolase gene (225 bp; Genbank No. U33177; nucleotides 1006-1230) flanking the cDNA sequences encoding human MTF cDNA (Genbank No. 19686).

Alternatively, the cDNA sequence is constructed using the cytomegalovirus promoter-containing expression vector pBK (Stratagene, California) or any other vector that might be used to promote secretion of expressed gene products. Additionally, signal peptide/targeting peptide sequences may be included in the construct to direct trafficking appropriately through the secretory pathway.

As in Example 4, the plasmid DNA (5-7 ug) is precipitated onto 1-1.25 mg of 1.6 um of gold particles providing sufficient material for one bombardment. Further experimental details are described by the manufacturer (Bio-Rad) and by Sanford et al., (1993) Methods Enzymol. 217:438-509. Adult S. mansoni are collected from by perfusion of the liver and placed in a Petri dish containing culture medium, such as RPMI 1640, containing an additional 2 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, 10% heat inactivated FBS, 100 units penicillin and 100 ug/ml of streptomycin for 15 min to 24 hrs before bombardment (for further information see Gregoire et al., (1987) J. Immunol. 139:3792-3801). After removal of the medium, the animals are bombarded with the DNA covered gold particles, using a chamber vacuum of 15 inHg, a target distance of 3 cm and 1500-2,00 p.s.i. of pressure for acceleration.

Female worms are gently separated from their male partners and discarded (Gupta and Basch (1987) J. Parasitol. 73:481-486). Male adult S. mansoni are placed in Petri dishes containing medium 199, supplemented with foetal calf serum (10%) and kept at 37°C in 5% CO₂ (95:5). At intervals, culture medium is decanted and MJF content is determined by use of a sandwich ELISA in which a monoclonal anti-MJF serves as the capture antibody, a polyclonal rabbit anti-mouse as the MTF detector and purified mouse rMIF as a standard (Bacher et al (1996) Proc. Natl. Acad. Sci. USA 93:7849-7854).

Alternatively, adult male worms may be incubated in anticoagulated whole human blood. Following incubation for 8 hrs or more, leukocytes and platelets are isolated and examined for the appearance of surface marker proteins in response to activation by MIF using FACScan cytometry (Becton Dickinson) (see, Bruinje- Admiraal, L.G., Modderman, P.W., Borne A. E. G. K., Sonnenberg, A. P-selectin mediates Ca2+-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry. (1992) Blood 80:134-142).

Example 7: Secretion of macrophage migration inhibitory factor (MIF) by adult Ascaris spp

A construct to express MTF inserts the cDNA sequence encoding human MJF cDNA (Genbank No. 19686) upstream of a pGL-3 plasmid containing Ascaris 3' UTR and polyadenylation genes (gene 12 = ATP synthase, GenBank accession No. M33757; nucleotides 3056 to 3342) (SL RNA gene, GenBank accession No. M27961), a translational elongation factor and fert-1 (GenBank accession No. U07350). To improve secretion of MTF, additional or alternative inserts can be included. Additionally, signal peptide/targeting peptide sequences may be included in the construct to direct trafficking appropriately through the secretory pathway, or MIF may be generated as a fusion protein with a secreted Ascaris protein, in a similar manner to that described in Example 4 and elsewhere herein.

Adult Ascaris are collected and embryos isolated from female uteri and allowed to develop by culture in a shaking water bath at 30 C. After development for 5 days at 30 C (to the 32-64 cell stage), egg shells were removed using 0.4 NKOH/1.4% sodium hypochlorite for 90 minutes at 30 C. Plasmid DNA is precipitated onto gold microcarriers and introduced into batches of Ascaris embryos by biolistics (as described by Davis et al., (1999) Proc. Natl. Acad. Sci. USA 96:8687-8692, and in a similar manner to that set out in Example 4). Animals continue to grow during cultured in nematode blastomere medium and samples of culture fluid are collected at daily intervals and assayed for MIF as described in Example 6.

Example 8: Use of mice with gene knockouts to demonstrate secretion of transfected peptides or proteins in host organisms

Transfected peptides or proteins that are secreted by Schistosomes, e.g. nematodes will often share identity with endogenous peptides or proteins within mammalian hosts. Hence, animals with gene knockouts are used in order to demonstrate unequivocally that transfected organisms secrete peptides or proteins whilst resident within a host. Animals with gene knockouts lack the capacity to generate specific substances. For instance, by targeting embryonic stem cells it has been possible to generate a mouse strain whose members are unable to generate MIF (Bozza, M., Satoskar, A.R., Lin, G., Lu, B., Humbles, A.A., Gerard, C, David, J.R. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis. J. Exp. Med. (1999) 189:341-346). There are numerous mouse strains which lack specific genes and these may be identified by reference to an alphabetical listing (see http://www.bioscience.org /knockout/alphabet.htm).

As described in Example 6, gene constructs which determine expression of MIF can be introduced into adult male S. mansoni. Groups of transfected S. mansoni are introduced into a syringe and injected into ileocolic veins of mice, whose abdomens have been opened during brief anaesthesia with ether. On recovery, peripheral blood collected from such animals is tested at daily intervals for secretion of MIF which is measured by ELISA as described in Example 6. In such animals, functional evidence of MIF secretion is evidenced as an increased sensitivity to the lethal effects of high dose bacterial lipopolysaccharide.

As described in Example 7, gene constructs which determine expression of MIF can be introduced by gavage into embryo Ascaris worms. Groups of such worms are suspended in buffered saline and introduced by gavage into the stomach of mice which lack the gene for MEF. In such animals, recovery from MJF deficiency is evidenced by increased susceptibility to the lethal effects of high dose bacterial lipopolysaccharide. Sequences that encode other local hormones may be used in a similar manner to demonstrate secretion in trematodes such as S. mansoni and in Ascaris or other nematodes. Local hormones may be other cytokines (including IL-1, IL-2, IL-4, IL-5, JL-6, JL-7, IL-8, JL-10, IL-13, transforming growth factor beta, lymphotoxin and tumour necrosis factor alpha), cell maturation factors (including granulocyte- macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (GCSF), erythropoetin, JL-1 beta JL-3, thrombopoetin, nerve growth factor, platelet- derived growth factor, insulin-like growth factors, epidermal growth factor alpha and beta transforming growth factors), alpha, beta and gamma interferons and interferon regulatory factors; hormones including insulin, glucagon, amylin, calcitonin, calcitriol, thyroid hormone, parathyroid hormone, hypothalamic hormones (including growth hormone-releasing factor, somatostatin, thyrotrophin releasing hormone, corticotrophin releasing factor, gonadotrophin releasing factor, anterior pituitary hormones (including growth hormone, prolactin, corticotropin, melanocyte-stimulating hormone), adrenal hormones (including aldosterone, hydrocortisone and corticosterone), sex hormones (including testosterone, progesterone, oestradiol, oestriol, follicle stimulating hormone and luteinising hormone), gut hormones (including gastrin and secretin), blood proteins (including clotting factors NM and IX, angiotensinogen, kininogen, fibrinogen, low density lipoprotein, high density lipoprotein, very low density lipoprotein), circulating enzymes (including angiotens in-converting enzyme, kinin releasing enzymes, plasminogen activator, neutral endopeptidase), cell surface markers (including CD4 and CD8), neurotransmitters and their precursors (including substance P, substance K, neurokinin B, neuropeptide Y, leu-encephalin, dynorphin A, met-encephalin, β- endorphin, pre-proencephalin, neural nitric oxide synthase) and therapeutic biopharmaceuticals (including streptokinase, urokinase). For many of these substances, gene knockout strains of mice have already been identified and such recipients can be used to test the capacity of transgenic constructs to be expressed in trematodes or nematodes, including schistosomes and to secrete specific peptides or proteins within a host.

Claims

1. A transgenic eukaryotic vector organism comprising a heterologous coding sequence which encodes (a) a therapeutic polypeptide, or (b) a polypeptide capable of generating a therapeutic agent, which vector organism is capable of living non- pathogenically within a mammalian or avian host organism, and of expressing the coding sequence as a therapeutic polypeptide.

2. A vector organism according to claim 1 which is a helminth.

3. A vector organism according to claim 2 which is a nematode or trematode.

4. A vector organism according to claim 3 which is a schistosome.

5. A vector organism according to claim 4 selected from Schistosoma mansoni and Schistosoma j aponicum.

6. A vector organism according to claim 5 which is Onocera volvus, or Ascaris.

7. A vector organism according to any one of the preceding claims which is male.

8. A vector organism according to any one of the preceding claims which is capable of secreting the expressed polypeptide when the organism is living within mammalian or avian host.

9. A population of vector organisms wherein some or all of the vector organisms are transgenic vector organisms as defined in any of the preceding claims; in which population all the individuals are of one sex.

10. A population according to claim 8 wherein all the individuals are male.

11. A pharmaceutical composition comprising a vector organism according to any one of claims 1 to 7 or a population according to claim 8 or 9, and a pharmaceutically acceptable carrier.

12. A vector organism according to any one of claims 1 to 8 or a population according to claim 9 or 10 for use in a method of treatment of the human or animal body.

13. Use of a vector organism according to any one of claims 1 to 8 or a population according to claim 9 or 10 in the manufacture of a medicament for the treatment of the human or animal body.

14. A method of treatment of the human or animal body which comprises administering to a subject in need thereof an effective amount of vector organisms according to any one of claims 1 to 8, a population according to claim 9 or 10 or a pharmaceutical composition according to claim 11.

15. A nucleic acid construct comprising a therapeutic coding sequence, which sequence is capable of being expressed when the construct is introduced into a vector organism as defined in any one of claims 1 to 8.

16. A construct according to claim 15 wherein the coding sequence is operably linked to a promoter capable of directing its expression in a vector organism according to any one of claims 1 to 8.

17. A construct according to claim 15 or 16 further comprising a nucleic acid sequence capable of being expressed as a polypeptide signal sequence operably linked to the coding sequence such that, on expression, the encoding polypeptide is secreted from the vector organism.

18. A cell harbouring a construct according to any one of claims 15 to 17.

19. A cell according to claim 17 which is a cell of a eukaryotic vector organism as defined in any one of claims 1 to 8.

20. A process of producing a vector organism according to any one of claims 1 to 8 or a population of vector organisms according to claim 8 or 9, which process comprises (a) transforming or transfecting a vector organism with a nucleic acid construct according to any one of claims 15 to 17; (b) propagating thus obtained vector organism; and (c) recovering a transformed vector organism or population thereof.

21. A process according to claim 20 comprising transforming or transfecting the vector organism at the sporocyst stage of its life cycle, and optionally propagating the transformed sporocyst in vitro.

22. A process according to claim 21 comprising transforming or transfecting the vector organism at the adult stage of its life cycle.

23. A process according to any one of claims 20 to 22 additionally comprising propagating the vector organisms in vivo in a gastropod host organism and recovering a vector organism therefrom, at the sporocyst, schistosomula or cercaria stage of its life cycle.

24. A process according to any one of claims 20 to 23 comprising propagating the transformed vector organisms by introducing one or more miracidia into a gastropod host organism.

25. A process according to claim 24 wherein a single miracidium is introduced, so that propagation generates population of vector organisms of one sex only, as defined in claim 8 or 9.

26. A process according to any one of claims 20 to 25 comprising propagating the vector organisms by culturing them in vitro at the schistosomula stage of their life cycle.

27. A transgenic vector organism according to claim 1 substantially as hereinbefore described.

28. A population according to claim 9 substantially as hereinbefore described.

29. A pharmaceutical composition according to claim 11 substantially as hereinbefore described.

30. A vector organism according to claim 12 substantially as hereinbefore described.

31. Use according to claim 13 substantially as hereinbefore described.

32. A method according to claim 14 substantially as hereinbefore described.

33. A nucleic acid construct according to claim 15 substantially as hereinbefore described.

34. A process according to claim 20 substantially as hereinbefore described.