WO1998023260A2

WO1998023260A2 - Non-ionic surfactant vesicles as a delivery agent for nucleic acid

Info

Publication number: WO1998023260A2
Application number: PCT/GB1997/003235
Authority: WO
Inventors: Katharine Christine Carter; Alexander Balfour Mullen; Alan James Baillie; John Douglas Ansell; William Gerard Murphy; Kay Samuel
Original assignee: University of Edinburgh; University of Strathclyde; Common Services Agency for Scottish Health Service
Current assignee: University of Edinburgh; University of Strathclyde; Common Services Agency for Scottish Health Service
Priority date: 1996-11-26
Filing date: 1997-11-26
Publication date: 1998-06-04
Anticipated expiration: 1999-05-26
Also published as: WO1998023260A3; JP2001510459A; EP0946148A2; GB9624536D0

Abstract

The present invention provides an aqueous formulation (especially an injectable formulation) comprising a polynucleotide and a carrier component suspended in an aqueous delivery vehicle. The formulation though not exclusively comprises DNA or RNA in combination with vesicles comprised of non-ionic surfactants.

Description

Non-Ionic Surfactant Vesicles as a Delivery Agent for

Nucleic Acid

The present invention relates to an aqueous formulation (such as an injectable formulation) comprising a polynucleotide and a carrier component suspended in an aqueous delivery vehicle. The formulation particularly though not exclusively comprises DNA or RNA in combination with vesicles comprised of non-ionic surfactants.

Many diseases, such as Factor IX deficiency, are genetic disorders in which there is an inability functionally to express the gene or genes responsible for the production of corresponding peptide (s) or protein(s) . For example, Factor IX deficiency (Haemophilia B) gives rise to a life long bleeding disorder, requiring therapeutic transfusion with the missing plasma-derived clotting factors. The clinical and economic cost of the disease to both patient and community is substantial. Factor IX is normally produced in the liver, although it has been shown that non-hepatic cells are capable of expression and post-translational modification of Factor IX. Genetic modification of haematopoietic stem precursors (which are more accessible than liver cells) after their transfection with exogenous nucleotides and transplantation back into the affected individual may induce high level, long term production of the protein by the derivative progeny, leading to clinical control of the disorder. In recombinant research "transfection kits" could be used as an in vitro , ex vivo or in vivo research tool.

It is known in the art that lipid vesicles have been used as drug carriers. USP 4217344 describes a process for the production of spheres which are capable of encapsulating water-soluble pharmaceuticals and the like, although, the entrapment of polynucleotides is neither described nor suggested.

USP 4235871 describes a method of encapsulating polynucleotides in synthetic, oligolamellar lipid vesicles or liposomes, however, the said vesicles or liposomes do not comprise non-ionic surfactants and there is no description of transfection efficiency or any indication of the stability of the system.

Other workers have reported the use of other methods for the transfection of cells with polynucleotides, particularly DNA. Such methods have included electroporation, use of free DNA, lipid coated plasmids, use of CaP0₄ and other chemical agents (Yoshida T. and Tsuda H. Biochem. and Biophys. Res. Comm (1995) Vol. 214, No. 2 pp701-708; Staedel C. et al Journal of Invest. Dermatol. (1994) Vol. 102, No. 5 pp 768-772; Yagi K. et al Biochem. and Biophys. Res. Comm (1993) Vol. 196, No. 3 pp 1042-1048; Fraley R.T. et al Proc. Natl. Sci. U.S.A. (1979) Vol. 76, No. 7 pp 3348-3352; Behr J-P et al Proc. Natl. Acad. Sci. USA (1989) Vol. 86 pp 6982-6986; Bahnson A.B. and Boggs S.S. Biochem. and Biophys. Res. Comm (1990) Vol. 171, No. 2 pp 752-757; and Sipehia R. and Martucci G. Biochem. and Biophys. Res. Comm. (1995) Vol. 214, No. 1 pp 206-211). A problem associated with the above referenced prior art has been that transfection efficiency, if reported, is relatively low and that gene expression tends to be transient. A further problem is that stability of integration of the nucleotide associated with transfection is generally poor.

Diverse non-ionic surfactants can be used to form vesicles with potential therapeutic applications such as drug delivery (Ozer et al., 1991) and immunological adjuvants (Brewer and Alexander, 1993) . We have already demonstrated (Carter et al., 1989a, b) that stibogluconate loaded non-ionic surfactant vesicles are as effective as drug loaded liposomes in the treatment of experimental visceral leishmaniasis.

It is an object of the present invention to provide DNA loaded non-ionic surfactant vesicle formulations of improved efficacy. This and other objects of the present invention will become apparent from the following description and examples.

Broadly stated, the present invention resides in the finding that improved transfection efficacy and/or stability of expression can be achieved by providing the polynucleotide carried by, non-ionic surfactant vesicles such as entrapped within and/or associated with the non- ionic surfactant vesicle walls. In an embodiment the polynucleotide may also be further located in the aqueous carrier vehicle. Thus, the present invention provides a formulation which comprises :- an aqueous vehicle; vesicles comprising a non-ionic surfactant suspended in the aqueous vehicle, and polynucleotide fragments carried by said vesicles.

It has now therefore been found that DNA carried by vesicles comprising non-ionic surfactants demonstrate better transfection efficiencies and/or greater stability of expression/integration than vesicles hitherto described in the art.

Use of the formulations of the present invention attain stable transfection efficiencies in vitro of between 7.5% to 96%, preferably between 20% to 90%.

Transfection efficiency may be taken to mean a percentage of cells in a given population which are transformed by a polynucleotide of interest.

It will be appreciated that the formulation of the present invention may be used to transfect cells in vitro or in vivo.

In another aspect there is provided formulations of the present invention for use in the preparation of medicaments for use in therapy.

The polynucleotide fragments carried by said vesicles may be entrapped within the vesicle aqueous phase and/or associated with the surfactant bi-layer(s) of the vesicle walls. Thus, the polynucleotide fragment may be located within the vesicle bi-layer(s) , in the inter-lamellar space (s), and/or it may be located on the surface of the vesicle bi-layers. The polynucleotide fragments may also be found in the aqueous vehicle. The concentration of polynucleotide in the aqueous vehicle may be the same, greater or lower than the concentration thereof carried by the vesicles. For convenience, the aqueous vehicle containing the polynucleotide fragments will generally be that which is used to load the polynucleotide fragments into and/or onto the vesicles, where such a method is used to introduce the polynucleotide fragments into and/or onto the vesicles.

In principle, the formulation containing the vesicles may be formed in any manner known in the art and appropriate to the polynucleotide fragments to be delivered. For example, vesicle formulations can be formed using either a "homogenisation" method or a "freeze-dried" method, both methods being known in the art. In the homogenisation method a required quantity of vesicle components, in a desired molar ratio can be processed in one of the following ways: Dry powdered vesicle components are hydrated with a solution of the polynucleotide for entrapment at a temperature which does not result in the denaturation of the polynucleotide fragments, for example, in the range from 0 up to 70°C and homogenised at the required speed and for the required length of time to produce the desired vesicle characteristics. Alternatively lipid material can be melted by the application of heat (e.g. temperature range 40-150°C) prior to hydration with the required solution at the necessary temperature. The suspension can then be homogenised at the required speed to produce vesicles having the desired characteristics.

Polynucleotide-containing vesicle suspensions can be produced using the homogenisation method outlined above by heating the vesicle constituents, for example, at 135°C. The molten lipid can then be cooled to, for example 70°C prior to hydration with preheated polynucleotide solution. Vesicle size reduction is achieved by mechanical homogenisation of the sample at a specific temperature. For example homogenisation at 8000 rpm for 15 minutes on a Silverson mixer result in vesicles suspensions with a mean diameter of 1030 ± 24.8 nm when hydrated with a solution containing 40 μg/ml of a hygro ycin plasmid construct.

In the freeze-dried method, a freeze dried preparation can be made in one of the following ways:

The required quantity of vesicle constituents in a desired molar ratio can be dissolved in an organic solvent (e.g. t-butyl alcohol) . This solution can then be frozen and freeze-dried for the time required for complete removal of organic solvent. The resultant lyophilised product can then be hydrated with a solution of the polynucleotide and agitated/homogenised at the required temperature to produce a vesicle suspension. Alternatively, vesicle suspensions are produced by the homogenisation process described above, and then lyophilised to remove the aqueous solvent. The resultant lyophilised product can then be hydrated with the required polynucleotide solution and agitated at the required temperature to produce a vesicle suspension.

A portion of the vesicles may become associated with a surface of the receptacle in which the vesicles are prepared. Thus, a vesicle preparation of the present invention may be obtained from a so-called "whole preparation" which comprises vesicles which remain in suspension, together with vesicles which have become associated with the surface of the receptacle; vesicles which have become associated with the surface of the receptacle may be removed by washing the surface of the receptacle and are thus termed the "flask wash" preparation; or vesicles which only remain in suspension.

The vesicles are preferably formed of a sterol such as cholesterol or ergosterol, together with a non-ionic surfactant. It is generally necessary to include a charged amphophile such as a fatty acid within the vesicle formulation in order to prevent vesicle aggregation. Suitable charged amphophiles include dicetylphosphate, stearic acid and palmitic acid.

It has been found particularly advantageous to employ a non-ionic surfactant. This may be a mono, di-, tri-, or poly (up to 10) glycerol mono- or di-fatty acid ester (e.g. a C₁₀ - C₂₀ fatty acid ester) such as triglycerol monostearate; or a polyoxyethylene ether preferably comprising from 1 to 10 oxyethylene moieties with a C₁₀ - C₂₀ normal or branched alkyl chain. Particularly preferred surfactants are Surfactant V (triglycerol monostearate) , Surfactant VI (hexaglycerol distearate) , Surfactant VII (diethylene glycol mono n-hexadecylether) , Surfactant VIII (tetraethylene glycol mono n-hexadecylether) .

According to a further aspect there is provided a formulation comprising; i) an aqueous vehicle ii) vesicles comprising non-ionic surfactants suspended in the aqueous vehicle, wherein the non-ionic surfactants are selected from the group consisting of mono-, di-, tri-, or poly (up to 10) glycerol mono- or di-fatty acid ester, or a polyoxyethylene ether comprising from 1 to 10 oxyethylene moieties with a C_I0-C₂₀ normal or branched alkyl chain; and iii) polynucleotide fragment (s) carried by said vesicles.

Preferably when the non-ionic surfactant is a fatty acid ester as defined above, the fatty acid ester conforms to general formula (I) :

R-[OCH₂-CH-CH₂]_n-OH

OR (I)

wherein R is independently selected from H or C₁₀-C₂₀ alkyl carbonyl and n is 1 to 10.

Preferably when the non-ionic surfactant is a polyoxyethylene ether as defined above, the polyoxyethylene ether to general formula (II) :

R-[OCH₂CH₂]_n-OH (II)

wherein R is a C₁₀-C₂₀ normal or branched alkyl chain and n is 1 to 10. Vesicle formulations of the invention may comprise non-ionic surfactant, cholesterol and dicetyl phosphate, or a fatty acid (for example, stearic or palmitic acid) , and these are advantageously preferably present in molar ratios of 1-5:1-5:0-3 respectively.

The mean vesicle diameter determined as described herein has now been found to be in the range of from 100 to 7000 nm and may therefore be considerably larger than 1000 nm. Preferably the vesicle mean diameter when applied in vivo lies in the range of from 200 to 1000 nm and more preferably from 300 to 600 nm.

The polynucleotide fragments may in principle be any polynucleotide fragments which may be effectively delivered in a vesicle suspension. Examples, of polynucleotide fragments include any naturally occurring or synthetic DNA molecules, cDNA molecules, and RNA molecules as well as mixtures thereof. The polynucleotide fragments can be selected from so-called sense, anti-sense and/or ambisense polynucleotide fragments or mixtures thereof. The polynucleotide fragments may be of any length from a few nucleotides up to about 10's of kilobases or base pair (b or bp) lengths. Generally, the length of the polynucleotide fragments will be from about 12 nucleotides in length up to about 30000 b or bp. Preferably, the length of the polynucleotide fragments will be in the range of from 400 b or bp up to about 30000 b or bp, more preferably still from about 400 b or bp up to about 6000 b or bp. Hydrophilic active agents will generally be soluble in the aqueous solution and entrapped (with) in intra- bilayer spaces, whereas those of a lipophilic nature will generally be present in the vesicular bilayer. The concentration of polynucleotide fragments in the vesicle phase is generally from 0.01 to 10% wt/wt.

The formulation is generally prepared by forming a mixture of the vesicle components- usually by melting these together and allowing to cool. In order to produce a vesicle suspension an aqueous liquid containing the polynucleotide fragments may be added to the melted vesicle formulation (e.g. at a temperature upto 70°C) followed by vigorous agitation. The formulation may be used as produced, or the concentration of polynucleotide fragments in the aqueous phase may be varied as required.

Unentrapped/unassociated polynucleotides may be removed from the vesicle formulation by column filtration techniques known in the art. However, this may lead to dilution of the vesicles and a possibility of contamination of the vesicle preparation. Alternatively therefore, the vesicle formulation may be centrifuged in order to pellet the vesicles whilst not substantially pelleting unentrapped/unassociated polynucleotide fragments, or non- ionic surfactant material which has not been suitably incorporated into the vesicles. The pellet comprising the vesicles and associated polynucleotide fragments may then be resuspended and used for transfection. The formulation may further be prepared by including a freeze/thaw procedure prior to centrifugation of the vesicle formulation. Typically, the vesicle formulation may be frozen at -20°C to -90°C for 18-24 hr and then allowed to thaw slowly at room temperature for 18-24 hr. Alternatively, a rapid freezing process, such as in liquid nitrogen, may be employed. The inclusion of such a freeze/thaw procedure has been found to increase the frequency of successful transfections.

It will be appreciated that the formulations of the present invention when used in vivo may include those adapted for oral, rectal, nasal, vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. Formulations can also be used for ex vivo cellular transfection, for example haemopoietic stem cells, lymphocytes, neoplastic cells, fibroblasts, keratinocytes, for reinfusion and/or reinplantation following transfection.

Embodiments of the invention will now be described by way of example only.

Examples Materials

The following chemically defined surfactants were used Surfactant V (triglycerol monostearate) and Surfactant VI (hexaglycerol distearate) purchased from Blagden Chemicals Limited, UK; Surfactant VII (diethylene glycol mono n- hexadecylether) , Surfactant VIII (tetraethylene glycol mono n-hexadecylether) , purchased from Chesham Chemicals Limited, UK. Vesicle suspensions were sized using a Malvern Zetasizer 4 (Malvern Instruments Limited, UK) . The cell line KGla (ECACC no: 88113006, human leukaemia cell line) was transfected with non-ionic surfactant vesicles containing the luciferase construct pG12-basic vector (about 5.4 kb) (Promega) to test for transient gene expression or a hygromycin construct (4.45 kb) (linear and circular plasmids, prepared inhouse by The Institute of Cell, Animal and Population Biology, Edinburgh University, using the pSP72 vector [Promega] with a pPGK-Hyg insert, to test for transient and stable integration. Cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal calf serum (Sigma) , or medium containing serum and 500μg/ml hygromycin B (Boeringher-Mannheim) for selection. Cells were also treated with non-ionic surfactant vesicles containing fluoroscein diacetate (FDA, Sigma) , a fluorescent stain, to confirm that non-ionic surfactant vesicles delivered a compound intracellularly. In some cases cells were grown in agar (base layer 0.6%, top layer containing cells 0.3%), prepared using cell culture medium containing hygromycin. Calf thymus DNA or DNA extracted from mouse spleen cells, stimulated with Concanavalin A in the presence of ^I4C-thymidine, was used in DNA entrapment/association efficiency studies. All other reagents were of analytical grade. For convenience the term "non-ionic surfactant vesicle" will be used throughout the examples. It will be understood that the term is taken to mean vesicles which comprise non-ionic surfactants. Example 1: Non-Ionic Surfactant Vesicle (NIV) Production

A vesicular melt was prepared by mixing surfactant, cholesterol and dicetyl phosphate (750 μmoles) in a 5:4:1 molar ratio, and heating the mixture at 130°C for 5 minutes. The melt was allowed to cool to 70°C prior to hydration with 5 ml of the appropriate solution (PBS for empty non-ionic surfactant vesicles or the appropriate concentration, μg/ml, of a gene construct or calf thy us DNA in PBS) at the same temperature. The hydrated lipid mixture was subjected to mechanical agitation using a Silverson mixer set at 8000 rpm for 15 minutes using a temperature of 70°C. Unentrapped/unassociated DNA was removed from vesicle suspensions by gel filtration using an 18 x 2.6cm Sephadex G50 column with PBS as the eluant. The cholesterol content of filtered non-ionic surfactant vesicle preparations was determined using a cholesterol assay kit (Sigma) to determine the amount the initial suspensions were diluted by filtration. In some cases dicetyl phosphate was replaced with palmitic or stearic acid. Cells for transfection were either treated with non- ionic surfactant vesicle suspensions as prepared or with non-ionic surfactant vesicle suspensions which had been gel filtered. Example 2: Determination of Non-Ionic Surfactant Vesicle DNA Content

In order to determine the amount of DNA entrapped/associated with non-ionic surfactant vesicle, 0.1ml of a columned vesicle suspension was diluted 1:1 with propanol to release the vesicular contents. The amount of DNA present in the resulting solution was determined using the method of Daxhelet et al . (1989, Analytical Biochemistry 179: 401-403) and the Hoechst 33258 DNA- specific fluorescent dye. Alternatively, ¹⁴C labelled DNA was used to determine DNA entrapment efficiency. Briefly, a 1ml aliquot of a non-ionic surfactant vesicle suspension, hydrated with PBS containing ¹⁴C-labelled DNA, was washed to remove unincorporated DNA and made up to the original volume with PBS. The entrapment efficiency was determined by comparing the amount of radioactivity present in a 1ml aliquot of the original non-ionic surfactant vesicle suspension and the aliquot of the "washed" non-ionic surfactant vesicle suspension.

Example 3: Transfection Method and Selection

Cells, at the required concentration (2 x 10⁵ - 2 x 10⁶/ml) , were incubated in 24 well tissue culture plates or 25 cm² culture flasks, with the appropriate suspensions, with or without gel filtration, of "empty" PBS loaded non- ionic surfactant vesicle or DNA loaded non-ionic surfactant vesicle, or a solution containing the equivalent amount of the relevant DNA construct. Cells were incubated at 37 °C in a humidified incubator (5% C0₂) for various time periods (3 hours - 48 hours) , harvested and washed twice with PBS and then incubated for 48 hours in fresh cell medium. Cells were then harvested and resuspended at the required concentration for cloning or for colony growth under selection in the presence of hygromycin if cells had been transfected with the hygromycin gene construct. Hygromycin selection was continued for up to 5 days then the cells were harvested, washed and resuspended in fresh medium. In some cases, cells were exposed to a second round of hygromycin selection. If cells had been transfected with the luciferase plasmid then the luciferase activity of the cell population was assessed at different time points post- transfection using a standard assay kit (luciferase assay system, Promega) . Cell number and cell viability was monitored throughout experiments. DNA was extracted from cells treated with DNA loaded NIV and untreated control cells to check for DNA integration. Briefly, cell suspensions were incubated with lysis buffer (lOmM Tris pH 7.4, lOmM EDTA, 150mM NaCl and 0.4% SDS) containing proteinase K (lmg/ml) at 56°C for up to 18 hours, and then treated with iso-propanol to precipitate the DNA. The suspension was then washed with 70% ethanol and the DNA was redissolved in TE buffer (Tris and EDTA pH8 buffer) . The DNA was digested with a restriction enzyme (Hindlll, up to 160U/sample) and then subjected to agarose electrophoresis and hygromycin sequences visualised by Southern blotting. Example 4: Cell Viability

The effect of non-ionic surfactant vesicle treatment on cell viability was assessed by flow cytometry (FACscan, Becton Dickinson) using the vital stain propidium iodide (Sigma) , or the characteristics of the cell population were compared with untreated controls.

RESULTS OF EXAMPLES 1 TO 4

1. Non-ionic surfactant vesicle suspensions contained vesicles with mean diameters in the range 400-900 nm.

2. Increasing the DNA hydrating concentration increased the amount of DNA entrapped. Non-ionic surfactant vesicles (5:4:1 molar ratio surfactant: cholesterol: dicetyl phosphate 150μmoles/ml) , hydrated with 40μg calf thymus DNA/ml gave 40% entrapment and hydrated with lOμg DNA/ml gave 20% entrapment. Non-ionic vesicles (5:4:1 molar ratio of Surfactant VII: cholesterol: dicetyl phosphate, 150μmoles/ml) , hydrated with 40μg spleen cell ¹⁴C labelled DNA/ml, had an entrapment efficiency of 4.7%. The detection limits of the assay method meant that it was impossible to determine the entrapment efficiency for a similar suspension hydrated with 4μg spleen cell ¹⁴C labelled DNA/ml. 3. Flow cytometry results showed that one hour incubation with gel filtered, FDA loaded non-ionic surfactant vesicles gave incorporation of the dye into 100% of KGla cells in the test suspension.

4. Transfection efficiency was approximately 9% of exposed cells for both linear and circular hygromycin plasmids (Table 1) .

Example 2 ; Effect of Treatment of KGla Cells with Non- Ionic Surfactant Vesicles loaded with Circular Hygromycin Plasmid on Transfection Efficiency

4 xlO⁵ KGla cells were incubated with 200μl gel filtered NIV (5:4:1 molar ratio of Surfactant VII: cholesterol: dicetyl phosphate, 150μmol/ml) , containing the circular hygromycin plasmid, (see the materials section) in 24 well culture plates containing 1.8ml medium. After 24 hours the cells were harvested, washed twice in PBS, resuspended in the original volume of fresh culture medium and incubated for a further 48 hours. The cells were washed as before, resuspended in culture medium (1 x 10⁵ cells/ml) supplemented with 0.3% agar and 500μg/ml hygromycin and plated over medium containing 0.6% agar in Petri dishes. Untransfected control cultures were also set up with/without hygromycin. After 5 days the number of colonies present was determined. Results are shown in Table 2. Example 3; Effect of Treatment of KGla Cells with Non- Ionic Surfactant Vesicles Loaded with Linear Hygromycin Plasmid on Transfection Efficiency.

2 x 10⁶ KGla cells were incubated with 2 ml gel filtered NIV (5:4:1 molar ratio of Surfactant VII: cholesterol: dicetyl phosphate, 150μmoles/ml) , containing a linear hygromycin plasmid, in 25 cm² tissue culture flasks containing 8 ml medium. After 24 hours the cells were harvested, washed twice in PBS, resuspended in the 10 ml fresh culture medium and cultured for a further 48 hours. The cells were then washed as before, and plated out at 1 cell/well in 96 well round-bottomed micro- titre plates containing 200μl culture medium supplemented with 500μg/ml hygromycin for selection. Untransfected control cultures were also set up with/without hygromycin. After 5 days the number of colonies present was determined. The contents of wells containing viable KGla cells which had been incubated with DNA loaded NIV were washed and transferred to wells of 48 well culture plates in 1ml medium. After 2 weeks samples were taken from each well and reselected with hygromycin. After 5 days the number of colonies present was determined. Results are shown in Table 3. Example 4 : Effect of Treatment of Kg-la Cells with

Various Non-Ionic Surfactant Vesicles Loaded with Plasmid Construct on Cell Viability lxlO⁷ KG-la cells were incubated in a 25 cm² flask with

500μl of the appropriate NIV formulation (3:3:1 molar ratio surfactant, cholesterol, dicetyl phosphate, lipid concentration 150μmol/ml, the preparation was hydrated with

40μg βgal construct/ml) , prepared by the Blood Transfusion

Service, Edinburgh, Scotland, using the pcDNA I vector (4.0 kb, Invitrogen) with a pSV-/3 galactosidase insert (3.8 kb,

Promega) and the volume made up to 10ml with medium (RPMI-

1640 containing 10% NBS, penicillin/streptomycin) . The flasks were incubated at 37 °C for 3 hours then 5ml of the cell suspension removed and 5ml fresh medium added. The recovered cells were washed twice with sterile PBS and suspended in 10ml medium in a fresh flask. Both washed and unwashed cells were cultured at 37°C for 72 hours. 4mls of cells were harvested from each flask, washed with PBS and then resuspended in 1ml fresh medium. The cells were counted and their viability determined. The cells were then pelleted, and 40μl of the cell pellet was incubated at

37°C with 40μl of substrate (fluorescein di-[3-D- galactopyranoside] used at 2mM) was added and the suspension incubated at 37 °C for 2 minutes. 2ml ice cold

PBS was added and the cell suspension incubated on ice for

30 minutes. The samples were analysed on a FACscan for green fluorescence. Rl and R2 are defined on the basis of the forward and side scatter distribution characteristics of the cell population. The distribution of cells between Rl and R2 was essentially the same for treated and untreated cells. Green fluorescence is obtained in untreated cells due to the intrinsic enzyme activity and was deducted from the results of treated cells. Empty NIV formulations (i.e. PBS loaded) gave results similar to those of untreated controls i.e. treatment did not cause an increase in the % positive cells or the peak fluorescence obtained (data not shown) .

Example 5 : Modified Production of Non-ionic Surfactant Vesicles (see flow chart 1, Figure 1)

Sufficient material for a single preparation was weighed and transferred into the vessel (sterile, stoppered, 50ml round bottomed flask) in which hydration and homogenisation steps were to be carried out. Components - Surfactant, cholesterol and dicetyl phosphate at a total molar concentration of 675μM, were present at a molar ratio of 4:4:1 respectively.

The mixture of components in the stoppered vessel was heated at 135°C - 140°C for 20 -30 minutes in a hot air melt oven.

The hydration solution, usually 5ml PBS alone or containing an appropriate concentration of DNA of interest was heated to 70°C in a waterbath for 15 - 20 minutes.

Melted vesicle components were immediately hydrated with the pre-heated hydration solution using a syringe and long form 19g needle, the flask was then re-stoppered.

The hydrated material was then loaded into a bench top shaker (Stuart Scientific) , such that the vessel was immersed in a waterbath at 70°C. The shaker was run at 800rpm for 2 hours, after which time the shaker and waterbath were switched off and the flasks left immersed in the waterbath to cool for 18 -24 hours, after which the contents of the flask was recovered.

Recovery of Non - ionic Surfactant Vesicle preparations

At harvest it was possible to discern that each preparation consisted of two separate recoverable fractions - the whole preparation (WP) and the flask wash (FW) . the whole preparation - this was recovered by pipetting as much material from the flask as possible. the flask wash - this was material closely associated with the inner surface of the flask following removal of the whole preparation.

It was recovered by washing the flask using 2ml PBS, the contents was mixed by swirling, left to stand for 10 minutes and collected by pipette.

Fractions were stored at room temperature until post - recovery steps were carried out.

Post - Recovery Steps

Initially unentrapped/unassociated DNA was removed from the vesicle preparation by column filtration, however, a consequence of this was dilution and the possibility of contamination of the preparation. A centrifugation step was therefore introduced to avoid these problems. lml of the whole preparation or the flask wash was diluted with 10ml PBS (at room temperature) for centrifugation in a Sorvall RC5C centrifuge. Centrifugation was carried out for 30 minutes at 10,000g at room temperature using a fixed angle rotor (SS-34) .

This process generated a pellet and supernatant. The supernatant was collected, whilst the pellet was resuspended in 10ml PBS (room temperature) and centrifugation repeated. Once again the supernatant was collected and finally the pellet was resuspended to the original volume using room temperature PBS.

Subsequent testing for transfection demonstrated that only treatment with the pellet fraction resulted in the transfection of cells in vitro (see Figure 2 which shows a series of experiments with empty/DNA loaded vesicles with each result represented by a peak on separate κ axes but with the same log scale) . This step also resulted in a marked reduction in toxicity as compared to using the unfractionated preparations (see Table 9) - probably due to a reduction in the total lipid cells were exposed to.

Generation of a pellet on centrifugation was not always seen - suggesting that the size of vesicles was not uniform from preparation to preparation.

This problem appears to have been overcome following the inclusion of a freeze/thaw step, prior to centrifugation. lml of the whole preparation or the flask wash was diluted with 10ml PBS (at room temperature) , this was frozen at -85°C for 18 - 24 hours, then allowed to thaw at room temperature for 18 - 24 hours followed by centrifugation as described above.

Inclusion of the freeze/thaw always generates a pellet from both whole preparation or the flask wash on centrifugation and results in an increase in the frequency of successful transfections.

Example 6 : Determination of DNA Content of NISV Method

Plasmid DNA (pEGFP) was labelled with ³²P-dCTP (Amersham) using a High Prime labelling kit (Boehringer) according to the manufacturers instructions.Unincorporated ³²P-dCTP was removed by centrifugation through a Sephadex G10 column (prepared in house) .

The ³P-labelled DNA was then mixed with 200μg cold (ie unlabelled) plasmid DNA and NISV prepared as described. Two experiments were carried out:

Experiment 1 - DNA labelled and vesicles prepared according to Example 1 were subsequently centrifuged and assayed, lml of preparation was diluted with 5ml PBS and centrifuged as described above ie 10, OOOg x 30 minutes, pellet and supernatant were collected and samples taken for analysis. See Table 5 for results.

Experiment 2 - DNA labelled and vesicles prepared according to Example 1, including freeze/thaw step and centrifugation. See Table 6 and 7 for results.

In experiment 1 20, 10 and 5μl of each fraction were spotted in triplicate into wells of filter plates (Hewlett Packard) , these were left to dry overnight, then 20μl of scintillant added to each well before counting in a Hewlett Packard Top Count β-counter. For experiment 2 lOμl of whole preparation and flask wash and 20μl of fractions following freeze/thaw and centrifugation were spotted in triplicate into wells of filter plates.

Example 7 ; FACS analysis of 4 NISV preparations for size distribution

Samples of empty or DNA loaded preparations were taken for analysis triplicate on a FACScan (Becton Dickinson) , forward scatter (a measure of size) data was collected and analysed by assessing the % of vesicles in each of 4 log decades (R1-R4) Rl and R2 < lOOn , R3 lOOOnm to < 7000 nm and R4 7000 or greater of increasing size, beads of known diameter were used as controls. See Figure 3 (a to i) which show histograms sharing the distribution of NISV over

4 log decades for forward scatter:

A. PBS alone ie control for hydration phase

B. l.lμm beads

C. 6.0μm beads

D. Whole Preparation, Empty, Freeze/thawed, not centrifuged

E. Whole Preparation, pEGFP loaded, Freeze/thawed, not centrifuged

F. Whole Preparation, Empty, Freeze/thawed, supernatant

G. Whole Preparation, pEGFP loaded, Freeze/thawed, supernatant H. Whole Preparation, Empty, Freeze/thawed, pellet I. Whole Preparation, pEGFP loaded, Freeze/thawed, pellet

The effects of rapid and slow cooling were also examined.

Preparation 1 Empty: Cooled rapidly

Preparation 2 pEGFP loaded: Cooled rapidly

Preparation 3 Empty Cooled slowly

Preparation 4 pEGFPloaded Cooled slowly

Cooled slowly: flasks left in waterbath and heater switched off.

Cooled quickly: flasks removed from waterbath and placed at room temperature.

Effect of freeze/thaw

As can be seen from table 8 freeze/thaw appears to increase the number of vesicles in Rl and R2 (smaller) for both whole preparation and flask wash as compared to that in untreated samples, when cooled quickly, but causes little change in distribution when cooled slowly. Effect of free/thaw on supernatant

Following centrifugation it can be seen that there is little difference in the distribution of vesicles in the supernatants of whole preparation and flask wash as compared to that of untreated samples, except in the presence of DNA when preparations are cooled quickly.

When preparations are cooled slowly it can be seen that there is a reduction in the % of vesicles in R4 ie largest vesicles. Effect of freeze/thaw on pellet

Examination of the pellets demonstrates that for whole preparation and flask wash, cooled quickly or slowly, the majority of vesicles are found in R3 and R4.

Freeze/thaw and slow cooling therefore do not appear to alter the size distribution of NISV, but do result in the production of a pellet containing a higher proportion of large vesicles on centrifugation, whilst leaving distribution in the supernatant little changed.

Removal of these smaller vesicles may account for the reduction in cytotoxicity observed when only pellets are utilised for transfection.

Example 8; Comparison of the effects of vesicle fractions on viability of K562 cells in vitro

K562 cells were cultured in 24 well tissue culture plates as described previously, with the exception that cells were not washed after 3 hours. As can be seen, in table 9, separation of preparations into pellet and supernatant following freeze/thaw reduces toxicity of the whole preparation considerably - whilst flask wash preparations are generally non-toxic even before the freeze/thaw and centrifugation steps.

If supernatants are added to cells at an equivalent concentration to that of the pellet, viability is markedly reduced, however, when added at a dilution of 1 in 10 their toxicity is greatly reduced as shown in table 7. Suggesting that it is the large number of small vesicles which are responsible for toxicity.

Example 9; In vivo delivery of pEGFP to cells of haematopoietic origin using DNA loaded NISV

Groups of 3-5 mice were injected either ip or iv with 0.5ml washed pEGFP loaded vesicles or empty vesicles. After 18 hours mice were killed and haematopoietic tissues removed. After preparation of single cell suspensions and washing, samples were taken for the assessment of fluoresence using a FACScan (Becton Dickinson) .

As can be seen from Figure 4 expression of GFP was seen in both lymphoid and granulocytic cells in the peripheral blood and peritoneal exudate cells. In vitro transfection of cell lines using pEGFP loaded NISV

Cells were transfected as described previously, using pEGFP loaded NISV which were not harvested as whole preparation or flask wash fractions nor freeze/thawed, but were separated by centrifugation to give a pellet and supernatant.

As can be seen from Figure 4 expression of GFP is seen only following culture with the pellet fraction of the preparation, pEGFP DNA alone or mixed with empty vesicles or supernatant did not lead to expression, neither did incubation with the unseparated pEGFP loaded preparation. Transfection ability seems to be associated with the pellet fraction of DNA loaded NISV.

Figure 3 demonstrates the level of transfection achieved following incubation of cell lines with pEGFP loaded vesicles which were separated by centrifugation following a freeze/thaw step, the expression of GFP is increased possibly due to the reduced toxicity observed following inclusion of a freeze/thaw step.

In Figure 5 vesicles were prepared as described in Example 5 20μl of the pellet obtained by centrifugation following freeze/thaw were used to transfect COS-7 and ES cells.

Cells were plated at 1 x 10⁵ cells/well in 2ml complete medium (containing 10% NBS and antibiotics) and allowed to adhere to the plate overnight. The following day vesicles were added to cells and incubated at 37°C (in a humidified incubator with a 5% C0₂ atmosphere) for 3 hours after which time the medium was removed from each well and discarded. Wells were then rinsed twice with sterile PBS and fresh medium added. Cells were incubated for a further 48 hours then harvested (using trypsin/EDTA to release from the plastic) for FACS analysis to assess the expression of GFP. TABLE 1

Viable Cells (% of initial population) after Hygromycin Selection

Untransfβctβd Control Untransfected Control Transfected No Selection Selection Selection

First 100 0 13 selection

Second - - 9* selection

* % viable cells after the second hygromycin selection shows that some transfected cells expressing hygromycin resistance had transient expression of the inserted gene. However, importantly a high level of stable transfection was observed.

TABLE 2

Number of Colonies in Soft Agar after Hygromycin Selection untransfected Untransfected Transfected

Control Control

No Selection Selection Selection

203 0 74 (37%)

TABLE 3

Number of Viable Clones (% shown in brackets) after Hygromycin Selection

Untransfected Control Untransfected Control Transfected No Selection Selection Selection

1st Selection

Expt 1 100/100 0 (0) 27/200(13.5) Expt 2 100/100 0 (0) 21/200(10.5)

2nd Selection

Expt 1 23/27 (11.0) Expt 2 15/21 ( 7.5)

TABLE 4

Transfection Efficiencies (shown as % positive cells) for NIV Prepared using Surfactants V, VI, VII, and VIII

Determination of DNA Content of NISV

Table 5

Data represents the % of ³²P labelled DNA incorporated into the pellet and supernatant of NISV following centrifugation, as compared to that of the unseparated preparation. Samples of 20, 10 and 5μl were counted in triplicate to allow for sampling inaccuracies which may occur due to the nature of the material. Percentages wee calculated as:

% in fraction = mean (n=3) counts in fraction x 100 mean (n=3) counts in unseparated preparation Assessment of DNA content of whole and flask wash fractions of preparations

Table 6

Determination of DNA Content of NISV Following freeze/thaw and centrifugation.

Table 7

S a al sis o 4 NISV re arations for size dist u

Comparison of the effects of vesicle fractions on viability of K562 cells in vitro

Table 9

Supernatant results are for Supernatants diluted lin 10

Claims

1. A formulation comprising; (i) an aqueous vehicle;

(ii) vesicles comprising a non-ionic surfactant suspended in the aqueous vehicle, and

(iii) polynucleotide fragments carried by said vesicles.

2. A formulation according to claim 1 wherein the non- ionic surfactant is selected from mono-, di-, tri-, or poly (up to 10) glycerol mono-, or di- fatty acid esters and a polyoxyethylene ether.

3. A formulation according to claim 2 wherein the non- ionic surfactant is a polyoxyethylene ether comprising 1 to 10 oxyethylene moieties with a C₁₀-C₂₀ normal or branched alkyl chain.

4. A formulation comprising; i) an aqueous vehicle ii) vesicles comprising non-ionic surfactants suspended in the aqueous vehicle, wherein the non- ionic surfactants are selected from the group consisting of mono-, di-, tri-, or poly (up to 10) glycerol mono- or di- fatty acid ester, or a polyoxyethylene ether comprising from 1 to 10 oxyethylene moieties with a C₁₀-C₂₀ normal or branched alkyl chain; and iii) polynucleotide fragment(s) carried by said vesicles.

5. A formulation according to any one of claims 1,2 or 4 wherein the non-ionic surfactant is a fatty acid ester of general formula (I) :

R-(0CH₂-CH-CH₂)_n-0H (I)

OR

wherein R is independently selected from H or C₁₀-C₂₀ alkyl carbonyl and is 1 to 10.

6. A formulation according to any one of claims 1 - 4 wherein the non-ionic surfactant is a polyoxyethylene ether of general formula (II) :

R-(OCH₂CH₂)_n-OH (II)

wherein R is a C_I0-C₂₀ normal or branched alkyl chain and n is 1 to 10.

7. A formulation according to any preceding claim wherein the vesicles further comprise a sterol and a charged species.

8. A formulation according to claim 7 wherein the non- ionic surfactant, the sterol and the charged species are present in molar ratios of 1-5:1-5:0-3.

9. A formulation according to any of the preceding claims wherein the vesicle diameter lies in the range of from lOOnm to 1500nm.

10. A formulation according to any one of claims 7,8 and 9 wherein the sterol is cholesterol and the charged species is dicetyl phosphate.

11. A formulation according to any one of claims 7,8 and 9 wherein the charged species is a fatty acid.

12. A formulation according to claim 11 wherein the fatty acid is selected from stearic acid and palmitic acid.

13. A formulation according to any preceding claim wherein the polynucleotide is selected from i) naturally occurring DNA molecules; ii) synthetic DNA molecules; iii) cDNA molecules; iv) RNA molecules; and v) fragments and/or mixtures of i) to iv) .

14. A formulation according to claim 13 wherein the polynucleotide is selected from sense, anti-sense and/or ambisense polynucleotide or mixtures thereof.

15. A formulation according to claim 13 or claim 14 wherein the polynucleotide is in the range of 400 bases (b) or base pairs (bp) to 30000 bases (b) or base pairs (bp) in length.

16. A formulation according to claim 13 or claim 14 wherein the polynucleotide is in the range of 400 b or bp up to about 6000 b or bp in length.

17. A formulation according to any preceding claim wherein the concentration of the polynucleotide in the vesicles is from 0.01 to 10% wt/wt.

18. A transfection kit for use in transfecting cells, said kit comprising vesicles comprising a non-ionic surfactant suspended in an aqueous vehicle and a polynucloetide fragement to be carried by said vesicles.

19. Use of a formulation according to any preceding claim for improved transfection efficiency of the polynucleotide into cells.

20. Use of a formulation according to any one of claims 1 to 17 for improved stability of expression/integration of the polynucleotide within cells.

21. Use of an ameliorative polynucleotide in a formulation according to any one of claims 1 to 17 in the manufacture of a medicament for the treatment of disease.