CA2241830A1 - Polymers - Google Patents

Abstract

A composition for delivering a biologically active polyanionic molecule comprising a linear polymer with a backbone comprising amido and tertiary amino groups arranged regularly on the said backbone and said biologically active polyanionic molecule bound to said polymer. Preferably the linear polymer comprises a poly(amidoamine). Preferably the biologically active polyanionic molecule is a nucleic acid, more preferably DNA. The compositions are useful for delivering nucleic acids to a cell, for example in cell transformation or transfection and in gene therapy.

Description

POLYMEI~

The present invention relates to polymers, in particular to polymers for the delivery of nucleic acid to a cell.

For many research applications in genetic manipulation and genetic engin~ering, it is n~ce~s~ry to express new or modified genes in living cells. However the uptake of DNA into cells is poor resulting in inconci~tent expression. Similarly, gene therapy, ~nti~en.se oligonucleotide 10 therapy and gene vaccination require that DNA and DNA analogues can survive in a hostile biological environment, penetrate biological barriers, be taken up into cells and move to the correct subcellular compar~nent to exert their therapeutic effects.

15 The identifi~tion of defective genes responsible for disease states, either through the overproduction of key proteins, the production of defective proteins or the defective control of gene production, offers new possibilities for the treatment of disease. By controlling the defect at the genetic level a range of diseases could now be treated effectively rather ~0 than by merely treating the symptoms of ~ese diseases. This has been achieved in some cases, or is believed to be achievable, by the expression of new competent genes, or by controlling the overproduction of unwanted gene products or by controlling the expression of genes. These processes could be achieved by the insertion of new DNA or by the ~ mini~stration 25 and uptake of complementary strands of DNA or DNA analogues which inhibit the production or control the production of existing genes [1]. In both of these strategies it is necessary to deliver to the cell sufficient DNA
to achieve modified cell expression. The DNA must also be delivered to the correct intracellular compartment to effect that change. While some 30 DNA is taken up naturally into cells, the amount taken up is small and inconsistent, and e~ s~ion of added DNA is poor. DNA is an inherently unstable material, partir~ rly in a biological environment where many specific enzymes capable of degrading DNA are found [2]. Either for ~erapeutic purposes, or for ~ es~ion of new or modified genes for 5 research purposes, a more efficient and reliable method of delivering DNA
is required and, in particular, protection of the DNA against metabolic effects is highly desirable.

A number of strategies have been proposed to achieve these aims. These 10 include the use of liposomes [3], cationic lipids [4~, which are often incorrectly referred to as 'cationic liposomes', and ~e use of cationic polymers such as polylysine [5] or polyornithin~ as DNA delivery agents.

Both oligonucleotides and DNA constructs, such as pl~mi(1s, have shown 15 improved activity by con~len~tion with polycations such as polylysine.
In the former case chemical conjugation of oligonucleotide to the polymer is required, whereas in the latter case complexation of polymer with DNA
also confers these effects. Poly-L-lysine (PLL) is believed to con-lçn~e the DNA into a smaller volume, and by the excess positive charge of the 20 complex, bind to negatively charged cell surfaces to facilitate interaction with the cell surface and uptake into the cell.

The effectiveness of polylysine-DNA complexes has been enh~n~e~l by coupling lip~n~ls to the polylysine which further facilitate binding and 25 uptake into cells [6~. Membrane destabilising agents have been added to DNA preparations to facilitate exit of the DNA from the degradative endosomal compartments of the cell [7].

To date, few dir~l ell~ cationic polymers have been used in this work, and 30 the available polymers are deficient in a number of respects. Poly-L-lysine, the principal polymer presently used for ~is purpose, is known to be toxic above a small molecular weight [8], it does not interact stoichiom~trically with DNA, and the resulting complex is unreliable, ~lifflclllt to control and its properties strongly dependent on the ratio of 5 DNA to polymer.

Ranucci et al [15] describes the synthesis of poly(amidoamine)s and suggests their use as polymeric drug carriers using covalent ~ hm~nt of the drug molecule to the polymer.
Ranucci and Ferruti [12] describes hydrolyzable block copolymers cont~inin~ poly(ethyleneglycol) (P~G) and poly(amidoamine) (PAA) or poly(amido-thioether-amine) .

15 Haensler and Szoka [10] suggests that polyamidoamine cascade polymers (dendrimers pl~aled from branched chain poly(amidoamine)s) of a certain size may be useful in transfection of cells ~n culture and states that linear polycations in general are relatively cytotoxic and by themselves not very efficient, which limits their usefulness for transfection of cells in culture.
Duncan et al [16] describes poly(~mi~o~mint?)-Triton X-100 conjugates which may be useful for drug delivery.

Katayose and Kataoka tl7] suggest that a PEG-poly(lysine) block 25 copolymer as a potential DNA delivery system.

Attaching PEG chains to macromolecules and colloidal particles has been described for many biomedical products [11].

30 There remains a need for polymers which have i~proved properties for use in DNA delivery systems.

A first aspect of the invention provides a composition for delivering a biologically active polyanionic molecule, the composition comprising a 5 linear polymer with a backbone comprising amido and tertiary amino groups arranged regularly on the said backbone and said biologically active polyanionic molecule bound to said polymer.

The said linear polymers are cationic and, depending on their n~ture, as 10 ~ cllsse(l more f~lly below, the polymers have a range of physicochemir~l properties.

Conveniently, the said linear polymer comprises a poly(amidoamine) (PAA). Suitably, said linear polymer consists of a poly(~mitlo~min~).
15 PAAs are degradable in water since they contain hydrolyzable amidic bonds in their main chain together with nucleophilic tertiary-aminic functions in the ,B position. The polymers can be synthesised from a wide variety of primary mono~min~s or secondary bi~min~s which enable full control to be exercised over the spacing and pKa of the c~tionic groups L9]
20 for optimi~tion of ~e interaction wi~h a suitable biologically active polyanionic molecule. Preferably, PAAs are water soluble, and thus facilitate the solubility of the complex. Further solubilisation of complexes can be achieved by use of the copolymers cont~inin~
hydrophilic PEG chains. (Reference [93 is incorporated herein by 25 reference.) It is pLe~lled if the pKa of the cationic groups is between 7 and 8. It has been found that PAA with a low pKa binds DNA less well than PAA with a high pKa. It is more preferred if the pKa of the PAA is around 8.

Thus, in a ~ led embo(liment, said linear polymer tilrther comprises ethylene glycol or poly(ethyleneglycol). It is particularly ~r~re~ d if the linear polymer is a poly(e~yleneglycol)-poly(~ o~min~) block copolymer or ethyleneglycol-poly(~mitlo~min~) block copolymer.

Preferably said linear polymer is a block copolymer with the structure [poly(amidoamine)-(ethyleneglycol)y]x wherein x is from 1 to 50 and y is from 1 to 200, wherever it may occur.

10 Also ~ler~lably said linear polymer is a block copolymer with ~e structure (ethyleneglycol)y-poly(amidoamine)-(ethyleneglycol)y wherein each y is independently 1 to 200.

Suitably the linear polymer consists of or comprises a PAA which has the 15 formula:
(a) O O
Il 11 CH~C~CN~?~NCC~C~N
Rl Rl R3 z or (b) O O
11 i CH2CH2C INR7N~C~CH2 INR~I --30 or said linear polymer comprises a PAA such as with the formula:

CA 0224l830 l998-06-l6 (C) O O O O
Il 11 11 11 .
N--CH2CH2CNR2NCCH2CH2N--CH2CH2CNR2NCCH2CH2NR~N
Rs R5 Rl Rl R3 z Rl Rl R5 R5 or (d) o o o o 1~ 11 11 11 --OEkC~C~n~NCC~C~2Nn~N-C~C~C~n~NCC~ OE~Nn~N
R~ R5 R~ E~ R~ F~ p~ ~ R5 R5 Z
s or (e) wherein --N-R2-N-1--N-R4-N- or--N-R6-N- in (a) or (b) 10 Rl R, R3 R3 Rs R5 are replaced by CHCH
- N N -CHCH
~o R~

and, in any case, z is from 0 (or 1 as a~ro~liate) to 70 and each R~ is independently H or a linear or branched hydrocarbon chain -CnH2n+, with 20 n 2 1-4 whenever it occurs; each R2 is independently a linear or branched alkylene chain -C,,H2n- with n = 1-4 whenever it occurs; each R3 is independently a linear or br~nrh~-l hydrocarbon chain -C~,H2n+, with n =

W O 97~5067 PCT/GB97/00022-1~ whenever it occurs; each R4 is independently a linear or branched alkylene chain -CnH2n- with n -- 2~ whenever it occurs; each R5 is - independently a linear or branched hydrocarbon chain -C~H2n+l with n =
1-4; each R6 is independently a linear or branched alkylene chain -CnH2n-5 wi~ n = 2-4; and wherein R7, R8, Rg and Rlo are independently H or linear or branched hydrocarbon chain -CnH2n~, wi~h ~ = 1-3 whenever they occur.

Preferably Z is from 30 to 70.
It is ~l~ft;ll~d if the Mn of ~e PAA is greater than 10 000; more preferably greater than 15 000.

Conveniently the linear polyrner has the formula:
O O
Il 11 --[-PAA--CO--(CH2CH20)yC--] ,~--20 wherein PAA has the formll~ as defined above, x is from 1 to 50 and y is from 1 to 200.

Preferably, the linear polymer has the formula O O

2~
CH3O~-CH2CH2O)y--C--PAA~--O(-CH2CH2O)y~CH3 wherein PAA has the formula as defined above and y is from 1 to 200.
The preferences for ~e degree of polymerisation of the PAA in the PAA-30 PEG copolymers is the same as for the PAA polymers (ie preferably Z is from 30 to 70).

The biologically active poly~ni(ni~ molecule rnay be any suitable such molecule but ~rerelably said molecule comprises a regular array of negative charges.

5 It is ~lel~lled if the molecule comprising a regular array of negative charges is a nucleic acid or a derivative thereof.

It is less ~ler~lled if the molecule is heparin.

10 The nucleic acid or derivative thereof may be DNA or RNA.

The nucleic acid may be an antisense nucleic acid. The nucleic acid is conveniently an oligonucleotide such as an antisense oligonucleotide.

15 If the molecule is an oligonucleotide it is ~lere,led that the PAA has a relatively low degree of polymerisation. If the molecule is a larger DNA
or RNA molecule it is preferred that the PAA has a relatively high degree of polymerisation.

20 The nucleic acid, as discussed below, is ~lefe~ably a therapeutic nucleic acid useful in gene therapy, nucleic acid vaccination, antisense therapy and the like. As tli.~cll~se-1 in more detail below, the nucleic acid may comprise natural (phosphate) phosphodiester linkages or it may comprise non-natural linl~ages such as those including phosphorothioates. It is 25 ~reJ~lled if the nucleic acid is DNA or a derivative thereof.

Antisense oligonucleotides are single-stranded nucleic acid, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-30 DNA duplex is formed. These nucleic acids are often termed "antisense"

because they are complement~ry to the sense or coding strand of the gene.
Recently, forrnation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the 5 DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites.

10 Clearly, ~e sequence of the ~nti~en~e nucleic acid or oligonucleotide can readily be d~L~ ed by rer~r~llce to the nucleotide sequence of the gene whose function is to be ill~lreled with.

In a still further embodiment the nucleic acid delivered to a target cell 15 encodes an ~nti~çnse RNA.

An antisense RNA includes an RNA molecule which hybridises to, and interferes with the expression frolm a rnRNA molecule encoding a protein or to another RNA molecule within the cell such as pre-mRNA or tRNA
20 or rRNA, or hybridises to, and i~ el~s with the e~l~~ession from a gene.

Conveniently, a gene expressing an antisense RNA may be constructed by inserting a coding sequence encoding a protein adjacent a promoter in the appropriate orientation such that the RNA complementary to mRNA.
25 Suitably, the antisense RNA blocks expression of ~m(l~irable polypeptides such as oncogenes, for example ras, bd, src or tumour suppressor genes such as p53 and Rb.

It will be appreciated that it may be sufficient to reduce expression of the 30 undesirable polypeptide rather than abolish the expression.

It will be further appreciated that DNA sequences suitable for expressing as antisense RNA and for designing other antisense nucleic acids may be readily derived ~rom publicly accessible ~l~t~ es such as GenR~nk and EMBL.
s Oligonucleotides are sub.~ect to being degraded or inactivated by cellular endogenous nucleases. To counter this problem, it is possible to use modified oligonucleotides, eg having altered internucleotide linkages which retain a negat*e charge, in which the naturally occurring phosphodiester linkages have been replaced with ano&er linkage. For e~ample, Agrawal et al (1988) Proc. Nall. Aca~. Sci. USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-l using oligonucleotide phosphor~mi-l~tes and phosphorothio~es. Agrawal et al (1989) Proc.
Natl. Acad. Sci. USA 86, 7790-7794 showed inhibition of HIV-l replication in both early-infected and chronically infected cell cultures, using nucleotide sequence-specific oligonucleotide phosphorothioates.
Leither et al (1990) Proc. Natl. Acad. Sci. USA 87, 3430-3434 report inhibition in tissue culture of inflnen7~ virus replication by oligonucleotide phosphorothioates .
Oligonucleotides having artificial linkages have been shown to be resistant to degradation in vivo. For example, Shaw et al (1991) in Nucleic Acids Res. 19, 747-750, report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3' end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.

A detailed description of the H-phosphonate approach to synthesizing oligonucleoside phosphorothioates is provided in Agrawal and Tang (1990) Tetrahedron Letters 31, 7541-7544, the te~ching.s of which are hereby incoIpo~ated herein by rerele~ce. Syntheses of oligonucleoside phosphororlitllio~tes and phosphor~mi~l~tes are lcnown in the art. See, for example, Agrawal and Goodchild ~1987) Tetrahedron Letters 28, 3539;
Nielsen et al (1988) Te~rahedron Letters 29, 2911; Jager et al (1988) S Biochemistry 27, 7237; U7.nz~n.~ki et al (1987) Tetrahedron Letters 28, 3401; Ba~ w~ll (1988) Helv. Chim. Acta. 71, 1517; Crosstick and Vyle (1989) Tetrahedron Letters 30, 4693; Agrawal et al (1990) Proc. Natl.
Aca~. Sci. USA 87, 1401-1405, the te~( hing~ of which are incorporated herein by rererellce. Other methods for synthesis or production also are possible. In a l)lere,led embodiment the oligonucleotide is a deoxyribonucleic acid (DNA3, although ribonucleic acid (RNA) sequences may also be synth~si~e-l and applied.

The oligonucleotides useful in the invention plerel~bly are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of re~ ce-l length. Such breakdown products are more likely to engage in non-specific hybri~ ti-~n and are less likely to be effective, relative to their filll-length coun~ alL~. Thus, it is desirable to use oligonucleotides that are resi~t~nt to degradation in the body and which are able to reach the targeted cells. The present oligonucleotides can be rendered more resi~t:~nt to degradation in vivo by substihltin~ one or more internal artificial intermlcleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulphur in the linkage.
The synthesis of oligonucleotides having one or rnore of these linkages substituted for the phosphodiester internucleotide linkages is well known in the art, including synthetic pathways for producing oligonucleotides having rnixed nlL~ ucleotide linkages.

30 Oligonucleotides can be made resistant to extension by endogenous CA 0224l830 l998-06-l6 W 0 97/25067 PCT/GB97tO0022-enzymes by "capping" or incorporating similar groups on the 5' or 3' terminal nucleotides. A reagent for capping is cornmercially available as Amino-Link II~M from Applied BioSystems Inc, Foster City, CA.
Methods for capping are described, for example, by Shaw et al (1991) S Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991) Proc. Natl.
Acad. Sci. USA 88(17), 7595-7599, the te~chin~.c of which are hereby incorporated herein by lei~le"ce.

A further method of m~king oligonucleotides re~i~t~nt to mlcle~e attack is for them to be "self-stabilized" as described by Tang et al (1993) Nucl.
Acids Res. 21, 2729-2735 incorporated herein by ~ef~lellce. Self-stabilized oligonucleotides have hairpin loop structures at their 3' ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I and fetal bovine serum. The self-stabilized region of the oligonucleotide does not illlel~le in hybridization with complemen~ry nucleic acids, and pharmacokinetic and stability studies in mice have shown increased in vivo persistence of self-stabilized oligonucleotides with respect to their linear c~ul~ alL~.

It is preferred that the oligonucleotides contain phosphodiester linkages.

However, the polymers of the invention can aid compaction and stabilisation of the nucleic acid and, it is believed, can protect the nucleic acid from degradation.
2~
We have found that this family of cationic polymers, and the complexes formed, have superior properties for use as DNA delivery systems in comparison with other cationic polymers previously reported for this purpose.

Complexes bet~,veen the polymers as defined, and in particular polyarnido~min~s and copolymers thereof, and DNA are readily formed by simple mixing at the required ratio of DNA to polymer. In contrast to complexes of poly-L-lysine and DNA, which are invariably insoluble and S form colloidal particles in the size range of 100 nm to several ~4m in ~ m~t~r, the PAA-DNA complexes remain soluble under some conditions.

The use of PEG cont~ining polymers increase the range of conditions 10 under which soluble complexes are seen. C~enerally PEGylation has the effect of reducing interaction with scavenger receptors and cells, so prolonging the circulation half-life and reducing immllnr~genic responses.
In the case of complexes with DNA this is also expected to reduce the metabolism of DNA by serum enzymes. The advantages of having the 15 PEG bound to the polymer rather than directly to the DNA is that it reduces the possibility that the hydrophilic PEG will i~lL~lrele with the uptake of DNA into the cell through hydrophobic membranes and hence location to the correct intracellular compartment. The synthesis of PAA
and PAA-PEG, however still allows for the further conjugation of other 20 biologically active recognition sequences to fur~her improve the uptake of the DNA and its transfer to the correct intracellular colll~a~ ent.

A ~refelled embodiment is wherein the polymer further comprises a biologically active recognition signal. The saidL signal may aid the 25 targeting, uptake or intracellular loc~ tion of the composition and thelerol-e the said biologically active polyanionic molecule.

Suitable said recognition signals include lig~n-l~ for binding and endocytosis especially of DNA delivery systems such as trall~llill, for 30 example see E. Wagner, M. Cotten, R. Foisner and M.L. Bernstiel (1991) CA 0224l830 l998-06-l6 W O 97~5067 PCT/GB97/00022-Proc. Natl. Acad. Sci. US~ 88, 4255-4259; carbohydrate residues, for example galactose, or mannose residues to target to hepatocytes or macrophages respectively. (G. Ashwell and J. Harford (1982) Ann. Rev.
Biochem. 51, 531-54 describes carbohydrate specific receptors of the liver S and use of asialoglyco~roteill receptor in gene targeting with ~ çhmP~nt of asialo-orosomucoid to PLL-DNA constructs is described in G.Y. Wu and C.H. Wu (1988) Biochemistry 27, 887-892.); folate receptors as described in C~.P. Leamon and P.S. Low (lg91) Proc. Natl. Acad. Sci.
USA 88, 5572-5576 and G. Citro, (:~. Szczylik, P. Ginobbi, G. Zupi and 10 B. Calabretta (1994) Br. J. Cancer 69, 463-464; monoclonal antibodies, especially those selective for a cell-sur~ace antigen; and any other ligand which will m~ te endocytosis of macromolecules.

Monoclonal antibodies which will bind to many of these cell surface 15 antigens are already known but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fr~gment [ScFv3). Suitable monoclonal 20 antibodies to selected antigens may be ~l~aled by known t~chniques~ for example those disclosed in "Monoclonal Antibodies: A manual of techniquesn, H Zola (CRC Press, 19883 and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982).
Chimaeric antibodies are (~ CllSSt"1 by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).

Suitably ~reL,ared non-human antibodies can be "hllm~ni7e~" in known 30 ways, for ex~mple by inserting the CDR regions of mouse antibodies into the framework of human antibodies.

Suitable endosome disrupting agents such as viral fusogenic peptides and adenoviral particles have been described in J-P. Bongartz, A-M. Aubertin, P.G. ~ilh~ l and B. Lebleu (1994) Nucleic Acids Research 22, 4681-4688 and M. Cotten, E. Wagner, K. Zatloukal, S. Phillips, D.T. Curiel, M.L. Bernstiel (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098.

All of these journal articles are incorporated herein by lere~ ce.
~omplexes have been formed between polymers and short single stranded DNA with both phosphodiester and phosphorothioate backbones, and high molecular weight double stranded I)NA. The complexes formed have been characterised by microcalorimetry, DNA melting profiles, gel-shift electrophoresis and photon correlation spectroscopy.

It is most preferred if the biologically active polyanionic molecule is a therapeutic molecule such as a therapeutic nucleic acid.

Th~ uLic nucleic acids include any nucleic acid that it is useful to deliver to a patient, and includes nucleic acid vaccines.

Preferably the nucleic acid is suitable for gene therapy.

In one embodinnent, the nucleic acid encodes a molecule having a directly or indirectly cytotoxic function. By "directly or indirectly" cytotoxic, we mean that ~e molecule encoded by the gene may itself be toxic (for example ricin; tumour necrosis factor; interleukin-2; illlelrelon-gamma;
ribonuclease; deoxyribonuclease; Pseudomonas exotoxin A) or it may be metabolised to form a toxic product, or it may act on something else to CA 0224l830 l998-06-l6 form a toxic product. The sequence of ricin cDNA is disclosed in Lamb et al (1985) Eur. J. Biochem. 148, 2~5-270 incorporated herein by lererence.

S For example, it would be desirable to deliver to cancer cells within apatient a nucleic acid encoding an enzyme using the compositions of the invention, the enzyme being one that converts a relatively non-toxic prodrug to a toxic drug. The enzyme cytosine ~ min~e converts S-fluorocytosine (SFC) to S-fluorouracil (SFU) (Mullen et al (1922) PNAS
10 89, 33); the herpes simplex enzyme thymidine kinase sensitises cells to treatment with the antiviral agent ganciclovir (C~CV) or aciclovir (Moolten (1986) Cancer Res. 46, 5276; F7.7e(1ine et al (1991) New Biol 3, 608).
The cytosine ~ min~e of any org~ni~m, for example E. coli or Saccharomyces cerevisiae, may be used.
Thus, in one embodiment of the invention, the nucleic acid encodes a cytosine ~e~min~e and the patient is concomit~ntly given 5FC. By "conco~ ly", we mean that the 5FC is ~mini~t~red at such a time, in relation to the transformation of the tumour cells, that SFC is converted .20 into SFU in the target cells by the cytosine cle~min~e expressed from the said gene. A dosage of approximately 0.001 to 100.0 mg SFC/kg body weight/day, preferably 0.1 to 10.0 mg/kg/day is suitable.

Components, such as 5FC, which are converted from a relatively non-25 toxic form into a cytotoxic form by the action of an enzyme are termed "pro-drugs".

In a further embodiment the nucleic acid delivered to the target cell is or encodes a ribozyme capable of cleaving targeted RNA or DNA. The 30 targeted RNA or D~A to be cleaved may be RNA or DNA which is essential to the function of the cell and cleavage thereof results in cell death or the RNA or DNA to be cleaved may be RNA or DNA which encodes an lln-lesirable protein, for example an oncogene product, and cleavage of this RNA or DNA may prevent the cell from becoming S cancerous.

Ribozymes which may be encoded in the genomes of the viruses or virus-like particles herein disclosed are described in Cech and Herschlag "Site-specific cleavage of single stranded DNA" US 5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P" US 5,168,053, Cantin et al "Ribozyme cleavage of HIV-l RNA" US 5,149,796; Cech et al "RNA
ribozyme restriction endoribonucleases and methods", US 5,116,742;
Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods, US 5,093,246; and Been et al "RNA
15 ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterification", US 4,987,071, all incorporated herein by ler~lellce.

In another embodiment of the invention, the nucleic acid replaces the 20 function of a defective gene in the target cell.

There are several thousand inherited genetic ~ e~es of m~mm~
including h~ n~, that are caused by defective genes. Examples of such genetic diseases include cystic fibrosis, where there is known to be a 25 m~lt~tion in the CFTR gene; Duchenne muscular dystrophy, where there is known to be a mutation in the dystrophin gene; sickle cell disease, where there is known to be a mutation in the HbA gene. Many types of cancer are caused by defective genes, especially protooncogenes, and tumour-suppressor genes that have undergone mutation.

W O 97t25067 PCT/G B97/00022 -The following table shows ~;ullell~ targets for gene therapy.

Diseases caused by single gene defects: current targets for gene therapy Disease Defective gene Tmmlmodeficiency Adenosine ~lc,.~ e Purine nucleoside phosphorylase Hypercholesterolaemia LDL receptor Haemophilia Factor IX
Factor VIII
Gaucher's disease Glucocerebrosidase Mucopolysaccharidosis ,B-glucuronidase Emphysema ~1-antitrypsin Cystic fibrosis Cystic fibrosis tr~n~mP.mhrane regulator PhenyLketonuria Phenyl~l~nin~ hydroxylase Hyperarnmonaemia Ornithine transcalb~lllylase Citl~lllin~mi~ Arginosuccinate synthetase Muscular dystrophy Dystrophin Th~ s~emia ,B-globin Sickle cell anaemia ~-globin Leukocyte adhesion deficiency CD-18 25 This list indicates the principal ~;UllellL targets for gene therapy. Many ofthe (1;~ç~es listed can be caused by defects in more than one gene; the gene defect llsted is the defect targeted by current research.

Thus, it is preferred that the composition of the invention, which may be 30 useful in the treatment of cystic fibrosis, contains a functional CFTR gene to replace the ffinction of the defective CFTR gene. Similarly, it is e~~ d that the virus or virus-like particle of the invention, which may be useful in the treatment of cancer, contains a functional protooncogene, or tumour-suppressor gene to replace the function of the defective 35 protooncogene or tumour-suppressor gene.

Examples of protooncogenes are r~s, src, bcl and so on, examples of tumour-suppressor genes are p53 and Rb.
~. .
The nucleic acid may contain introns, or it may be a gene or a fragment 5 thereof, or cDNA, or fragment thereof.

Nucleic acids suitable for use in vaccines of the present invention include those described in Volurne 12(16) of Vaccine which is a special collrelcllce issue of ~e WHO meeting on nucleic and vaccines, and is incorporated 10 herein by lerel~--ce. Nucleic acidl vaccines for tuberculosis, influenza, hepatitis B, Lei~hm~ni;~i.c and HIV have been considered.

The nucleic acid, especially DNA, which is bound to the polymer in the composition of ~e invention may be any suitable size. Preferably the 15 nucleic acid is from 10 to 10 million bases or base pairs. Suitably oligonucleotides are from 10 bases to 200 bases, more suitably 10 bases to 100 bases.

Conveniently RNA and DNA molecules are from 100 to 1 million bases 20 or base pairs.

More preferably the nucleic acid is from 20 to 1 million bases or base pairs, still more preferably the nucleic acid is from 1000 to 500,000 bases or base pairs and most ~lerel~bly the nucleic acid is from 5000 to 150,000 25 bases or base pairs.

The nucleic acid may conveniently be plasmid DNA whether supercoiled, open circle or linearised plasmid DNA.

30 It is believed that the nucleic acid binds to the polymer non-covalently.

A second aspect of the invention provides a composition according to the first aspect of the invention for use in medicine.

A third aspect of the invention provides a composition according to the 5 first aspect of the invention in the m~mlf~ct~re of a me~ rnent for treatment of a disease.

A fourth aspect of the invention provides a ph~rm~relltir~l composition comprising a composition according to the first aspect of the invention and 10 a pharn ~ce~lti~-~lly effective carrier.

The formnl~tions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of ph~ cy. Such methods include the step of bringing into association the 15 active ingredient (composition of the invention) with ~he carrier which con~ti~ltes one or more accessory ingredients. In general the formulations are prepared by uniformly and intim~t~ly bringing into association the active ingredient wi~ liquid carriers or finely divided solid carriers or both, and then, if necess~ry, shaping ~e product.
Form~ ti~ns in accordance wi~ the present invention suitable for oral mini~tration may be presented as discrete units such as capsules, cachets or tablets, each cont~ining a pred~te~ ed amount of the active ingredient; as a powder or granules; as a solution or a suspension in an 25 aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one 30 or more accessory ingredients. Compressed tablets may be prepared by CA 0224l830 l998-06-l6 compressing in a suitable m~chin~ the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inertdiluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked S povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by mouldmg in a suitable m~chinf~ a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be forrn~ terl so as to provide slow or controlled release of the active 10 ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formlll~tions suitable for topical ~-lmini~tration i~n the mouth include lozenges comprising the active ingredient in a flavoured basis, usually 15 sucrose and acacia or tr~c~nth; pastilles comprising ~e active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Formulations suitable for parenteral ~lmini.~tration include aqueous and 20 non-aqueous sterile injection solutions which may contain anti-oxi~l~nt~, l~url~ls, bacteriostats and solutes which render the form~ ti-)n isotonic with the blood of the int~nlle~l recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The fonm~l~tions may be presented in unit-dose or multi-dose 25 containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously 30 described.

Preferred unit dosage forrm-l~ti-)n~ are those cont~inin~ a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.

5 It should be understood that in addition to the ingredients particularly mentioned above the fc)rmnl~tion~ of this invention may include other agents conventional in the art having regard to the type of forml-l~ti~n in question, for example those suitable for oral ~(lminictration may include flavouring agents.
A fifth aspect of the invention provides a method of m~king a composition according to the ~Irst aspect of the invention comprising contacting said biologically active polyanionic molecule with said linear polymer.

lS Preferably, the biologically active polyanionic molecule and the said linear polymer are simply mixed together, preferably in solution, more preferably in aqueous solution. They may be mixed together quickly or slowly. Preferably, the biologically active polyanionic molecule is a nucleic acid, more ~e~el~bly DNA.
When nucleic acid is used, it is convenient to mix the nucleic acid and polymer in a high salt solution and dialyse against water. This method is particularl~ ~ref~l,ed for large nucleic acid molecules such as those > lkb. It is also convenient to heat the mixture of polymer and nucleic 25 acid and to cool the ~ Ule slowly. Preferably, the mixture is a solution, more ~reÇel~bly an aqueous solution.

A sixth aspect of the invention provides a method of delivering a biologically active polyanionic molecule to a host, the method comprising 30 ~flminictering to said host an effective amount of a composition according to the first aspect of the invention.

The host is suitably a patient to be treated wi~ the biologieally aetive polyanionie moleeule.

The host may be, for example, a eell in eulture in vitro or it may be an experiment~l animal.

Preferably, the biologieally aetive polyanionie moleeule is a nueleie aeid, 10 more ~lefelably DNA.

When the host is a eell in eulture the method ean be used to transfeet or transform the eell with the nueleie aeid, preferably DNA.

15 A seventh aspeet of the invention provides a method of delivering a biologieally aetive polyanionie moleeule to a eell in an environment7 the method eomprising ~lmini~tering to said environment a composition as defined in the first aspeet of the envilo~ L.

2~ The environment may be a patient to be treated or an experiment~l animal to be treated or a culture m~ m cont~inin~ eells.

Preferably the environment is a culture medium cont~ininsg cells. When the biologieally active polyanionic molecule is a nucleic acid the cells may 25 be transfected or transformed using this method by ~rlmini~tering a suitable composition to the culture medium.

By "cells" we include both prokaryotic and eukaryotic cells. Thus, the cells include eells of baeteria, yeast, fungi, plants, vertebrates (sueh as 30 m~mm~ n eells) and invertebrates (such as insect cells).

Preferably the nucleic acid to be del*ered is DNA.

Preferably in this method the said composition is contacted with said cell.

S Preferably for ~he methods described in ~e fifth, sixth and seventh aspects of the invention the biologically active polyanionic molecule is a therapeutic molecule and more l~lereLably the therapeutic molecule comprises a nucleic acid.

10 An eighth aspect of the invention provides a method of treating, preventing or ameliorating a disease in a mlllticellular org~ni.cm which mlllticellular or~nism benefits from the ~tlminislTation of a biologically active polyanionic molecule, the method comprising ~lminictering to the patient a composition as defined in the first aspect of the invention.
Preferably the biologically active polyanionic molecule comprises a therapeutic nucleic acid or derivative thereof The multicellular org~ni~m may be an anirnal or human or a plant, 20 ~ler~rably an animal especially a m~mm~l and more preferably a hllm~n The an~mal or human is therefore a patient.

The aforementioned compositions may be ~lmini~tered to the plant in any 25 suitable way.

The aforementioned compositions of the invention or a form~ tion ~ereof may be ~-lrnini~tered to the patient by any conventional method including oral and parenteral (eg subcutaneous or intr~mnsc~ r) injection. The 30 treatment may consist of a single dose or a pluralit,v of doses over a period of time.

Whilst it is possible for a composition of t:he invention to be ~mini~tered alone, it is yr~rerable to present it as a pharmaceutical form~ ti-)n, together wi~ one or more acceptable carriers. The carrier(s~ must be "acceptable" in the sense of being comp~tihle with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline or l~urr~ïed solutions ~ elably wherein the buffer buffers in a physiological pH range which will be sterile and 10 pyrogen free.

A ninth aspect of the invention provides a polymer with the formula (ethylene glycol)y-poly(amidoamine)-(ethyleneglycol)y wherein each y is independently from 1 to 200.
Preferably the compound has the formula O O
Il 11 CH3O~-cH2cH20-)y~--PAA~(-cH2cH2o-)y~H3 wherein PAA is a poly(~mi-lo~mine) as defined above and each y is independently from 1 to 200.

This polymer can be used as the polymer in all previous aspects of the 25 invention and it may also be useful in binding drugs generally for delivery to a patient to be treated.

The invention will now be described in more detail with respect to the following Figures and Examples wherein:
Figure 1 shows a gel-shift assay comparing the interaction of (a) poly-~-lysine and (b) PAA wilh phosphorothioate oligonucleotide (15 mer) at various oligonucleotide:polymer ratios (the abbreviation ON means oligonucleotide, and the abbreviation P means polymer whenever it occurs in the Figures relating to gels);
Figure 2 shows ~e result of isothenn~l titration microcalorimetly experiments comparing ~e interactions between PAA and DNA (b) and poly-L-lysine and DNA (a);

10 Figure 3 shows an analysis of the ligand binding characteristics of the curves generated in Figure 2;

Figure 4 shows ~e T"~ plotted ~g~in.~t the molar ratio showing ~he differences between PLL and NG23 binding in buffer and salt;
Figure 5 shows the titration curve for herring sperm DNA (iIlteraction of PAA with DNA);

Figure 6 shows the results of a gel shift assay using NG30;
Figure 7 shows gel shift assays using phosphorothioate oligonucleotide for both the random block copolymer NG32 (a) and the triblock copolymer NG33 (b);

25 Figure 8 shows ~e results of microcalorimetry experiments using herring sperm DNA for both the random block copolymer NG32 (a) and the triblock copolymer NG33 (b).

Figure 9 shows the serum stability of DNA polycation complexes in 30 comparison with free DNA. PLL--poly(L-lysine); PAA = bis acryloyl W O 97~5067 PCT/GB97/00022 pipera~ine-2-methyl piperazine PAA; and x is the degree of polymerisation.

F,Y~-nrle 1: General scheme for polvmers and polvmer synthesis and S description of analvtical methods used in subsequent ~ ,c Polyamidoamines Linear polyamidoamines are obtained by hydrogen transfer polyaddition 10 reaction of primary monoamines (Scheme î.a) or bis(secondary-amine)s (Scheme l.b) to bisacryl~mi~l~s ~9]:

Scheme l.a Il 11 x CH2= CHC-N-R2-N-CCH--CH2 + x H-N-H ~
I
R, Rl R3 o o - CH~CH~CN ~ CC~CH~N
~ ~ 1?~
X

25 Scheme l.b O O
Il 11 x CH2--CHC-N-R2-N-CCH=CH2 + x H-N-R4-N-H
S Rl Rl R3 R3 o o Il 11 --c~c~ ~ NCC~C~
~ ~ R3 wherein x is from 1 to 70;

each Rl is independently H or a linear or branched hydrocarbon chain -CnH2n+l with n = 1-4 whenever it occurs; each R2 is independently a 15 linear or branched alkylene chain -C"H2n- with n = 1-4 whenever it occurs; and each R3 is independently a linear or branched hydrocarbon chain -CnH2n+ l with n = 1-4 whenever it occurs; each R4 is independently a linear or branched alkylene chain -CnH2n- with n = 2-4 whenever it occurs.
Preferably x is from 1 to 70.

Reference [9~ is incorporated herein by lerelellce.

25 1 mole of bisacrylamide and 1 mole of a primary mono~mine or a secondary bi~mine are mixed in the presence of water (about 2-4 rnl/g of the sum of the monomers~ or alcohols. It is advisable to add a small amount of radical inhibitor in order to avoid radical polymerization of the acrylic monomer. It is better to carry out the reaction under nitrogen 30 atmosphere, but it is not strictly necessary. The preferred reaction temperatures range from 20 to 50~C. The reaction times vary from 1 to W O ~7~5067 PCT/GB97/0002Z

7 days, depending on the reaction temperature and the nature of the monomers (steric hindrance on the aminic nitrogen greatly affects the polymerization rate). Isolation is usually performed by solvent/non solvent precipitation or ultrafiltration or dialysis in water. In order to 5 increase the shelf stability of PAA's, it is better to transform them into their hydrochloride salts by aci(lific~tion of ~e reaction ~ e with diluted HCl before isolation.

Poly(amidoamine)-poly(ethylene glycol) block copolymers Poly(~miflo~min~)-poly(e~ylene glycol) block copolymers ~12] are yl~aled by copolymeri_ing ~min~tetl PEG's, that is PEG's functinn~li7.~
at both ends with secondary arninic units [12], with another amine, in the presence of a bisacrylamide. In practice, the amine~ the ~min~te(l PEG
1~ and bisacrylamide are mixed together in the presence of water or alcohols, taking care that the sum of the moles of amine and ~min~te~l PEG equals the moles of bisacrylamide. The reaction conditions are exactly the same as in the PAA synthesis. Isolation is usually performed by ultrafiltration or dialysis in water.
Reference [1y is incorporated herein by lerelellce.

Poly(amidoamine~-poly(ethylene ~lycol) triblock copolvmers 2~ They are pLcyaled by a two-step procedure involving:

a) synthesis of a polyamidoamine end-capped with acrylamido groups;
~) saturation of the latter by addition of PE~G ~min~te(l at one end with secondary arnino groups.

W O 97/~5067 PCT/GB97/00022-The first step is accomplished following the general procedure for ~e synthesis of PAA's, but using an excess of bisacrylamide. The proper excess is selected on the basis of the Flory's theory on stepwise polymerizations [13] in order to obtain a PAA roughly of the desired S length. Reference [13] is incorporated herein by r~;re~ ce.

In the second step 2.1 moles of mono~min~te~ PEG per mole of excess bisacrylamide are added to the reaction ~ ule together with the proper amount of solvent and allowed to react for 3-5 days under the same 10 conditions. Isolation is usually performed by ultraf~tration or dialysis in water.

After isolation, the products have been characterized by gel permeation chromatography (GPC), intrinsic viscosity measurement and 15 potentiometric titration.
t GPC analysis has been rlm m~kin~ use of TSK-GEL G3000 PW and TSK-GEL G4000 PW columns connected in series, using TRIS buffer pH 8.09 as mobile phase (flow: 1 ml/min) and a UV detector operating at 230 mn.
20 From the GPC chromatograms the number- and weight-average molecular weights (Mn and Mw, respectively) have been calculated starting from a calibration curve purposely calc~ t~-l for PAA~s L14], Intrinsic viscosities have been m~cllred at 30~C in TRIS buffer pH 8.09 by means of Ubbelohde viscometers. Potentiometric titrations with HCl have been 2~ performed on copolymers in order to determine the percentage of PAA
and the molecular weight o~ the PAA segments. Reference [14] is incorporated herein by reference.

CA 0224l830 l998-06-l6 W O 97/2~067 PCT/GB97/00022-Interaction of DNA wi~h Cationic Polymers The interaction of DNA with polyamidoamine polymers can be described in general terms by its interaction with oligonucleotides. In the S experimental work presented here, a number of dirrclclll DNA's have been used for characterisation. These are: a 15mer phosphorothioate oligonucleotide, Herring sperm DNA, a short chain length phosphodiester oligonucleotide of about 10-3() nucleotides leng~h which is about 80%
single stranded in nature, and calf thymus DNA which is double stranded 10 phosphodiester DNA of high molecular weight. All work described below m~ lres the relative amounts of binding between DNA and polymer by l~r~ ce to the input DNA:polymer ratio calclll~te(l by DNA bases (or phosphate groups) per monomer of the polymer.

15 The mtq~ m~ in which the experiment is conducted has a ~i~nific~nt effect on the interactions between DNA and polycations. Interactions are dependent upon the salt concentration, the buffer ion, and the pH of the ionisable groups. Experiments have therefore been carried out in various media to understand the effects which may be observed in biological fluids 20 which are much less easy to measure.

A number of dirrercllt techniques have been used to define the interactions between DNA and polycations. These include electrophoresis gel shift assays, isothermal titration microcalorimetry, determination of DNA
25 melting points, circular dichroism (CD) and photon correlation spectroscopy (PCS).

Gel shift electrophoresis 30 If DNA is added to a central well on an agarose gel, and a potential WO 97/2~067 PCT/GB97/00022 diffel~llce applied to the gel, the DNA will migrate to ~e anode. If cationic polymer is applied it will migrate to the cathode. Complexes which are electrically neutral and or relatively large do not move from the central well. If complexes are incomplete, or are weakly bound they may 5 migrate more slowly to either anode or cathode depending on overall charge of the complex. Electrophoresis is carried out in Tris borate buffer pH7.6.

Isothermal titration microcalorimetry Virtually all chemical reactions and interactions results in either the liberation or abstraction of heat. With a sensitive enough calor~meter, these heat changes (enthalpies) can be measured even for small amounts of biological materials. Titrating one component against another by small 15 additions from a microsyringe results in a series of heat oul~uls which reach zero when the reaction is complete. Analysis of the heat evolved over the titration can give measures of the strength of reaction, the equilibrium constants and other information about the complexation reaction. The results presented for isothermal titration microca}orimetry 20 have been carried out in TBE buffer so they are directly comparable to the gel shift assays.

Determination of DNA meltin~ points 25 When heat is applied to double stranded DNA, the DNA splits into single strands. The temperature at which this occurs depends on the base composition of the DNA, and is related to the strength of interaction of the two strands. This event can be readily measured by the accompanying increase in absorbance at 260 nm. The temperature at which 50% of the 30 absorbance increase occurs (50% strand separation) is known as the melting temperature (Tm) The presence of cationic polymer binding to DNA can stabilise the DNA thus increasing the Tm. These assays have again ~een carried out in TBE.

S Photon correlation spectroscopy (PCS) Tlle scattering OI laser light by colloids can be analysed to provide an accurate m~ rement of ~e size of the colloids. This technique has been used to assess the size of some of the complexes stll-lie-l The ~ollowing Examples relate to the characterisation of interactions between DNA and poly~mi(lo~mines.

F,~ le 2: BPMP2 ~Bisacryloyl Pi~l ~illC - 2 methyl piperazine) 15 copolvmer O O C~
Il /~ 11 / <
--CH2CE~C N NCCH~C~ N N--~/ ~
x wherein x is 1 to 70.

25 Synthesis Bisacryloyl piperazine (13AP) (2.718 g; 14.00 mmol) was dissolved into 0.1402 g/ml 2-me~yl~i~erazine (2MP) aqueous solution (10.00 ml; 14.00 mmol); after addition of distilled water (5 ml), the reaction ~ Ule was left for 5 days at 25~C and, finally, freeze-dried. Yield: 3.58 g.

GPC retention time = 825 sec. Intrinsic viscosity = 0.45 dl/g. Mw =
18000. Mn = 9000 Experimental The interaction between poly~mi-lo~min~s and DNA has been characterised mainly using a synthetic oligonucleotide lSmer with stable phosphorothioate linkages. These linkages are negatively charged as in the natural phosphodiester linked DNA. To establish the potential of PAA
10 as a cationic polymer suitable for DNA delivery, these interactions have been compared to poly-L Lysine (PLL), the most studied of the cationic polymers used in DNA delivery.

In Fig 1 a gel-shift assay is shown comparing the interaction of (a) poly-L-15 Iysine (PLL) and (b) PAA with phosphorothioate oligonucleotide (lSmer) at various oligonucleotide:polymer ratios. The samples were applied to the central wells of an agarose gel and electrophoresed for 30 mimltes at 50 volts. The poly-L-lysine complexes in Pigure la show either no uncomplexed DNA or polymer over a wide range of nucleotide to 20 monomer ratios. This probably results from the co-operative binding activity seen between PLL and DNA. The PAA/DNA complex Figure lb, shows a small range of DNA:PAA ratios (1:1 - 1:2) where polymer and oligonucleotide appear to be completely complexed.

2~ When these interactions were investi~ted using isothermal titration microcalorimetry, the net thermodynamic characteristics of the interaction can be seen following subtraction of the heat of dilution of the polymer from the titration of polymer into DNA. From these curves it can be seen that the interactions between PAA and DNA are dirÇerenl from those of 30 poly-L lysine and DNA (Figs 2 & 3). With poly-L-lys~ne (Fig 2a),~

interactions initially gave off smaller amounts of heat leading to a peak of heat output, and the reaction did not saturate so rapidly. These results strongly suggest co-operative binding. In contrast, PAA (Fig 2b) showed a simpler profile of heat changes in which saturation was achieved more 5 readily. This profile again appeared to be multiphasic. The ligand binding characteristics of these curves were analysed (Figs 3a and 3b).
These showed that ligand binding with PLL appeared to be a triphasic process with interactions at nucleotide to monomer ratios of 0.5, 1.0 and 1.~. The first interaction was weakly exothermic, the second endothermic 10 and the third more strongly exomprmic. In contrast PAA gave a single exothermic interaction with DNA at an oligonucleotide to polymer ratio of 0.92. Overall the PAA reaction was more exothermic than the PLL
reaction.

1~ In the melting temperature experiments, sense and antisense oligonucleotide was mixed with polymer. In this llli~t,Ule the sense and antisense strands will combine to give a double stranded piece of DNA
interacting with the polymer. Again the change in Tm was measured over a range of oligonucleotide to monomer ratios. In Figure 4 the Tm values 20 resulting from interactions of various ratios of polymer to DNA were m~mred in TBE buffer. PLL showed an increasing stability of the oligonucleotide complex with increasing amounts of polymer, the most stable complexes being achieved with a large excess of polymer. PAA
however showed a maximum stability at molar ratios of ON:Polymer 25 between 1:1 and 2:1.

The interaction of PAA with DNA of a number of dirrelelll types have been analysed by both gel-shift electrophoresis and isothermal titration microcalorimetry, and the interactions with herring sperm DNA and calf 30 thymus DNA are similar to those seen for the phosphorothioate ' oligonucleotides. The titration curve for herring sperm DNA is shown in Figure 5.

The particle size of ~e complexes will be very important in dete~ i"i"~
5 the biodistribution of the complexes in vivo. The size of ~he complexes pro~ cecl under various conditions have been m~ lred using photon correlation spectroscopy and can be found in Table 1. PLL complexes are usually insoluble after a 1:1 ratio (ie after charge neutralisation~, whereas oligonucleotide:PAA complexes were soluble below salulalillg levels of 10 polymer (ie 1:2 ON:polymer ratios). Generally, in buffer, aggregates with PLL are larger ~an those of PAA as measured by photon correlation spectroscopy (PCS). In water the PAA complexes are soluble and not rletect~ble by PCS.

15 Table 1: Size of Oligonucleotide-C.~tio~ic Polymer Complexes measured by PCS.
Polymer Ct mpl~ in Complex in TBE:
water PLL 198 nm > 3 ~bm PAA soluble 250 Dm PAA-PEG/PEG-PAA-PEG soluble soluble :Exam~le 3: Other polvamido~ n~ struetures Polyamidoamines of a wide variety of structures can be synthesised, which 25 will vary in their physicoçhe~nic~l properties such as pK of the amide groups, and the spacing between the charged amide groups which will govern the interaction wi~ DNA.

MBA-2MP (Methylene bisacryl~mi(l~2m~lhyl~ Jerazine)copolymer C~
O O
Il 11 r~
C~C~C I C~ I CC~CE~ N N
H H x wherein x is 1 to 70.

Synthesis N,N'Methylenebisacrylamide (MBA) (1.177 g; 7.63Inmol) was suspended in 0.0780 g/ml 2MP aqueous solution (10.00 ml; 7.79 mmol) in the presence of 4-methoxyphenol (6 mg); the reaction llli~lul~ was stirred at 20~C, under nitrogen atmosphere and in the dark, for 4 days.
15 Afterwards, HCl was added up to pH Z-3 and the resulting solution was ultrafiltered in water through a membrane with Mw cut off 3000 and freeze-dried. Yield = 1.30 g. GPC retention time--890 sec. Intrinsic viscosity = 0.24 dl/g. Mw = 8500. Mn = 5300-.

20 MBA-~A (Methylene bisac~ ~h..~ methylamine) copolymer o o Il 11 - CH~C~CNC~NCC~C~N -' H CH3x 25 wherein x is 1 to 70.

Synthesis MBA (1.668 g; 10.82 mmol) was suspended in 0.0343 g/ml methylamine aqueous solution (10.00 ml; 11.04 mmol) in the presence of 4-CA 0224l830 l998-06-l6 methoxyphenol (8 mg~; the reaction ~Lule was stirred at 20~C, under nitrogen atmosphere and in the dark, for 4 days. Afterwards, HCl was added up to pH 2-3 and the resulting solution was ultrafiltered in water through a membrane with Mw cut off 3000 and freeze-dried. Yield =
5 0.93 g. GPC retention time = 875 sec. Intrinsic viscosity = 0.24 dl/g.
Mw = 9500. Mn = 6000.

MBA-DMEDA (Methylene bis a~ i(l~rlim~il,yl~~hylene ~ ne) copolymer o o Il l!
--Cl~CI12CNCI~NCCI~CI~NCI~ N--H H C}~ Cl~
wherein x is 1 to 70.

15 Synthesis MBA (1.980 g; 12.84 m mol) was suspended in 0.148 g/n~ N,N'-dimethylethylen~ min~ aqueous solution (7.51 rnl; 12.58 mmol) in the presence of 4-methoxyphenol (11 mg); the reaction mixture was stirred at 20 25~C, under nitrogen atmosphere a~d in the dark, for 4 days.
Afterwards, HCl was added up to pH 2-3 and the reslllting solution was ultrafiltered in water through a membrane with Mw cut off 3000 and freeze-dried. Yield = 2.80 g. GPC retention time = 780 sec. Intrinsic viscosity = 0.53 dl/g. Mw = 21500. Mn = 9500 Experimental The binding properties of several dirr~lellL structures have been tested for their association with DNA by the gel shifL assay. All of the above 30 structures demonstrated an association with DNA which was similar to ~at seen wi~ NG23. An example of one of these gels using NG30 is shown in Fig. 6.

Two dir~ pes of PEG- polyamidoamine copolymers have been 5 syIlthesised. Firstly a random block copolymer (NG32), and seeondly a ~iblock copolymer (NG33). These are described in Examples 4 and 5.

Tnrle4: PI~BP2MP2l~olyethylenes~lycol-(Bisacrylov~ e - 2 methyl ~ilJe~ e)l block copolvmers ~G32 = PEG BP2 MP2 Structure:

Il 1~
--[-PAA--C~(CH2CH2O)yC--]x--where:
the number y is about 45 and x is 1 ~o 70.
-PAA- is:

11 A 1~l ~ ~ ~l A
--N N--CH2CH2CN NCCH2CH2N N~CH2CH2CN NCCII2CH2N N--Z

and ~e number z is about 6.6.
.

25 Synthesis 1,4-Bis(acryloyl)piperazine (1.003 g, 5.16 mmol), 2-me~ylpiperazine CA 0224ls30 Isss-06-l6 ~0.455 g, 4.54 mmol), piperazinyl formate of PEG 2000 (1.536 g, 0.69 m m ol), prepared as described in [18], and 4-methoxyphenol (5 mg) were dissolved in distilled water (5 ml) and allowed to stand at 25~C, under inert atmosphere and in the dark, for 3 days. After ultrafiltration in water 5 through a membrane with Mw cut of~ 10,000 the solution was ~eeze-dried.
Yield = 1.93 g. GPC retention ~ime = 785 sec. Intrinsic viscosi~ =
0.55 dl/g. Mw = 36000. M" = 10000. Percentage of PAA = 53%
(w/w). Mn of the PAA se~m~-nt~ = 2300.

10 li',Y~lnplc 5: PEG-BP2MP-PEG rPE~(Bisacrvloyl pip~l ~.e - 2 methyl ~ ...e)-PEG triblock copolvmers NG33 = PEG-BP2MP-PEG

1~ Structure:
O O
Il 11 CH30--(-CH2CH20-)y--C--PAA--C--0--(-CH2CH20-)y--CH3 2~) where:
the number y is about 43 -PAA- is:

O O CH3 - i~i A ~

and the number z is about 6.7.

Synthesis 1,4-Bis(acryloyl)piperazine (1.081 g, 5.56 mmol), 2-methylpiperazine (0.491 g, 4.90 mmol) and 4-methoxyphenol (7 mg) were dissolved in S distilled water (4 rnl). The resulting solution was le~ at 25~C, under inert a~nosphere and in the dark, for 2 days, then piperazinyl formate of PEG
1900 monomethyl ether (2.978 g, 1.47 mmol), prepared as described in 118~, and distilled water (20 rnl) were added, and the reaction ll~ Ule allowed to react for fur~er 4 days under the same conditions. Finally, the solution was ultrafiltered through a membrane with Mw cut off 10,000 and ~reeze-dried. Yield = 2.85 g. GPC retention time = 875 sec. Intrinsic ~iscosity = 0.20 dl/g. Mw = 11400. Mn a 3300. Percentage of PAA
= 37% (w/w~. Mn of the PAA segments = 2350.

15 Experimental Both gel shift assays (Fig 7) using phosphorothioate oligonucleotide and microcalorimetric titration analysis (Fig 8) using herring sperm DNA are ' presented for both Lhe random block copolymer NG32 (a) and the triblock 20 copolymer NG33 (b). If it is ~.s~lmt?~l that the DNA interacts with t~e PAA but not the PEG moiety, the interaction appears to be identical to that of the PAA alone and therefore complexation was unaffected by the addition of a PEG moiety.

2~ Photon correlation spectroscopy (Table 1 in Example 2) could not detect the presence of particles in either of the PAA-PEG complexes at any ratios of oligonucleotide to polymer, in either buffer or water. This is believed to be of great si~nific~nce for DNA delivery.

~,Y~m rle 6: Sl-m m ~ry of ~ndin s ~or interaction of PAAs ~th oli~onucleotides These polymers have been shown to interact with DNA in a dif~re.ll way 5 to that described for other linear cationic polymers such as poly-L-lysine to form complexes with improved properties for DNA delivery systems.
Binding is not co-operative, is pH dependent over a lower range than that seen with polymers of primary amines such as PLL. The binding of PAA
to DNA is not co-operative in nature occurring over a more defined range 10 of nucleotide to monomer ratios. Below the pK of the polymers the binding is stronger for PAA than that described for PLL, also the complexes with PAA are more soluble than those seen for PLL, and the PEG-PAA polymers appear to be soluble at all nucleotide to monomer ratios. Either as a repeating block copolymer structure or as an ABA
15 block copolymer skucture, inclusion of PEG into the polymer iInproves the solubility of the complex without affecting ~e binding of DNA to the polymer. Complexation is observed with both natural (phosphodiester) and synthetic (phosphorothioate) DNA for a range of polyamidoamine structures.
F~mple 7: PEG-PAA-PE~l Structure:
O O
CH3~(-CH2CH2O-)y--C--PAA--C~(-CH2CH20-)y--CH3 where:
the number y is about 43 30 -PAA- is:

W O 97/2~067 PCT/GB97/00022 --N N-- C~2CHzCN NCCH2CH2NC~CH2N--CH2CH2CN NCCH2CH2N N--and the number ~ is about 6.4.

Syn~hesis:

S It was prepared by the same procedure as NG33, substi~1tin~ N,N'-dimethylethylene rli~mine (0.527 ml; 4.90 mmol) for 2-me~ylpiperazine.
Yield = 2.74 g. GPC retention time = 880 sec. Intrinsic viscosity =
0.18 dl/g. Mw = 10300. Mn = 3500. Percentage of PAA = 36%
~w/w). Mn of the PAA segments = 2170.
F,x~ )le 8: P~:G-PAA-PEG-I~

Structure:

O O
Il 11 CH30~-CH2CH20-)y--C--PAA~(-CH2CH20-)y--CH3 where:
20 the number y is about 43 -PAA- is:

--N N---C~CI~CNCI~NCCI~CH2N N--CE~CE~CNICH2lCC~CH2N N--and the number z is about 4.2.

CA 0224l830 l998-06-l6 Synthesis:

It was prepared by the same procedure as NG33, substhlting N,N'-methylenebisacrylamide (0.922 g; 5.98 mmol~ for 1,4-5 bis(acryloyl)piperazine. Yield = 3.08 g. GPC retention time = 890 sec.
Intrinsic viscosity = 0.15 dl/g. Mw = 9600. Mn = 2500. Percentage of PAA = 26% (w/w). Mn of the PAA segment~-- 1350.

rle 9: PEG-PAA-PEG-m O O
Il 11 C~30 (-CH2CH20-)y~--PAA~-CH2CH20-)y~H3 15 where:
the number y is about 113 -PAA- is:

--N~ N--CH2CH2C~ NCCH2C~2N N--CH2CH2CN NCCH2CH2N N

and the number z is about 6.3.
Syn~esis:

It was prepared by the same procedure as NG33, substitllting piperazinyl formate of PEG 5000 monomethylether (7.534 g, 1.47 mmol), prepared as described in [18], for piperazinyl formate of PEG 1900 monomethyle~:her. Yield = 3.83 g. GPC retention time = 840 sec.
Intrinsic viscosity = 0.30 dl/g. Mw = 25000. Mn--8000. Percentage of PAA = 18% (w/w). Mn of the PAA segments = 2200.

~Y~mrle 10: PEG-PAA-I

S O O
Il 11 --[-PAA--CO (CH2CH2O)yC--Ix--where:
10 the number y is about 45 -PAA- is:

1~l R o 1~l --N N--t~2cH~cN~NccEI2c~2NcH2cH2N--C~IzCH2CN NCCH2CEIzN N--and the number z is about 6.9.

15 Syn~esis:

It was ~l~aled by the same procedure as NG32, substih-tin~ N,N'-dimethyle~ylene fli~min~ (0.489 ml; 4.54 mmol) for 2-methylpiperazine.
Yield = 2.12 g. GPC retention time = 780 sec. Intrinsic viscosi~ =
0.58 dl/g. Mw = 42000. Mn = 11500. Percentage of PAA = 54%
(w/w). Mn of ~e PAA segments = 2310.

mrle 11: PEG-PAA-II

O O
Il Jl --[-PAA--CO--(CH2CH2O)yC--3x--CA 0224l830 l998-06-l6 where:
the number y is about 45 and x is 1 to 70.
-PAA- is:

o o ~ o o C~CI~zCNCH2NbC~CE~ N N--C}~ CNC~Z I CCE~ zh~\N_~
H H H H

S and the number z is about 4.3.

Synthesis:

It was prepared by the same procedure as NG32, substhlltin~ N,N'-10 methylenebisacrylarnide (0.854 g; 5.54 mmol) for 1,4-bis(acryloyl)piperazine. Yield = 1.83 g. GPC retention time = 795 sec.
Intrinsic viscosity = 0.49 dl/g. Mw = 34000. Mn = 10500. Percentage of PAA = 42% (w/w). Mn of the PAA segments = 1420.

15 Exa~ple 12: PE~PAA-m St~ucture:

O O
--[-PAA--CO--(CH2CH2O)yC--]x--where:
the number y is about 90 and x is 1 to 70.
25 -PAA- is:

- N N - CHlCH2CN NCCH~CH2N N - CH2CH2gN NCCH2CEN N -and ~e number z is about 6.5.

Synthesis:

S It was prepared by the same procedure as NG32, substil~ltin~ piperazinyl formate of PEC~ 4000 (2.915 g, 0.69 mmol), prepared as described in ~18~, for piperazinyl formate of PEG 2000. Yield = 2.94 g. GPC
retentio~ time = 760 sec. Intrinsic viscosity = 0.64 dl/g. Mw = 58000.
Mn = 16000. Percentage of PAA = 3~5% (w/w). Mn Of ~e PAA
10 segments = 2250.

~,Y~nrle 13: A-lmini~tration of Ha-ras antisense se~uence to T24 bladder carcinoma cells 15 Polymer NG23 (PAA-PEG) is mixed wi~ Ha-ras antisense oligonucleotide to form a polymer-oligonucleotide composition as in the previous Examples.

The composition is added to T24 ~ladder carcinoma cells growing in culture.

This leads to a reduction in viability of the cells and an inhibition of Ha-ras protein production.

~,y~rnple 14: Transfection of m~mm~ n cells with a chloramphenicol acetvl transferase (CAT) ~en~cont~inin~ plasmid Polymer NG23 (PAA-PEG~ is mixed with a plasmid which expresses the S CAT gene in m~mm~ n cells to form a polymer-DNA composition as in the previous Examples.

The composition is added to suitable m~mm~ n cells growing in culture.
The cells are transfected by the plasmid and CAT gene expression is 10 me~ured using standard methods with l4C-labelled chloramphenicol.

F,Y~n~l)le ~ (lmin;~tration of CFTR (cystic fibrosis transmembrane re~ulator) cDNA to a cystic rlbrosis patient 15 Polymer NG23 (PAA-PEG (which has been m~nllf~ctured and kept under sterile conditions) is mixed with sterile and pyrogen-free plasmid DNA
encoding, and capable of expressing in human lung cells, the C~TR
cDNA. The resulting composition is prepared into a sterile, pyrogen free formulation suitable for ~clmini~tration to the lungs. An effective amount 20 of the formulation is ~rlmini.ctered to the lungs of the CF patieht.

rle 16: Serum stabilitv of DNA polvcationic complexes in comparison with free DNA

A DNA plasmid (PCT0297L, 30 ,ug) was mixed at optimal ratios with polymers (DNA:PAA, 1:2, DNA:PLL l:l.S) to form a DNA polycation complex. The complex was then added to newborn calf serum and incubated for various time periods (total volume 100 ,ul, 64% serum) at 37~C. After incubation the samples (10 ,ul) were snap frozen to prevent further degradation, and when all samples had been taken, were loaded onto an agarose electrophoresis gel prestained with ethi(lillm bromide.
The gel buffer was a NaOHIKCI/EDTA system pH12.5 which allowed the complex to dissociate and the DNA to separate according to molecular weight. The bands were visualised using a UV light box recorded using S a CCD camera and the images sc~nn~cl using a Shim~ ie~itometer at 550 nm to give a ~ ve estimate of the amount o~ undegraded plasmid in comparison to untreated complex.

The results are shown in Figure 9. Free DNA is degraded within 5 10 min--t~s in this system. A poly-L-lysine complex has some protective effect, but is not as effective as complexes with polyamido~min~s. We note from ~his worl~ that poly~mi~o~mines with a higher degree of polymerisation are more ef~ective in protecting from degradation.

REFERENCES

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[4] L. };elgner, T.R. Gadek, M. Holm, R. Roman, H.W. Chan, M.
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[8] J-P. Clarenc, G. Degols, J.P. Eeonetti, P. l~ilh~ l and B. Lebleu (1993) Anticancer Drug Design 8, 81.
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[10] J. Haensler and F.G. Szoka (1993) Bioconjugate Chem. 4, 372-379.
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Rev. 6, 133.
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2~ tl3] P. J. Flory, "Principles of Polymer Ch~ try", Cornell University Press, Ithaca, New York (1953).
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Key to polvmer codes and structures NG23 = BPMP2 (Bisacryloylpiperazine-2 me~yl piperazine) NG28 = MBA-2MP (Me~hylenebisacrylamide-2 methyl piperazine) 5 NG29 = MBA-MMA (Methylenebisacrylamide-methylamine) NG30 = MBA-DMEDA (Methylenebisacrylamide-dime~ylethylene fli~min~) NG32 = PEG BP2MP2 (Polyethyleneglycol-(Bisacryloyl-piperazine-2 me~ylpiperazine) block copolymer 10 NG33 = PEG BP2MP2-PEG triblock copolymer Abbrevi~ n~

PAA is poly(ami-lo~min~); PLL is poly(-L-lysine); PEG is 15 poly(e~yleneglycol).

Claims (29)

1. A composition for delivering a nucleic acid or a derivative thereof comprising a linear polymer with a backbone comprising amido and tertiary amino groups arranged regularly on the said backbone and a nucleic acid or derivative bound to said polymer.

2. A composition according to Claim 1 wherein said linear polymer comprises a poly(amidoamine).

3. A composition according to Claim 1 or 2 wherein said linear polymer consists of a poly(amidoamine).

4. A composition according to Claim 1 or 2 wherein said linear polymer further comprises ethylene glycol or poly(ethylene glycol).

5. A composition according to Claim 4 wherein said linear polymer is a poly(ethylene glycol)-poly(amidoamine) block copolymer or ethylene glycol-poly(amidoamine) block copolymer.

6. A composition according to Claim 5 wherein said linear polymer is a block copolymer with the structure [poly(amidoamine)-(ethylene glycol)y]x wherein x is from 1 to 50 and each y is independently from 1 to 200 whenever it occurs.

7. A composition according to Claim 5 wherein said linear polymer is a block copolymer with the structure (ethylene glycol)y-poly(amidoamine)-(ethylene glycol)y wherein each y is independently 1 to 200.

8. A composition according to Claim 2 or 3 wherein said poly(amidoamine) has the formula:
(a) or (b) or (c) or (d) or (e) wherein are replaced by and, in any case, z is from 0 or 1 as appropriate to 70 and each R1 is independently H or a linear or branched hydrocarbon chain -CnH2n+1, with n = 1-4 whenever it occurs; each R2 is independently a linear or branched alkylene chain -CnH2n- with n = 1-4 whenever it occurs; each R3 is independently a linear or branched hydrocarbon chain -CnH2n+1 with n = 1-4 whenever it occurs;
each R4 is independently a linear or branched alkylene chain -CnH2n- with n = 2-4 whenever it occurs; each R5 is independently a linear or branched hydrocarbon chain -CnH2n+1 with n = 1-4; each R6 is independently a linear or branched alkylene chain -CnH2n- with n = 2-4; and wherein R7, R8, R9 and R10 are independently H or linear or branched hydrocarbon chain -CnH2n+1 with n = 1-3 whenever they occur.

9. A composition according to any one of Claims 4 to 6 wherein the linear polymer has the formula:

wherein PAA is a poly(amidoamine) as defined in Claim 8, x is from 1 to 50 and y is from 1 to 200.

10. A composition according to any one of Claims 4, 5 or 7 wherein the linear polymer has the formula wherein PAA is a poly(amidoamine) as defined in Claim 8 and y is from 1 to 200.

11. A composition according to any one of the preceding claims wherein the nucleic is DNA or a derivative thereof.

12. A composition according to any one of Claims 1 to 10 wherein the nucleic is RNA or a derivative thereof.

13. A composition according to Claim 11 or 12 wherein the nucleic acid derivative comprises a phosphorothioate linkage.

14. A composition according to any one of the preceding claims wherein said nucleic acid or derivative is a therapeutic molecule.

15. A composition according to any one of the preceding claims wherein the polymer further comprises a biological recognition signal.

16. A composition according to any one of the preceding claims for use in medicine.

17. Use of a composition according to Claim 14 in the manufacture of a medicament for treatment of a disease.

18. A pharmaceutical composition comprising a composition according to any one of Claims 1 to 15 and a pharmaceutically effective carrier.

19. A method of making a composition according to any one of Claims 1 to 15 comprising contacting said biologically active polyanionic molecule with said linear polymer.

20. A method of delivering a biologically active polyanionic molecule to a host, the method comprising administering to said host an effective amount of a composition according to any one of Claims 1 to 15.

21. A method of delivering a biologically active polyanionic molecule to a cell in an environment, the method comprising administering to said environment a composition as defined in any one of Claims 1 to 15.

22. A method according to Claim 21 wherein said composition is contacted with said cell.

23. A method according to any one of Claims 19 to 22 wherein said nucleic acid or derivative thereof is a therapeutic molecule.

24. A method of treating, preventing or ameliorating a disease in a multicellular organism, which multicellular organism benefits from the administration of a biologically active polyanionic molecule, the method comprising administering to the patient a composition as defined in any one of Claims 1 to 15.

25. A method according to Claim 24 wherein the biologically active polyanionic molecule comprises a therapeutic nucleic acid or derivative thereof.

26. A polymer with the formula (ethylene glycol)y-poly(amidoamine)-(ethyleneglycol)y wherein each y is independently from 1 to 200.

27. A polymer according to Claim 26 with the formula:

wherein PAA is a poly(amidoamine) as defined in Claim 8 and y is from 1 to 200.

28. Any novel polymer or polymer composition as disclosed herein.

29. Any novel use of a polymer or polymer composition as disclosed herein.