WO2009056961A2

WO2009056961A2 - Animal model

Info

Publication number: WO2009056961A2
Application number: PCT/IB2008/002918
Authority: WO
Inventors: Herman Pieter Spaink; Christoph Peter Bagowski
Original assignee: Leiden University
Priority date: 2007-10-30
Filing date: 2008-10-30
Publication date: 2009-05-07
Also published as: GB0920971D0; GB0721197D0; GB2463587A; EP2224844A2; WO2009056961A9

Abstract

The present invention provides an animal model which may be used to test the effects, monitor the transport, and/or determine the properties of drugs/biomolecules. More specifically, the animal model is a transparent non-human animal model which may be used to detect, analyse and assess the uptake of drugs/biomolecules at the single cell level, or even at an intra-cellular organelle level, or the effect of particular drugs/biomolecules on certain tissues/organelles and/or how treatments may be developed.

Description

ANIMAL MODEL FIELD OF THE INVENTION

The present invention provides an animal model which may be used to test the effects, monitor the transport, and/or determine the properties of drugs/biomolecules. More specifically, the animal model is a transparent non-human animal model which may be used to detect, analyse and assess the uptake of drugs/biomolecules at the single cell level, or even at an intra-cellular organelle level, or the effect of particular drugs/biomolecules on certain tissues/organelles and/or how treatments may be developed. BACKGROUND

A major obstacle to the success of a number of drugs is delivery to the desired place of action. Equally important is the stability of the drug, its half-life and excretion rate. Poor delivery, absorption, distribution, metabolism and excretion are often reasons for the failure of otherwise potent drugs. The delivery of peptide based and non-peptide based drugs into cells is crucial to the success of many biopharmaceutical drugs, indeed the drug not only has to enter the target cells but first has to reach the particular organ and tissue. Moreover, drugs which have an intra-cellular mode of action often need to be transported to particular organelles or sub-compartments of the cell. For instance, in the case of gene therapy, the therapeutic material must be delivered to the nucleus. The majority of small molecule drugs reach their targets because they are able to passively diffuse through the cell membrane and this requires that the drug is soluble in the polar extracellular milieu and in the non-polar cell membrane environment. For drugs that are not soluble in both the extracellular milieu and/or membrane, various methods are used to transport bioactive substances to the cell, the organelles and/or sub-compartments thereof.

There are a number of means by which macromolecules may be delivered into cells. These have been studied extensively and the transduction of nucleic acids into cells has been established as a routine and efficient process for many different types of cells. The transduction of peptides and proteins has progressed, but is far from being a standard lab technique yet.

It is important to note that most of the experiments have been carried out in in vitro systems and the results from these experiments do not necessarily translate to the situation in a living vertebrate organism.

Peptide and protein transduction is often attempted using transporter peptides such as, for example, the TAT protein from HIV, the VP22 protein from Herpes simplex virus and the Antennapedia protein from Drosophila (also known as Penetratin). The exact mechanism of protein transduction is still unclear and in the case of the TAT peptide, the sequence can be scrambled or the D-isomers of the amino acids used, suggesting that a specific ligand/receptor mediated mechanism is not responsible. As such, an unknown physical interaction between the peptide and the cell membrane is thought to permit cellular entry.

Protein transduction techniques are capable of delivering all kinds of substances, for example, small molecules, oligonucleotides, peptides, proteins, iron beads and liposomes to cells, although there are some limitations to these techniques.

Firstly, peptide delivery does not work in all cell types and toxicity has been observed with the use of different transporter peptides. Antibodies and humanized antibodies show great promise and are already used as successful drugs (e.g. Herceptine, Genentech). In addition, peptide aptamers have been developed as drugs and can be identified by phage display techniques and in high throughput randomized screens. Peptide aptamers are molecules selected for their intracellular binding to a specific target protein. Peptide aptamers provide a basis for the development of novel diagnostic and therapeutic strategies, with implications for a broad variety of different diseases, including cancer, viral infections, metabolic diseases and neurological disorders.

In addition to the above-described protein/peptide-based drugs, RNA/DNA, large/small molecule, gene therapy and chemical based drugs are used and are being continually developed. Recent advances in RNA/DNA based drug discovery include RNAi based drugs, ribozymes and oligonucleotide aptamers.

Fluorescence microscopy is the most sensitive technology for detection of molecular dynamics in living cells. Were it not for the fact that the tissues of patient/test organisms are not easily accessed by fluorescent light sources, this technology would be highly suited to applications requiring imaging of therapeutics. In the case of skin, there is very a limited depth of imaging possible even when 2- photon technology is applied. Therefore, imaging data of therapeutics is either obtained from tests on cell cultures or using bio-luminescence or radio-activity-based technologies on whole test organisms. However, the latter technology is insufficiently sensitive to allow imaging at the single cell level in living animals. Furthermore, using prior art technology, high doses are required in order to be able to obtain a signal. Therapeutic compounds are usually active at a low concentration and as such, the use of high doses represent an artificial and physiologically irrelevant situation. Additionally there are requirements for developing animal models of disease, so that disease progression and/or potential therapies can be developed.

It is among the objects of the present invention to obviate or mitigate the abovementioned problems with the prior art. SUMMARY OF THE INVENTION

The present invention is based upon the discovery that certain non-human transparent organisms may be used as a model in which to test the effects, monitor the transport and/or determine the properties of drugs/biomolecules into and around whole organisms and especially into and around tissues, cells, organelles and/or sub- cellular compartments (or regions). In addition, it has been found that in contrast to prior art methods, it is possible to detect, analyse and assess the uptake of drugs/biomolecules at the single cell level, or even at an intra-cellular organelle level in an entire living vertebrate organism in real time by non-invasive methods.

Thus in a first aspect, there is provided a method of assessing the cellular uptake of a drug/biomolecule, said method comprising the steps of:

(a) administering a drug/biomolecule to a transparent non-human organism; and

(b) detecting the cellular uptake of said compound in the transparent non- human organism. In a second aspect, there is provided a use of a transparent non-human organism in a method for assessing the cellular uptake of a drug/biomolecule.

In a further aspect there is provided use of a transparent non-human organism in a method for assessing infection by an infectious agent, in order to detect disease progression and/or potential utility of treatments. It is to be understood that the term "cellular uptake" should be taken to encompass the active or passive delivery, ingress, absorption, localisation, endocytosis, passage, penetration, transfer and/or diffusion of compounds into cells. In other words, the methods described herein permit the user to determine whether or not a particular compound or compounds is/are taken-up by cells. In addition, the term "cellular uptake" may encompass the subsequent passage or movement of compounds into sub-cellular compartments/regions and/or into or towards cell organelles such as, for example the nucleolus, nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus, vesicles, mitochondria, lysosomes and the like. As such, the methods disclosed herein may concern cellular, sub-cellular and/or organelle uptake and/or localisation.

In view of the above, the methods described herein may find particular use in methods of assessing and/or improving the delivery of drugs/biomolecules to cells and/or to specific regions/compartments and/or structures/organelles within cells. It is to be understood that the terms "drug(s)" and/or "biomolecules" should be taken to include (but not to be limited to) molecules, for example proteins (such as antibodies and/or fragments thereof), peptides, glycopeptides and nucleic acids. In addition, these terms may encompass pathogenic organisms, such as viruses (such as those used as viral vectors) and nucleic acids. By way of example, viral vectors which may be included within the scope of this invention may include, for example, adenoviral and/or lentiviral vectors. It has been observed by the present inventors that pathogenic organisms, such as viruses, may be taken up by zebrafish embryos, even where the viruses are not normally associated with zebrafish infection and the viruses visualised. This can allow for the study of movement of a pathogenic organism within the transparent non-human organism, such as zebrafish, or indeed as a potential model for testing possible treatments for infection. One of skill in the art would understand that the term "nucleic acids" is to include DNA, RNA and/or plasmids and/or antisense and/or inhibitory nucleic acids derived from either. Other compounds which may be subjected to the methods of the present invention may also include nucleic acid mimetics, such as, for example, morpholinos or PNAs and/or monosaccharides and/or polysaccharides.

"Drugs" and/or "biomolecules" may also be known as therapeutic compounds, medicaments or medicines. As such, the invention may concern methods of assessing or improving the cellular uptake (or cellular delivery) of, for example, chemotherapeutic, antiviral, antibiotic, antifungal, steroidal, analgesic, antipyretic and/or anti-inflammatory compounds and the like. The invention may also include methods involving therapeutic agents for use in treating certain genetic disorders such as, for example, compounds consisting or comprising of nucleic acids or compounds such as vectors (plasmid/viral vectors). It is to be understood that the list of drugs/biomolecules provided above is not exhaustive and one of skill in the art would readily be able to determine those drugs/biomolecules not listed here but which may also be subjected to the methods described herein.

In this regard, it is important to note that the methods described herein may be particularly useful in assessing and/or improving the cellular uptake/delivery of drugs/biomolecules which are not normally or not easily taken up by cells.

The present invention represents a clear and surprising advantage over the prior art as, using methods of cellular and sub-cellular detection, drugs/biomolecules may be detected within the cells of transparent non-human organisms- i.e. in a whole, live organism and at the single cell and sub-cellular (organelle) level, even when used at very low concentrations. This is particularly advantageous when the compound is a drug/biomolecule as it ensures that studies into the cellular uptake or delivery of that drug/biomolecule are conducted at physiologically relevant concentrations.

It is to be understood that the term "transparent non-human organism" may encompasses a number of non-human species including, for example, adult and/or embryo vertebrates, amphibians, reptiles and/or fish. In addition "transparent non- human organisms" should be taken to encompass any species the life cycle of which comprises a stage during which the organism is transparent - the various transparent life cycle stages may find application in the methods described herein. More specifically, the invention relates to organisms such as Zebrafish (Danio rend), Tetraodon, Medaka {Oryzias latipes), Glassfish or the like and/or species of frog such as, for example Xenopus laevis. In one embodiment, the transparent non-human organism may have been genetically altered so as to comprise, for example, transparent skin or the like and as such organisms such as the newly developed "sunroof amphibian" (Rena japonicά) may be used.

The techniques which may be used to administer a drug/biomolecule to a transparent non-human organism are well known to one of skill in this field and may include, for example, the injection of the drug/biomolecule into the organism or yolk during adult and/or early embryonic stages. Additionally or alternatively, the drug/biomolecule may be injected directly into certain cells, tissues, organs, structures or cells and/or administered by electroporation, canulation of the bladder/intestines, viral infection and/or vector, coating to a carrier composition and or inclusion in porous beads. In a further embodiment, a drug/biomolecule may be administered by adding it to food consumed by the organism or to some other substrate that the organism ingests and/or breathes. The compound may be injected into, for example, the developing embryo at the single cell stage, into the blood island cells or into the tail region. Additionally, or alternatively, the drug/biomolecule may be added to the water in which the organism bathes such that when respiring and or feeding, the transparent non-human organism takes in the compound/biomolecule. Preferably, when the drug/biomolecule is administered to the transparent non- human organism, it is alive. Even more preferably, when detecting the cellular uptake of the drug/biomolecule, the transparent non-human organism is also alive. In this way, it may be possible for the user to assess the cellular uptake of a drug/biomolecule in vivo, in a whole organism and under normal physiological conditions. Nevertheless, one of skill in the art will appreciate that certain methods of detecting drugs/biomolecules in vivo may not be suitable for use on live organisms. Accordingly, while the organism may be alive when the drug/biomolecule is administered, prior (preferably immediately prior) to detction of the drug/biomolecule, the organism may be killed. There are a number of ways in which the cellular uptake of the drug/biomolecule may be detected in the transparent non-human organism and preferably the method is a method of visual/optical detection. Advantageously, the drug/biomolecule may be labelled, conjugated or tagged to an optically detectable moiety. Optically detectable moieties which may be considered as suitable for use in the methods described herein may include (but are not limited to), for example, fluorescent compounds, quenching fluorescent compounds, Fluorescence resonance energy transfer (FRET) donors, Bioluminescence Resonance Energy Transfer (BRET) donors, luminescent compounds, enzymatic compounds, optical contrast compounds and/or iron contrast compounds. More specifically, compounds such as, for example, fluorophore dyes (Cyanine and the like), green fluorescent protein (GFP), rhodamine red and TAMRA may all be suitable for use.

Accordingly, by labelling a drug/biomolecule with an optically detectable moiety such as those described above, it may be possible to detect the labelled drug/biomolecule using techniques such as, for example fluorescence, electron and/or atomic force microscopy techniques.

In view of the above, and as already stated, the invention may relate to drug/biomolecule delivery/infection and in particular to methods of improving drug/biomolecule delivery or studying infection. In one embodiment, the methods provided by this invention may be used to develop or further develop drugs/biomolecules for use in treating disease with particular emphasis on ensuring that the drug/biomolecule is taken up by cells involved in or associated with a particular disease or condition.

One of skill in the art will appreciate that certain diseases or conditions may best be treated by drugs/biomolecules which specifically target certain tissues, organs and/or cells or which are specifically taken up by or delivered to certain cells. By way of example, renal or hepatic disorders may be expected to be best treated by drugs/biomolecules taken up by cells comprising the kidney or liver. As such, the methods described herein may be used to identify drugs/biomolecules which upon administration are taken up by or accumulate within (i.e. targeted or delivered to) certain cell types, or to identify compounds which, when administered with (either separately or bound, fused or conjugated thereto) drugs/biomolecules, modulate the cellular uptake and/or delivery thereof. By subjecting drugs/biomolecules and/or compounds to the methods described herein, it may be possible to identify drugs/biomolecules and/or compounds which once administered are specifically targeted to certain cells, tissues, structures or organs.

By making modifications to the drug/biomolecule and/or compound and subjecting the modified drug/biomolecule and/or compound to the methods described herein and comparing the results with those obtained using an unmodified form of the same drug/biomolecule and/or compound, it may be possible to identify modifications which have a positive effect on the cellular uptake of the drug/biomolecule and/or compound. Drugs/biomolecules and/or compounds identified as having improved cellular uptake may be regarded as exhibiting an improved bioavailability. It is to be understood that cells "involved in or associated with" the disease or condition may be those comprising a diseased tissue, organ or structure. For example, cells involved in or associated with a cancer may be considered as those cells which comprise a tumour.

The phrase "drug/biomolecule delivery" may be considered an aspect of a drug/biomolecule's efficacy, its pharmacokinetic properties and/or its bioavailability. It is to be understood that the term "bioavailability" may be taken to mean the in vivo and/or cellular distribution of a drug/biomolecule after administration. More specifically, the "bioavailability" of a drug/biomolecule may be taken to relate to the number of organs, tissues or cells a particular drug/biomolecule reaches, or may be detected in, after administration. Similarly, the bioavailability of a drug/biomolecule may relate to the extent to which a drug/biomolecule can penetrate a cell - for example a drug/biomolecule which is capable of penetrating the nucleolus may be considered as more bio-available than a drug/biomolecule which only penetrates as far as the cytoplasm of the same cell. In addition, it is to be understood that after administration, certain drugs/biomolecules may only be taken up by a few cells of certain organs or tissues - such drugs/biomolecules may be regarded as having a limited bioavailability/infectivity. Other drugs/biomolecules may be taken up by many more cells and as such may be considered to exhibit a wider degree of bioavailability/infectivity. In addition, the term "bioavailability" may encompass the total concentration of a drug/biomolecule taken up by cells after administration. A drug/biomolecule with a high bioavailability may be considered as one in which a high concentration of the drug/biomolecule administered to an organism is taken up by the cells of that organism.

Thus, in a further embodiment, the present invention may provide methods which permit the user to determine whether or not the bioavailability/infectivity (i.e. the intra-cellular delivery or sub-cellular localisation) of a particular drug/biomolecule is modulated by one or more other (or "secondary") compounds. For example, drugs/biomolecules may be administered in combination with one or more secondary compounds, either together as a single composition or separately. In a further embodiment, the one or more secondary compound(s) may be bound, conjugated to, or associated with the drug/biomolecule.

Accordingly, the present invention may provide a method for determining whether or not a compound (i.e. a secondary compound) is capable of modulating the bioavailability/infectivity of a drug/biomolecule, said method comprising the steps of:

(a) administering the compound and the drug/biomolecule to a transparent non-human organism; and

(b) detecting the uptake of the drug/biomolecule by the cells of the transparent non-human organism; wherein a compound capable of modulating the bioavailability/infectivity of a drug/biomolecule modulates the uptake of the drug by the cells of the transparent non- human organism.

As stated, it is to be understood that the combination of a "compound and drug/biomolecule" as mentioned above, should be taken to include instances where a drug/biomolecule is administered in combination with a secondary compound - either separately or together in a single composition or, more preferably, instances where the compound is conjugated or otherwise bound or fused to the drug/biomolecule.

In addition, by making modifications to the compound and/or drug/biomolecule and subjecting the modified compound and/or drug/biomolecule to the above described method and comparing the results with those obtained using an unmodified form of the same compound and/or drug/biomolecule, it may be possible to identify modifications which have a modulatory, such as a positive effect on the cellular uptake of the compound and/or drug/biomolecule. Compounds and/or modifications identified as increasing the delivery/cellular uptake of a drug/biomolecule may be useful in the treatment of certain diseases and/or condition.

Preferably, and as stated above, the compound (or secondary compound) is a compound administered together with, or in combination with a drug/biomolecule (either as a separate entity or bound, conjugated or fused thereto). Almost any type of compound may be used in this way and as such, the term "compound" should be taken to include (but not be limited to) for example, small organic molecules, proteins, peptides, amino acids, polysaccharides, monosaccharides, carbohydrates, antibodies (or fragments thereof), nucleic acids, nucleic acid mimetics and/or metal ions. In a further embodiment, the present invention may provide a method of determining whether or not a modification made to a drug/biomolecule modulates the cellular uptake/delivery of the drug/biomolecule, said method comprising the steps of:

(a) administering a modified drug/biomolecule to transparent non-human organism; and

(b) detecting the cellular uptake of the modified drug/biomolecule in the transparent non-human organism ; wherein, by comparing the results with those obtained using an unmodified version of the same drug/biomolecule, it may be possible to determine whether or not the modification made to the drug/biomolecule modulates the cellular uptake/delivery thereof.

The term "modulated" as used herein should be taken to mean that the drug/biomolecule has, as a result of the modification made thereto and relative to an unmodified version of the same drug/biomolecule, an increased or decreased ability to be taken up by, or delivered to, a cell.

In a further embodiment, it may be possible to use any of the abovementioned methods in an automated and/or high throughput fashion. For example, individual non-human transparent organisms may be cultured in, for example, the wells of a microtiter plate and contacted with drug/biomolecules tagged or labelled with optically detectable tags and optionally combined, conjugated, bound or fused to secondary compounds (as described above). In this way, the methods described herein and the cellular uptake/delivery of a large number of drugs/biomolecules can be practiced/assessed at any one time. Additionally or alternatively, and with relevance to each of the methods described herein, the drug/biomolecule administration procedures, culture protocols and/or labelled drug/biomolecule detection methods may be fully automated.

In one embodiment, the methods do not concern transgenic non-human transparent organisms wherein certain genes have been altered or mutated or heterologous genes added to render the non-human transparent organism or certain structures/cells present within, detectable using any of the optical detection methods described above.

For example, non-human transparent organisms which have been genetically altered so as to render one or more structures/cells thereof fluorescent may not be included in the methods described herein.

As stated above, the term "drugs/biomolecules" as used herein, may include certain vectors such as, for example, viral vectors and specific examples may include adenoviral and/or lentiviral vectors, as well as infection agents such as viruses. These vectors or viruses may be administered to transparent non-human organisms either alone (as a drug/biomolecule) or they may further comprise a drug/biomolecule which is to be delivered to one or more cells or sub-cellular compartments/regions of the transparent non-human organism. This is particularly useful where the drug/biomolecule to be delivered to a cell is a nucleic acid. As such, the present invention may provide a method of assessing the cellular uptake of a drug/biomolecule, said method comprising the steps of:

(a) administering a vector such as a viral vector, comprising the drug/biomolecule to a transparent non-human organism ; and

(b) detecting the cellular uptake of said vector in the transparent non- human and/or effect of the drug/biomolecule on the transparent non-human. In a further aspect, there is provided a use of a viral vector for assessing and/or determining the cellular uptake of a drug/biomolecule in a transparent non-human organism.

One of skill in the art will immediately appreciate that such methods may be particularly useful when the drug/biomolecule is a nucleic acid to be used in some form of gene therapy.

In another embodiment transgenic non-human organisms can be used. These transgenic non-human organisms can be obtained via viral delivery of a gene SiRNA, antisense molecule or the like as described herein. One of skill in the art will appreciate that it may be possible to combine a method for assessing the cellular uptake of a drug/biomolecule with the administration of a vector, such as a viral vector, as described herein. This is particularly beneficial as it may provide a rapid means for marking or detecting successful transport of a drug/biomolecule to a cell and/or to a sub-cellular compartment/region thereof. For example, in the case of the transport of a drug/biomolecule to the nucleus of a cell, it may be desirable to first administer to the transparent non-human organism, a vector (for example a viral vector) that encodes a moiety capable of binding the drug/biomolecule. In this way, the moiety capable of binding the drug/biomolecule may become expressed on the cytoplasmic and/or nuclear membrane of a cell and hence facilitate the delivery of the drug/biomolecule to the nucleus.

Thus, in one embodiment, the present invention provides a further method of assessing the cellular uptake of a drug/biomolecule, said method comprising the steps of:

(a) introducing a nucleic acid encoding a moiety capable of binding the drug/biomolecule to a cell of a transparent non-human organism; (b) administering said drug/biomolecule to the transparent non-human organism; and

(c) detecting binding between the drug/biomolecule and the moiety capable of binding thereto. Preferably, the nucleic acid is introduced using a vector such as, for example, a viral vector. In one embodiment, the viral vector may be an adenoviral, lentiviral vector or the like.

Advantageously, the vector may further comprise a reporter element such as, for example lacZ or GFP. One of skill in the art will readily appreciate that the observation that viral vectors, such as adenoviral and other virus vectors can be used in this way represents a surprising finding and provides a wealth of opportunities within the medical research field.

Binding between the drug/biomolecule and the moiety encoded by the vector can for example be monitored by, for example fluorescence resonance energy transfer

(FRET) methodology. By way of an illustrative and non-limiting example, an adenoviral vector encoding a 14-3-3 gene fused to optically detectable moieties may first be administered to a transparent non-human organism. Subsequently, a peptide labelled with an optically detectable tag, for example a R18-TAMRA peptide may be administered such that when the Rl 8 binds 14-3-3, a FRET signal is generated.

An alternative to the use of FRET based detection techniques may require the detection and/or measurement of the level of activation/inhibition of a reporter transcript. Advantageously, the reporter transcript may be under the control of the moiety capable of binding the drug/biomolecule encoded by the vector. One of skill in the art will appreciate that since the moiety capable of binding the drug/biomolecule may be present in the nucleus, binding between the drug/biomolecule and the moiety will signal the delivery of the peptide even if the drug/biomolecule is not fluorescently labelled. The fact that the drug/biomolecule need not have to be tagged with an optically labelled moiety offers distinct advantages over the prior art methods for assessing the cellular uptake of a drug/biomolecule.

DETAILED DESCRIPTION

The present invention will now be described by reference to the following Figures which show:

Figure 1 : Confocal microscopy showing cellular uptake of PNA-Tamra in the Zebrafish. (A-E) PNA-Tamra injected at one-cell stage; (F) PNA-Tamra administered by bathing protocol; (G, H) PNA-Tamra injected directly into blood islands.

Figure 2: Confocal microscopy showing cellular uptake of R18-Tamra in the Zebrafish. (A-B) R18-Tamra injected at one-cell stage; (C-D) R18-Tamra administered by bathing protocol; (D) R18-Tamra administered by tail injection; (E) Rl 8-Tamra administered by injection into blood islands.

Figure 3: Adenovirus vector injected at one cell stage and embryos stained at various times after injection; (A) 2 days; (B-E) 5 days post injection. Also shown is a control sample that shows there is no background staining with X-gal in zebrafish.

Figure 4 shows MHV virus injected in the head, showing cells infected in the head region above the otic vesicle.

Figure 5 shows MHV virus bathed at sphere stage with 1:100 MHV paricles diluted in egg water shows cells on the miotomes. Material and methods Animal care and handling Zebrafish (Danio rerio) (Tuebingen line and AB strain) were handled in compliance with local animal care regulations and standard protocols. Fish were kept at 28° C in aquaria with day/night light cycles (lOhours dark versus 14hours light periods). The developing embryos were kept in an incubator at 28° C. Injection of zebrafish embryos

The stock of the respective compound was thawed and diluted in water to the working condition of which 1 nl was injected into the yolk of 1-cell stage embryos, in the blood islands of embryos at 24 hours of development, or directly in tail tissue of 24 hours post fertilization embryos. Fluorescent microscopy

Pictures of live embryos at the respective stages were taken either in a BioRad MRC 1024ES confocal microscope (Carl Zeiss, Jena) or imaging was done with the BioRad CSLM (BioRad 1024ES; Software: BioRad Laser sharp 2000). Compounds used. (i) TAMRA-Rl 8 peptide

The Rl 8 amino acid sequence NH₂-PHCVPRDLSWLDLEANMCLP-COOH labelled with tetramethylrhodamine (TAMRA) at the N-terminus was synthesised by

Sigma (Sigma-Genosys Ltd, Cambridge, UK). MS spectra showed no degradation of the peptide. It was stored at -20°C as lyophilized powder. The peptide was dissolved to 46 μM in 20 mM HEPES pH 7.01 , aliquoted and stored in the dark at -20°C.

(ii) TAMRA-PNA

A peptide nucleic acid oligonucleotide was organically synthesized. At the 5 prime terminus of the single stranded oligonucleotide a peptide was coupled that consists of the following amino acids: NH2CO-valine-lysine-arginine-lysine-lysine- lysine-proline. The oligonucleotide sequence (called pi 8) was as follows TAGCCGGTAGTCCAA. TO the 3 prime end of this oligonucleotide a TAMRA dye was coupled. The total MW of the compound is 4088.59 and it was dissolved at a concentration of 139.7 micromolar.

(iii) Adenoviral vector The viral vector used was described by Driesse et al. Staining of lacZ was performed using X-gal in a standard procedure as described by Lin et al. In several cases sections were made to visualize the blue staining. Example 1 Injection of the PNA Tamra coupled compound and its uptake in zebrafish embryos. When PNA-Tamra was injected in the yolk of a one cell-stage embryos (Fig.l

A and B) at later stages of development, in this case at the tail bud stage (about 10 hours after injection), the compound was detected in nearly all areas of the embryo although with different intensities. In all areas of the embryo it is apparent that cells have taken up the compound (Fig. IA). Furthermore, a higher resolution picture shows that in areas of the embryo in a high number of cells which have taken up PNA-Tamra, a signal is detected in the cell nucleus (Fig. IB). This result shows the in vivo function of the nuclear localization signal present in PNA. At a later stage of development, at 24 hpf the PNA-Tamra signal is still detected distributed throughout the embryo in the head region (Fig. 1C), weakly in the myotomes and the somites (Fig. ID), in the notochord and the tail region (Fig. IE). In a bathing experiment, the embryos at 17.5 hpf were bathed in a watery solution containing PNA-Tamra for half an hour and subsequently washed three times. After 6 hours live imaging using confocal fluorescent microscopy was performed. Clear signals are detected throughout the embryo also in further developed parts of the embryo showing cellular uptake of the compound (Fig. IF). The PNA-Tamra was further injected in the blood islands at 24 hpf and 4 hours later the PNA-Tamra signal was detected by confocal microscopy. Head regions show cellular uptake (Fig. IG) and also parts of the myotomes and the somites show PNA-Tamra incorporation (Fig. IH). Example 2 Injection of the Rl 8 Tamra coupled peptide

When the Rl 8 peptide coupled to Tamra was injected in the yolk of a one cell- stage embryos at the tail bud stage (about 10 hours after injection), the compound was detected well distributed in nearly all areas of the embryo although with different intensities (Fig.2A). In all areas of the embryo it is apparent that cells have taken up the compound in contrast to the PNA-Tamra compound described above the higher magnification picture of the live embryo reveals a positive signal more around the perimeter of the cells, indicating membrane localization for the Rl 8 peptide. In a similar bathing experiment as described above, we see cellular uptake and distribution throughout the embryo (Fig.2C and D). An R18-Tamra signal is detected in the head region in specific structures like the proliferative cell layer and other structure (Fig. 2C), a signal is further observed in the myotomes and the somites (Fig. 2D). The Rl 8- Tamra peptide was further injected directly in the tail of a 24 hpf embryo and in the blood islands at 24 hpf. 4 hours later the PNA-Tamra signal was detected by confocal microscopy. In both cases, injection directly in the tail region as well as in the blood islands the R18-Tamra is taken up by distant cells and can be detected in different organs and cellular structures (Fig.2E and F, respectively. Example 3 Injection of an adenoviral vector.

We injected the viral vector IG.Ad.CMV.LacZ as described by Driesse et al. into 1 cell stage embryos using standard procedures using titers as mentioned by Driesse et al. The results are shown for a two day embryo in figure 3a and for later stage embryos in figure 3b and 3c. Clear staining was observed in various parts of the fish body whereas no staining was detectable in control experiments, demonstrating the functionality of the viral vector in zebrafish. Example 4

Three different RNA viruses were tested for their ability to infect zebrafish cells:

1. Mouse hepatitis virus (MHV) Background: Mouse hepatitis virus is probably the most important pathogen of laboratory mice. Although the infection generally causes no overt clinical signs, it can cause profound changes in the immune system, affecting the interpretation of a wide variety of experimental results. It is an ssRNA virus of the family Coronaviridae. Approximately 25 strains or isolates of MHV have been described. Transmission: MHV is extremely contagious and is transmitted primarily via aerosol, direct contact, fomites , and, experimentally, via transplantable tumors and via the placenta.

2. Equine arteritis virus (EAV) Background: Equine arteritis virus is restricted to the Equidae. Antibodies to this virus have been found in horses, ponies, and zebras, and outbreaks have occurred in horses and ponies. The prevalence of the virus can vary significantly among horse breeds; Standard breeds are particularly susceptible.

Transmission: Equine arteritis virus can be transmitted by both the respiratory and the venereal route. Acutely affected horses excrete the virus in aerosols; aerosol transmission predominates when horses are gathered at racetracks, sales, shows, and other events. Venereal transmission from carrier stallions is particularly significant on breeding farms. Stallions appear to be the only carriers for the virus; carrier states have not been seen in mares, geldings, or sexually immature colts. Equine arteritis virus can also be carried on fomites. Mares infected late in pregnancy may give birth to foals infected in utero.

3. Sindbis Virus (SINV)

Background: Sindbis Virus (SINV) is a member of the Togaviridae family, in the alphavirus subfamily. The virus was first isolated in 1952 in Cairo, Egypt. The virus is transmitted by mosquitoes (Culex spp.) SINV causes sindbis fever in humans and the symptoms include arthralgia, rash and malaise. Sindbis fever is most common in South and East Africa, Egypt, Israel, Philippines and parts of Australia. Sindbis virus is an "arbovirus" (arthropod-borne) Transmission: Sindbis Virus is transmitted between vertebrate (bird) hosts and invertebrate (mosquito) vectors. Humans are infected with Sindbis virus when bitten by an infected mosquito.

Materials and Methods Zebrafish embryos at Odpf and 4dpf were either injected with the virus or bathed with virus. An injection of virus

1 - EAV pure virus injected in the yolk of 8 cell stage zebrafish embryos

2 - EAV pure virus injected in the cell of 1 cell stage zebrafish embryos 3 - EAV pure virus injected in the yolk of 4dpf zebrafish embryos 4 - EAV pure virus injected in the hindbrain of 4dpf zebrafish embryos

5 - MHV pure virus injected in the yolk of 8 cell stage zebrafish embryos

6 - MHV pure virus injected in the cell of 1 cell stage zebrafish embryos

7 - MHV pure virus injected in the yolk of 4dpf zebrafish embryos 8 - MHV pure virus injected in the hindbrain of 4dpf zebrafish embryos

9 - Sindbis pure virus injected in the yolk of 8 cell stage zebrafish embryos

10 - Sindbis pure virus injected in the cell of 1 cell stage zebrafish embryos

11 - Sindbis pure virus injected in the yolk of 4dpf zebrafish embryos

12 - Sindbis pure virus injected in the hindbrain of 4dpf zebrafish embryos

B - Bathing of Zebrafish embryos in egg water containing virus

Results

For all three viruses tested Embryos bathed in egg water with viral particles showed signs of infection (GFP signal inside zebrafish cells). Also when the different viruses were injected either ectopically (in the yolk) or directly in the embryo at different locations, signs of infection were detected for all three different RNA viruses. For both means of viral delivery, examples are given for the MHV virus in Figure 4 and 5.

References

Driesse MJ, Kros JM, Avezaat CJ, Valerio D, Vecht CJ, Bout A, Smitt PA. (1999) Distribution of recombinant adenovirus in the cerebrospinal fluid of nonhuman primates. Hum Gene Ther. 10(14):2347-54.

Lin S., Yang S., Hopkins N.(1994) LacZ expression in germline transgenic zebrafish can be detected in living embryos. Dev £ιo/.;161(l):77-83.

Wang, B., H. Yang, Y. C. Liu, T. Jelinek, L. Zhang, E. Ruoslahti, and H. Fu. 1999. Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. Biochemistry 38:12499-12504.

Claims

Claims:

1. A method of assessing the cellular uptake of a drug/biomolecule, said method comprising the steps of: (c) administering a drug/biomolecule to a transparent non-human organism; and

(d) detecting the cellular uptake of said drug/biomilecule compound in the transparent non-human organism.

2. Use of a transparent non-human organism to test the effects, monitor the transport and/or determine the properties of drugs/biomolecules.

3. The method of claim 1, wherein the method provides a means of assessing or improving the delivery of drugs/biomolecules to cells and/or to specific structures/organelles within cells.

4. The method of claim 1 for providing a model of infection, wherein an infectious or pathogenic agent can be visualised in said transparent non-human organism.

5. The method according to claim 4 wherein the infectious or pathogenic agent is a virus.

6. The method or use of any preceding claim, wherein the "transparent non- human organism" is an adult and/or embryo selected from the group consisting of:

(i) vertebrate; (ii) amphibian,

(iii) reptile; and (iv) fish.

7. The method or use of any preceding claim, wherein the drug/biomolecule is administer to a live transparent non-human organism.

8. The method or use of any preceding claim wherein the assessment of cellular uptake is performed in a live transparent non-human organism.

9. The method or use of any preceding claim, wherein the drug/biomolecule is a viral vector, or virus.

10. The method or use of any preceding claim, wherein the drug/biomolecule is labelled, conjugated or tagged to an optically detectable moiety.

1 1. The method or use of claim 10, wherein the optically detectable moiety is selected from the group consisting of:

(i) fluorescent compounds; (ii) quenching fluorescent compounds; (iii) fluorescence resonance energy transfer (FRET) donors or acceptors; (iv) bioluminescence Resonance Energy Transfer (BRET) donors; (v) luminescent compounds; (vi) enzymatic compounds; and

(vii) optical contrast compounds and/or iron contrast compounds.

12. The method or use of claims 8 or 9, wherein the optically detectable tag is selected from the group consisting of:

(i) fluorophore dyes; (ii) green fluorescent protein; (iii)rhodamine red; and

TAMRA.

13. The method or use of claims 8 -12, wherein the labelled drug/biomolecule is detected by fluorescence, electron and/or atomic force microscopy techniques

14. A method for determining whether or not a compound is capable of modulating the bioavailability of a drug/biomolecule, said method comprising the steps of:

(c) administering the compound and the drug/biomolecule to a transparent non-human organism; and

(d) detecting the uptake of the drug/biomolecule by the cells of the transparent non-human organism; wherein a compound capable of modulating the bioavailability of a drug/biomolecule modulates the uptake of the drug by the cells of the transparent non- human organism.

15. The method of claim 14, wherein the compound is bound, conjugated to, or associated with the drug/biomolecule.

16. A method of determining whether or not a modification made to a drug/biomolecule modulates the cellular uptake/delivery of the drug/biomolecule, said method comprising the steps of:

(a) administering a modified drug/biomolecule to transparent non-human organism; and (b) detecting the cellular uptake of the modified drug/biomolecule in the transparent non-human organism ; wherein, by comparing the results with those obtained using an unmodified version of the same drug/biomolecule, it may be possible to determine whether or not the modification made to the drug/biomolecule modulates the cellular uptake/delivery thereof.

17. The method or use of any preceding claim, conducted in an automated and/or high through-put fashion.

18. A method of assessing the cellular uptake of a drug/biomolecule, said method comprising the steps of:

(a) administering a vector comprising the drug/biomolecule to a transparent non-human organism ; and

(b) detecting the cellular uptake of said vector in the transparent non- human .

19. Use of a viral vector for assessing and/or determining the cellular uptake of a drug/biomolecule in a transparent non-human organism.

20. A method of assessing the cellular uptake of a drug/biomolecule, said method comprising the steps of:

(a) introducing a nucleic acid encoding a moiety capable of binding the drug/biomolecule to a cell of a transparent non-human organism;

(b) administering said drug/biomolecule to the transparent non-human organism; and

(c) detecting binding between the drug/biomolecule and the moiety capable of binding thereto.

21. The method of claim 20, wherein the nucleic acid is introduced using a vector such as, for example, a viral vector.

22. A method of studying infection of an agent, said method comprising the steps of: a) introducing an infectious agent to a cell of a transparent non-human organism; and b) optically observing whether or not infection occurs in said transparent non-human organism.

23. The method according to claim 22 for studying infection of agents not generally associated with infection of said non-human transparent organism.

24. The method according to claims 22 or 23 for use in studying disease progression.

25. The method according to claims 22 or 23 for use in testing agents/compounds for treating and/or preventing infection by the infectious agent.