JP2002544126A - Membrane disrupting peptides oligomerized by covalent bonds - Google Patents

Abstract

(57) Abstract The present invention relates to modified membrane disrupting peptides. The invention also relates to a delivery conjugate comprising the modified membrane disrupting peptide, and uses of the delivery conjugate. The modified membrane disrupting peptide is modified to form covalently linked multimers. The present invention also provides a method of treating a genetic disorder, comprising administering to a patient in need of such treatment an effective dose of a delivery conjugate, wherein the conjugate comprises a therapeutic nucleic acid and a compound of the invention. Includes a modified membrane disrupting peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a modified membrane disrupting peptide (membrane disruptive peptide).
v. peptide). The invention also relates to a delivery conjugate comprising the modified membrane disrupting peptide, and uses of the delivery conjugate.

[0002] In the field of gene therapy, there is a need for simple non-viral delivery agents that can be used to efficiently transfect cells. Cationic peptides have been used to concentrate DNA by electrostatic interactions and to prepare simple and reproducible peptide: DNA formulations. Methods for their preparation and purification are well described in the literature. Cationic peptides also
It has been used as a chemical anchor for the introduction of additional peptidic or non-peptidic entities to assist in transporting the packaged gene from the extracellular location to the nucleus of the desired target cell.

[0003] According to some groups, when cationic peptides are used as delivery agents in vitro, transfection is an intracellular vehicle in which the delivered gene encapsulates the gene during or after cell uptake. It has been demonstrated to be optimal only in the presence of exogenous drugs which are thought to work by increasing the ability to escape from. Examples of such exogenous agents are chloroquine or membrane disrupting peptides. Destabilization of biological membranes is thought to be central to the role of these agents. Disruption in the structure of any membrane that acts as a barrier to transport of the delivery complex increases its ability to access the nucleus. However, for many in vivo applications, apart from complicating the formulation, the use of exogenous drugs (e.g., small organic chloroquine) can result in different clearance mechanisms in vivo and a difference between small and large molecules. It makes sense to assume that it will be hindered by the spreading factor found. A fusogenic or membrane disrupting peptide (eg, Wagne
r (Wagner et al., PNAS 89, 7934-8, 1992) and Smi
th (peptide as described by WO 97/35070)) can be anchored either to the enriched structure prior to complex formation or to a preformed peptide: DNA complex. It also complicates the manufacture of the formulation. Furthermore, such peptides have been shown to be less efficient than adenovirus particles in enhancing gene transfer (Gottschalk et al.,
Gene Therapy 3, 448-457, 1996; Wagner et al.
PNAS 89, 7934-8, 1992), and the search for more efficient peptides continues.

[0004] Peptides that can concentrate DNA and disrupt biological membranes can simplify formulations and lead to more efficient transfection.

[0005] Melittin is a well-known membrane disrupting peptide; however, despite being one of the most extensively studied peptide sequences, it appears that there are only two examples (herein). , Its use in gene delivery has been described). First, dioleoylphosphatidylethanolamine linked to melittin
In the form of N- [3- (2-pyridyldithio) propionate] (DOPE derivative) (Legendre et al., Bioconjugate Chem. 8: 5).
7-63, 1997), and secondly, U.S. Pat. No. 5,547,93.
In No. 2. In Legendre et al., Conjugation of lipids is an expensive and complicated process, and problems with the use of DOPE in vivo have been reported. In U.S. Pat. No. 5,547,932, a formulation of an endosomolytic agent conjugated to a nucleic acid binding agent is described. The described formulation forms a relatively inefficient transfection formulation, which is also probably toxic to the transfected cells. Indeed, the toxicity of melittin may explain why it has not been used as a single peptide in successful non-viral formulations.

[0006] The present invention provides modified membrane disrupting peptides, wherein the membrane disrupting peptides have been modified to form covalently linked multimers.

[0007] Preferably, the modified membrane disrupting peptide is further modified to form a substantially continuous α-helix.

[0008] By modifying the peptide so that it forms a multimer, and preferably such that the peptide forms a substantially continuous α-helix, Has been found to provide reduced transfection levels when the peptide is used to deliver nucleic acids to cells.

[0009] Without being bound by any theory, the membrane-disrupting peptide must be in monomeric form so that it can be inserted into a membrane, where the membrane-disrupting peptide can aggregate to form pores. It has been proposed. Pore formation is considered toxic to cells. This is because it increases passive ion permeability. Multimerization of the peptide prevents the formation of monomers, thereby preventing the formation of pores and reducing cytotoxicity. The multimer still retains some membrane disruption properties due to the predominant non-polar amphipathic α-helix. By modifying the peptide to obtain a substantially continuous alpha helix, the membrane disruption properties of the peptide are increased. This is because a substantially continuous α-helix is effective in interacting with the cell membrane. Multimerization of a peptide also has the advantage that the peptide is less likely to be dissociated from any bound nucleic acid.

The term “membrane-disrupting peptide” refers to a peptide that can promote membrane destabilization and reduce the energy required by a molecule to cross a membrane. Assays (eg, erythrocyte lysis assays) can be used to determine whether a peptide is a membrane disrupting peptide; however, different membrane disrupting peptides have different cell specificities and are different for erythrocytes The cell type can be lysed. Therefore, different cell types should be used in the lysis assay to determine if the peptide is a membrane disrupting peptide. The membrane disrupting peptide is preferably a toxic membrane disrupting peptide. It is further preferred that the membrane disrupting peptide is toxic by inserting itself into the membrane in monomeric form. The membrane-disrupting peptide is a toxic α-helix membrane-disrupting peptide (eg, melittin, cecropin A, cecropin P1, cecropin D, magainin 2, bombolitin or paradaxin (S).
aberwal et al., Biochimica et Biophysica Ac.
ta, 1197, 109-131, 1994).
The membrane disrupting peptide preferably forms an amphipathic α-helix with one surface rich in cationic residues (which allows for DNA enrichment) and a hydrophobic surface capable of interacting with membrane lipids. . It is also preferred that the membrane disrupting peptide comprises an α-helical region and a basic region at the C-terminus of the peptide, which can concentrate nucleic acids. Preferably, the membrane disrupting peptide is a human bactericidal / permeability enhancing protein (ba
tericidal / permeability-increasing p
protein (BPI) (Gray et al., J. Biol. Chem. 2).
64, 9505, 1989 and U.S. Pat. No. 5,856,302).
. Preferably, the membrane disrupting peptide is melittin.

The term “peptide” as used herein refers to a polymer of amino acids having a chain length between 10 and 150 amino acids. The term does not refer to or exclude peptide modifications (eg, glycosylation, acetylation, and phosphorylation). Included in this definition are peptides comprising one or more analogs of amino acids (including, for example, unnatural amino acids), peptides having substituted linkages, and other variants known in the art (naturally occurring amino acids). Both modified and synthetic modifications).

The term “modified to form a multimer” means that the membrane disrupting peptide is modified such that the peptide is covalently linked to one or more other membrane disrupting peptides. . Preferably, the multimer is a dimer, trimer, or tetramer, more preferably, a dimer or tetramer, and most preferably, a dimer. The membrane-disrupting peptides forming the multimer may be the same or different.

[0013] The membrane disrupting peptides may be linked together at any point along the length of the peptide;
However, it is preferred that the peptides are linked together at the N-terminus and / or C-terminus of each peptide, and most preferred that the peptides are linked together at the N-terminus of each peptide.

Preferably, the membrane disrupting peptide is an amino acid that forms a covalent bond with an amino acid of another modified membrane disrupting peptide either directly via a disulfide bond or via a linking group, Is modified to form a dimer. Alternatively, amino acids that form a covalent bond directly through a disulfide bond or through a linking group can be added to the membrane disrupting peptide. Preferably, the amino acid added to or substituting for an amino acid of a peptide is a cysteine amino acid, which can form a disulfide bond with a cysteine residue of another peptide. The dimer-forming membrane-disrupting peptides need not be identical; however, it is preferred that the dimer-forming peptides be identical.

[0015] Preferably, the membrane disrupting peptide comprises two or more amino acids that form a covalent bond with the amino acid of another modified membrane disrupting peptide either directly via a disulfide bond or via a linking group. It is modified to form a trimer or tetramer by substituting an amino acid to form a trimer or tetramer. Or,
Two or more amino acids that form a disulfide bond, either directly or via a linking group, can be added to the membrane disrupting peptide. The membrane-disrupting peptides that form a trimer or tetramer need not be identical; however, the peptides that form a trimer or tetramer are preferably identical.

[0016] The membrane disrupting peptides can be linked together via a linker, such as a commercially available linker (including a bismaleimide linker or a bisvinylsulfone linker).

The term “substantially continuous α-helix” forms an α-helix and α
It refers to the region of the membrane disrupting peptide that does not include one or more amino acids that disrupt helix formation. Preferably, a substantially continuous helix is at least 10 amino acids long. Preferably, the substantially continuous helix is at least 5% of the length of the modified membrane disrupting peptide, more preferably between 40% and 90% of the length of the modified membrane disrupting peptide, and most Preferably, it is formed at about 80% of the length of the modified membrane disrupting peptide. The presence of the α helix in the peptide
It can be easily measured using standard circular dichroism analysis.

[0018] Amino acids that can disrupt α-helix formation include proline, glycine, tyrosine, threonine, and serine. However, as will be appreciated by those skilled in the art, the ability of an amino acid to disrupt α-helix formation depends on the overall sequence of the peptide and other factors, such as the pH of the solution in which the peptide folds.

Preferably, the amino acid capable of disrupting α-helix formation is proline.

Preferably, the membrane disrupting peptide comprises an amino acid that disrupts α helix formation,
Substitutions with amino acids that do not disrupt helix formation are modified to form a substantially continuous α-helix.

In a preferred embodiment, the modified membrane disrupting peptide of the invention has the amino acid sequence CIGAVLKVLTTGLAALISWIKRKRQQ. It is further preferred that the modified membrane disrupting peptide forms a dimer via a direct disulfide bond between the N-terminal cysteine amino acids.

The modified membrane disrupting peptides of the present invention may further comprise other functional groups (eg, lipids),
It can be modified by the addition of targeting groups, such as antibodies or antibody fragments that target the peptide to specific cell types, antigenic peptides, sugars, and neutral hydrophilic polymers such as PEG and PVP. Suitable functional groups that can be added to the modified membrane disrupting peptides of the present invention are described in International Patent Application WO 96/41606, as well as methods of attaching such groups to the peptides.

Preferably, the modified membrane disrupting peptides of the present invention are further modified by the addition of amino acids to a substantially contiguous α-helical region of the peptide, wherein the additional amino acids are substantially contiguous α Extend the length of the helical region. Preferably, the substantially continuous α-helical region is extended such that the α-helical region is at least 10 or more amino acids, preferably at least 20 or more amino acids in length.

[0024] Preferably, the modified membrane disrupting peptides of the present invention are further modified by the addition of a basic amino acid to the C-terminal region of the peptide.

The C-terminal region of the modified membrane disrupting peptide of the present invention is a region extending from the C-terminal amino acid to a region that forms a substantially continuous α-helical region.

[0026] Basic amino acids are well known to those skilled in the art, and include lysine, arginine, and histidine. Preferably, the C-terminal region is modified by the addition of between 1 and 50 basic amino acids, more preferably between 5 and 15 basic amino acids.

The present invention also provides functional homologs of the modified membrane disrupting peptides of the present invention.

Preferred functional homologs of the modified membrane disrupting peptides of the invention still retain their activity and preferably have at least 80% of the peptide of the invention.
More preferably, it has 90%, and most preferably, 95% homology. Preferably, such functional homologs (including fragments of the peptides of the invention) differ by as little as 1 to 10 amino acids. More preferably, the amino acid changes are conservative. A conservative change is a change that replaces one amino acid with one from its family of related amino acids in its side chain. For example, substitution of leucine with isoleucine or valine, substitution of aspartic acid with glutamic acid, substitution of threonine with serine, or similar conservative substitutions of amino acids with structurally related amino acids will result in the biological activity of the peptide. Has no major effect on

However, it is often preferable to change amino acids to change the biological activity of the peptide. For example, mutations that disrupt or enhance one or more functions of the peptide may be particularly useful. Such mutations can generally be made by changing any conserved sequences in the peptide. Such a homolog may be present in the protein of the invention or a fragment thereof at least 80%, more preferably 9%.
It is preferred to have 0% and most preferably 95% homology. Preferably, such altered proteins or fragments thereof differ by 1 to 10 amino acids.

The present invention also provides the use of a modified membrane disrupting peptide of the invention in a delivery complex for delivering a nucleic acid to a cell.

[0031] The modified membrane disrupting peptides of the present invention can be used to deliver negatively charged polymers, preferably nucleic acids, to any cell type. Preferred cell types include E. coli. and prokaryotic cell types such as E. coli, and mammalian cells, including ex vivo primary cells (eg, including HUVEC and DC cell mammalian cell lines, including HeLa, HepG2, CHO and melanoma cell lines), and lower eukaryotes. Biological cell type (
Eukaryotic cell types such as yeast. Preferably, the modified membrane disrupting peptides of the present invention are used to deliver nucleic acids to mammalian cells.

The present invention also provides a delivery conjugate comprising the modified membrane disrupting peptide of the invention and a nucleic acid.

[0033] Preferably, the delivery complex consists of the nucleic acid to be delivered and the modified membrane disrupting peptide of the invention.

[0034] The delivery complex of the invention can be any delivery complex comprising the nucleic acid to be delivered and the modified membrane disrupting peptide of the invention.

[0035] Numerous delivery complexes for delivering nucleic acids to cells are well known to those skilled in the art. Peptides derived from the amino acid sequence of the viral envelope protein were used for gene transfer when administered with a polylysine DNA complex (Plant et al.,
1994, J.A. Biol. Chem. , 269: 12918-24); Trub.
etsky, et al., 1992, Bioconjugate Chem. , 3:32
3-327; Mack et al., 1994, Am. J. Med. Sci. , 307: 1
38-143). This suggests that co-concentration of polylysine bound with cationic lipids may lead to improvements in gene transfer efficiency. WO95 / 02
698 discloses the use of viral components to attempt to increase the efficiency of cationic lipid gene transfer.

In a preferred embodiment, a delivery complex of the invention comprises a modified membrane-disrupting peptide and a nucleic acid encapsulated within nanoparticles or microparticles such as, for example, polylactide glycoside particles and liposomes.

In a further preferred embodiment, a delivery complex of the invention comprises a nucleic acid, a nucleic acid enriched peptide and a modified membrane disrupting peptide of the invention.

[0038] The nucleic acid enriching peptide can be any peptide that enriches nucleic acids, including polylysine and histone-derived peptides. Preferred nucleic acid enriching peptides are described in International Patent Applications WO 96/41606 and WO 98/35984.

[0039] Preferably, the delivery complex is formed by concentrating the nucleic acid using a nucleic acid enriching peptide to form an enriched nucleic acid complex. The enriched nucleic acid complex is then coated with the modified membrane disrupting peptide of the invention.

The present invention provides a method for forming a delivery complex according to the present invention, comprising the following steps: 1. concentrating the nucleic acid using the nucleic acid enriched peptide to form an enriched nucleic acid complex; Coating the concentrated nucleic acid complex with the modified membrane disrupting peptide according to the invention.

The presence of serum during transfection was found to increase the level of transfection. Preferably, the serum is fetal calf serum. Other suitable sera that can be used are well known to those of skill in the art and include normal human serum and normal mouse serum.

[0042] The modified membrane disrupting peptides of the present invention can be used to deliver therapeutic nucleic acids to cells in vivo, in vitro and ex vivo treatments. Therapeutic uses of nucleic acids in various diseases are well known to those skilled in the art.

The therapeutic nucleic acid delivered to the cells can be any form of DNA or RNA vector, including plasmids, linear nucleic acid molecules, ribozymes, and deoxyribozyme and episomal vectors. Heterologous gene expression is expressed in muscle (Wolf)
fJ. A. Et al., 1990, Science, 247: 1465-1468; C
arson D. A. Et al., US Patent No. 5,580,859), thyroid (Syk).
es et al., 1994, Human Gene Ther. , 5: 837-844).
, Melanoma (Vile et al., 1993, Cancer Res., 53: 962-9).
67), skin (Hengge et al., 1995, Nature Genet., 10).
161-166), liver (Hickman et al., 1994, Human Gen).
e Therapy, 5: 1477-1483) and after exposure of airway epithelium (Meyer et al., 1995, Gene Therapy).
y, 2: 450-460).

Useful therapeutic nucleic acid sequences include those encoding receptors, enzymes, ligands, modulators and structural proteins. Therapeutic nucleic acid sequences also include nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins,
Sequences encoding cell membrane-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoan antigens and parasite antigens. Therapeutic nucleic acid sequences useful according to the invention also include those encoding proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (eg, RNA such as ribozymes or antisense nucleic acids). Proteins or polypeptides encoded by nucleic acids include hormones, growth factors, neurotransmitters, enzymes, coagulation factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, structural proteins, viral antigens, parasites Insect and bacterial antigens. Specific examples of these compounds include proinsulin, growth hormone, dystrophin, androgen receptor, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding protein, epidermal growth factor, TGF-α
, TGF-β, PDGF, angiogenic factors (acid fibroblast growth factor, basic fibroblast growth factor and angiogenin), substrate proteins (type IV collagen, type VII collagen, laminin), phenylalanine hydroxylase, tyrosine hydroxy Rase, oncogene (ras, fos, myc, erb, src,
sis, jun), E6 or E7 transforming sequences, p53 protein, Rb gene product, cytokines (eg, IL-1, IL-6, IL-8) or their receptors, viral capsid proteins, and immunological responses. Includes proteins from viral, bacterial and parasite organisms that can be used to derive, and other proteins of importance of use in the body. Compounds that can be incorporated are limited only by the effectiveness of the nucleic acid sequence on the protein or polypeptide to be incorporated. One skilled in the art will recognize that as more proteins and polypeptides are identified, they can be incorporated into the selection of delivery complexes, transfected using the modified membrane disrupting peptides of the invention, and expressed. Recognize easily.

Nucleic acids delivered using the modified membrane disrupting peptides of the present invention include nucleic acids that encode proteins that may be patient deficient or that may be clinically effective at higher than normal concentrations As well as nucleic acids designed to eliminate translation of unwanted proteins. Nucleic acids for use in eliminating harmful proteins include antisense RNAs and ribozymes and the D-encoding them.
NA expression construct.

Hammerhead ribozymes are the smallest known ribozymes, and provide themselves for both invitro synthesis and delivery to cells (Sullivan
, 1994, J.A. Invest. Dermatol. , 103: 85S-98S
Usman et al., 1996, Curr. Opin. Struct. Biol. ,
6: 527-533).

The invention also provides a modified membrane disrupting peptide or a delivery conjugate of the invention for use in therapy.

The present invention further provides the use of a modified membrane disrupting peptide or delivery complex of the invention in the manufacture of a composition for the treatment of a genetic disorder.

Genetic disorders are defined as disorders that can be treated at the genetic level (ie, by delivering nucleic acids to a patient in need of such treatment). Genetic disorders include enzymatic deficiencies (eg, enzymatic deficiencies in the liver, digestive system, skin, and nervous system)
, Endocrine deficiency (eg, deficiency of growth hormone, reproductive hormone, vasoactive hormone and hydrostatic hormone), exocrine deficiency (eg, deficiency of pancreatic hormone secretion)
, Neurodegenerative disorders (such as Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Tay Sachs disease, etc.), cancer, muscular dystrophy and albinism.

The present invention also provides a method of treating a genetic disorder, comprising administering to a patient in need of such treatment an effective dose of a delivery conjugate, wherein the conjugate comprises a therapeutic nucleic acid and Includes the modified membrane disrupting peptides of the present invention.

(Examples) (Materials and Methods) (Peptide Synthesis and Purification) (Method 1: Multiple Peptide Synthesizer (MPS)) E. FIG. Biosystems Pio
Neer peptide synthesizer Synthesized with current version 1.7. This instrument was equipped with a single MPS unit mounted at column position 2 and was controlled by the workstation current software version 1.3.

Each synthesis was performed using Fmoc-PAL-PEG-PS resin (PE Biosystem).
ms) on a 0.05 mmol scale. For this synthesis, the extension, slow activation and coupling cycles provided by the instrument were used. Deprotection was performed using a solution of 20% piperidine in DMF. The following amino acid derivatives were used to fit this peptide; Fmoc-
L-Ala-OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-LC
ys (Trt) -OH, Fmoc-L-Gln (Trt) -OH, Fmoc-G
ly-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fm
oc-L-Lys (Boc) -OH, Fmoc-Nle-OH, Fmoc-L-
Pro-OH, Fmoc-L-Ser (tBu) -OH, Fmoc-L-Thr
(TBu) -OH, Fmoc-L-Trp (Boc) -OH, Fmoc-LV
al-OH, Fmoc-L-Lys (Fmoc) -OH. Activation of this amino acid was achieved with TBTU and DIPEA. In the final synthesis process,
The Fmoc group was removed, the solvent was subsequently exchanged for dichloromethane, and the resin was dried under a stream of dry nitrogen. The column containing the resin was then removed and further dried under vacuum at room temperature for 2-3 hours.

The peptide without the cysteine residue was cleaved from the resin using trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS) (95: 2.5: 2.5) and the cysteine residue was removed. The peptide containing the group is converted to trifluoroacetic acid (TFA
) / Water / triisopropylsilane (TIS) / ethanedithiol (EDT) (9
2.5: 2.5: 2.5: 2.5). An empty 5 ml syringe was attached to one end of each column, and a 2.5 ml syringe containing the cut mixture was attached to the other end. The cleavage mixture (1 ml) is injected into each column, and the column is allowed to stand for 30 minutes, then an additional 1 ml is injected, and after 30 minutes the last 0.1 ml.
5 ml were injected. After standing for 30 minutes, the empty 2.5 ml syringe was removed and the remaining cut mixture was drawn into a 5 ml syringe. The column was removed from the 5 ml syringe, inverted and replaced, then a new 2.5 ml syringe containing the cutting mixture was attached to the other end and the cutting procedure was repeated. The contents of the 5 ml syringe were then discharged into a 50 ml centrifuge tube containing diethyl ether (45 ml). The resulting precipitate was stabilized at room temperature for 1-2 hours and the supernatant was removed. The remaining diethyl ether was blown with a stream of dry nitrogen gas, and the pellet was further dried under vacuum for 2-3 hours.

(Method 2: Continuous Flow Synthesis of Peptide) E. FIG. Biosystems Pio
Neer peptide synthesizer Synthesized with current version 1.7. This was controlled by the workstation current software version 1.3.

Each synthesis was performed using Fmoc-PAL-PEG-PS resin (PE Biosystem).
ms) on a 0.2 mmol scale. For coupling of all amino acids except cysteine, the extension coupling cycle provided with this instrument was used. For cysteine coupling, a special elongation (1 hour coupling time) solvent exchange cycle was used with a 1: 1 coupling of DMF / DCM.
Used to occur. Deprotection was performed using a solution of 20% piperidine in DMF. The following amino acid derivatives were used to fit this peptide: Fm
oc-L-Ala-OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-
L-Cys (Trt) -OH, Fmoc-L-Gln (Trt) -OH, Fmo
c-Gly-OH, Fmoc-L-His (Trt) -OH, Fmoc-LI
le-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-
OH, Fmoc-L-Pro-OH, Fmoc-L-Ser (tBu) -OH,
Fmoc-L-Thr (tBu) -OH, Fmoc-L-Trp (Boc) -O
H, Fmoc-L-Val-OH, Fmoc-L-Lys (Fmoc) -OH.
Activation of amino acids except cysteine was achieved using TBTU and DIPEA in DMF. Activation of cysteine was determined by TBTU in DMF and Sym in DCM.
-Achieved using Collidine. In the last synthesis step, the Fmoc group was removed, followed by a solvent exchange for dichloromethane and drying of the resin under a stream of dry nitrogen. The resin was washed off the column, passed through a filter funnel, air-dried for several minutes, transferred to a previously weighed flask, and further dried under vacuum until constant weight was achieved.

Peptides without cysteine residues were converted to trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS) (95: 2.5: 2.5) at room temperature for 1.5 hours.
(20 ml) from the resin and the peptide containing cysteine residues was conjugated with trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS) / ethanedithiol (EDT) (92) at room temperature for 1.5 hours. .5: 2.5: 2.5: 2.
5) Cleavage was performed using (20 ml). In each case, the resin was filtered, washed with exactly TFA (3 × 5 ml), and collected, filtered and washed, and evaporated to a volume of about 3 ml. The remainder is converted to diethyl ether (45
ml) into a 50 ml centrifuge tube. The resulting precipitate was allowed to stabilize at room temperature for 1-2 hours and the supernatant was removed. The remaining diethyl ether was blown with a stream of dry nitrogen gas, and the pellet was further dried under vacuum for 2-3 hours.

(Purification of Peptide) This peptide was subjected to reverse phase h. p. l. Purified by c. Molar equivalents of peptide were subjected to a Shandon Hypersil using a gradient of water (0.1% TFA) acetonitrile (0.1% TFA) (typically 10-65% acetonitrile in water) at a flow rate of 24 ml / min for 20 minutes. SAS column (120Å, 10
μ, 150 × 21.2 mm) or water (at a flow rate of 6 ml / min for 30 minutes).
0.1% TFA) acetonitrile (0.1% TFA) gradient (typically
Phenomenex Jupi with 30-100% acetonitrile in water)
Purified on any of the ter C4 columns (120 °, 5μ, 250 × 10 mm).

[0058] Fractions containing the peptide of interest as pooled by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis and HPLC analysis were pooled and lyophilized.

Peptide Multimer Formation To achieve disulfide or cysteine bond-mediated dimerization or oligomerization, the pure peptide is dissolved in fresh 20 mM ammonium bicarbonate and allowed to stand at room temperature for 16 hours. I left it. Dimerization was performed by running Pharmacia Superdex Peptide HR with 20% acetonitrile in water containing 0.1% TFA at a flow rate between 0.8 ml / min and 1.0 ml / min.
Confirmed by analytical gel filtration chromatography using a 10/30 column.
The decrease in the elution time of the dimerized peptide was confirmed by monitoring the absorbance of the eluate at 214 nm.

For the synthesis of bisvinylsulfone conjugate (CP48), 2.0 mg Bio
link 6 (Molecular Biosciences, Colerod)
o) was dissolved in 100 μl acetonitrile and 25 mM HEPES, p
The volume was made up to 1.0 ml with H7.2. Then 200 μl of this solution was added to 8
Added to 15 mg of CP36 monomer (5.2 μmol) dissolved in 00 μl buffer. The reaction was monitored by MALDI and RP-HPLC and the reaction was judged complete after 1 hour at 24 ° C. The final product is reduced to 0.1 for 30 minutes.
% Acetonitrile in water with 30% TFA using a 30-100% gradient rise.
henomenex Jupiter C _{4 for} semi-separation (semiprepar
active) isolation by column purification. The mass of the final product is determined by MALDI-
Confirmed by TOF MS.

Erythrocyte Lysis Assay 1.0 ml 110 mM citrate (pH 5) was added to 9.0 ml blood to stop clotting. The blood was spun at 2000 rpm for 5 minutes. The plasma supernatant is aspirated and the pellet is mixed continuously with HBS at least 6 times,
Washed by centrifugation and aspiration. The clear supernatant was aspirated and the pellet was washed twice with the appropriate buffer: HBS (pH 7.4) or 1
Either 5 mM sodium acetate (pH 5) or 150 mM NaCl. The appropriate buffer is added to the blood pellet to make a total volume of 6 ml, and 1 ml is taken from the stock preparation, diluted 15-fold with buffer and the final working solution (work
solution was obtained. The peptide was tested as three serial dilutions in a 96-well plate by adding 75 ml of the blood solution in the appropriate buffer to 100 ml of the peptide solution in the corresponding buffer and mixing.
The blood was incubated with the peptide for 1 hour at 37 ° C. At this stage, 1% Triton X-100 was added to the peptide-free blood solution and served as a control for 100% lysis. The cells are transformed into 2500
Spin down for 5 minutes at rpm and 80 ml of supernatant was used for spectrophotometric analysis at 450 nm.

(Preparation of Complex) 40 μg / ml plasmid DNA in HBS (HEPES buffered saline) (pH 7.4) was immediately mixed with an equal volume of the appropriate concentration of transfection reagent in HBS, And incubated for 1 hour at room temperature. The concentration of the transfection reagent is determined for the peptide by the final desired charge ratio or by the final desired nitrogen (from PEI): phosphate (from DNA) ratio.
(Exgen-500, Euromedex, France). The charge ratio was determined using Felgner et al. (1997) (Nomenclature fo
r synthetic gene delivery systems. Ge
ne Therapy, (1997) 8: 511-512).

HepG2 Transfection Assay 1. β-Gal Transfection Protocol HepG2 was plated at 5 × 10 ⁴ cells / well in a 96-well plate in DMEM + 10% FCS (with antibody) before transfection. And incubated at 37 ° C. The next day, the cells were washed with 100 μl / well of PBS. 90 μl of HEPES buffered RPMI containing 10% FCS and antibody is added to the cells, followed by 10 μl of transfection complex containing plasmid pCMVβ reporter plasmid (plasmid encoding β-galactosidase), as described above. did. This complex was formed in the presence or absence of 5 mM DTT to reduce disulfide bonds of all peptides. Using appropriate controls, it was confirmed that the presence of DTT in the formation itself had no observable effect upon transfection. Cells were transfected three times with each complex. The cells are
Centrifuged at rpm and then incubated at 37 ° C. for 5 hours in a non-gas incubator under humid conditions. After 5 hours, the transfection medium was removed and the cells were washed with 100 μl / well of PBS, and then incubated for 19-20 hours in a CO ₂ gas incubator in DMEM / 10% FCS (with antibody) medium. . Finally, this cell is
Wash twice with BS, 30 ml 0.1% Triton, 250 mM Tris
(PH 8) and frozen at -20 ° C prior to β-gal assay.

2. β-Gal Assay The frozen lysed cells were thawed at room temperature and 10 μl was taken from each well for protein assay. The remaining cell lysate was purified using Galacton-Ssta
The β-gal reporter was assayed using the rβ-gal assay (TROPIX) and luminescence was measured using a 96-well TopCount scintillation counter in SPC format. DC protein assay kit (Bi
lysates were assayed for total protein content using oRad). Transfection numbers were determined using the β-gal standard curve generated in cell lysates from untransfected cells on the same 96-well plate as pgβ
-Gal / ng reported as total protein.

HUVEC Transfection Assay Luciferase Transfection Assay Primary HUVEC (Promocell, Germany) was prepared by using Endothelial Growth Medium (Promocell) with supplement (EGMS).
(Germany, Germany) at 1 × 10 ⁴ cells / well prior to transfection in 0.1% gelatin-coated 96-well plates and incubated at 37 ° C. in a CO ₂ gas incubator. The next day, cells were washed with PBS. Medium M199 containing 90 μl per well of 10% FCS and antibody
(Sigma) was added to the cells, followed by 10 μl of transfection complex per well prepared with the pCMVLuc reporter plasmid (plasmid encoding firefly luciferase). Cells were transfected three times with each complex. The cells are centrifuged at 1100 rpm for 5 minutes and then
Incubated for 1 hour at 0 C in a CO ₂ gas incubator. After 1 hour, the transfection medium was removed and the cells were added to 100 μl / well of P
Wash with BS, then 20-2 at 37 ° C. in 100 μl / well EGMS
Incubated for 4 hours in a CO ₂ gas incubator.

Luciferase Assay After 20-24 hours, luciferase expression was determined by LucScreen assay (T
ROPIX) and the emission was determined in 96-well T
The measurement was performed using an opCount scintillation counter (Packard).

GFP Transfection Protocol Primary HUVECs were seeded at 1.5 × 10 ⁵ cells / well before transfection in 2 ml EGMS in 6-well plates. The next day, the cells are washed twice with 2 ml of PBS per well and consist of 100 μl of transfection complex prepared with 1 ml of transfection solution (pCMVEGFP reporter plasmid (plasmid encoding green fluorescent protein) per well. ) Was added and 900 μl of M1 containing 10% FCS and antibody
And 99. Cells were transfected twice with each complex. Cells were incubated at 37 ° C. for 2 hours in a CO ₂ gas incubator, after which the medium was returned to 2 ml EGMS per well. The cells were further incubated at 37 ° C for 2 hours.
Incubated for 4 hours in a CO ₂ gas incubator.

GFP Assay After 24 hours, cells were washed twice with 2 ml of PBS per well, trypsinized and resuspended in M199 medium and 10% FCS. % Transfected cells were determined by FACS analysis.

In Vivo Delivery of PCMVLuc Using Peptides PCMVLuc at 500 μg / ml in HBS is mixed at appropriate concentration with an equal volume (typically 500 μl) of peptide or polylysine (127 mer) (Sigma) and Was obtained. The charge ratio is determined by Felgner et al. (Hum. Gene).
Ther. , 8 (5), 511-2, 1997). Peptide or polylysine was added to the DNA for 3-4 seconds while mixing with a vortex mixer at 800 rpm. The complex was incubated for 1 hour at room temperature. 300 μl of the conjugate was injected into the tail vein of CD-1 mice.
Twenty hours later, the mice were sacrificed and 80-200 mg of each organ was removed, wiped briefly to remove excess liquid, frozen in liquid nitrogen and stored at -80 ° C. The frozen tissue is weighed and then lysed in lysis buffer (1 mM
EDTA, 1% Triton X-100, 15% glycerol, 8 mM
Melt in MgCl ₂ , 0.5 mM PMSF, 10 mM sodium phosphate with 1 mM DTT and Mini Bead Beater 8 (Stratec
h Ltd) and 1 mm glass beads for 0.3-2 minutes. The homogenate is removed and the glass beads are washed with lysis buffer,
The wash was then combined with the homogenate. 13000 particulate matter
Remove by centrifugation at rpm for 5 minutes and 80 μl Berthold
0.1 mM luciferin, 0.44 mM in LB593 luminometer
ATP was assayed for luciferase activity using ATP and an acquisition time of 4 seconds. Results are shown as RLU corrected for mg of each tissue weight.

The following peptides were prepared as monomers or, if possible, as dimers or other multimers using the methods described above.

[0071]

Embedded image CP1 is melittin, the main toxic component of bee venom. CP2 is CP1 (G1 is C). This peptide was designed to form the N-terminal cysteine-linked dimer of CP1. CP18 is CP1 (P14 is A). This peptide was modified to form a substantially continuous helix. CP36 is CP1 (G1 is C; P14 is A). The peptide was modified so that it formed a dimer and had a substantially continuous helix. CP39 is CP36 (C1 is norleucine). This modification was induced to increase hydrophobicity at the N-terminus. Peptides CP41-CP45 were designed to study the effect of conservative amino acid changes on CP36 activity. CP37 was CP18 (I20 is L) and all lysines were replaced with glutamic acid. CP41 is CP36 (I20 is L). CP42 is CP36 (I17 is L; I20 is L). CP43 is CP36 (W for A14; L for I20). CP44 is CP36 (W for A15; L for I20). CP45 is CP36 (W for T11; L for I20). CP46 is a functional homolog of melittin containing conservative amino acid changes and has been described by DeGrado et al. Am. Chem. Soc. , 103, 67
9-681. CP47 is CP46 with an N-terminal cysteine added for dimer formation. CP49 is CP36 with a C-terminal cysteine added for multimer formation. CP50 is CP18 with a C-terminal cysteine added for multimer formation. CP51 is CP41 in which all lysine residues have been substituted with glutamic acid. CP52 is CP36 formed by bisamine modification of an additional C-terminal lysine.
Is a dimer of CP53 is a dimer of CP36 with the addition of a C-terminal lysine and a cysteine residue attached to CP36. CP54 is CP36 with LAALISW inserted at position 20 to increase the length of the alpha helix. CP55 is a heterodimer of CP36 and CP51. CP56 is a tetramer consisting of CP52 and two CP36 peptides. CP57 is CP1 (K23 is C). CP58 is a heterodimer of CP36 and CP50. CP59 is a tetramer consisting of a dimer of CP52 bound to two CP37 peptides. CP60 is a tetramer composed of a dimer of CP53. CP61 is a dimer consisting of CP36 and CP51, where a disulfide bond is formed between the N-terminal cysteines.

As determined by plasmid delay on agarose gel, melittin (C
P1) can bind to DNA effectively, but the melittin: DNA complex
It transmitted little or no transfection of the two cells and this melittin was observed to be toxic to cells. After incubating cells with such a complex, the total protein content per cell decreased with increasing ratio of melittin to DNA (FIG. 4). This suggests that the toxic effects caused by melittin's ability to form multiple structures are perturbed (
perturbing). The literature on the mechanism of melittin states that peptides exist as tetramers in solution at high concentrations and / or high ionic strength, but in membranes, where they can aggregate into pore-forming tetramers. Suggest that it must be able to separate into a monomeric form so that it can be inserted into The melittin pore-forming tetramer is thought to increase passive ion permeability and is considered toxic to cells. It has been proposed that dimerization or tetramerization of melittin should result in a less toxic construct for cells, and that dimerization or tetramerization effectively prevents inhibition of the formation of pore-forming tetramers. The dimer or tetramer of melittin also has membrane disruption properties, mainly due to the apolar amphipathic helix. By substituting the Pro residue at position 14 of melittin with a residue that allows helix continuity, which causes kinking in the helix, the interaction between melittin's amphipathic helix and the membrane surface is reduced. It was decided to optimize the action.

As indicated above, the dimer was assembled and its membrane disrupting activity was measured by a erythrocyte lysis assay. Surprisingly, the lytic activity of the dimer was higher than melittin at pH 7 and 5 (see FIGS. 1 and 2), but the dimer appeared to be less toxic to mammalian cell lines. (See FIG. 4). It is therefore proposed that pore formation is toxic, while other membrane destruction / instability mechanisms are non-toxic.

Helix extension and melittin dimerization as described results in a construct capable of binding DNA; the resulting DNA complex is detrimental to HepG2 cells as determined by protein content determination. Gender was significantly lower (see FIG. 4) and transmitted efficient transfection of many cell types in the absence of exogenous factors (see FIGS. 3 and 5). It was also found that the presence of fetal calf serum during transfection gave increased levels of transfection (see FIG. 5).

FIG. 9 shows that peptides CP36 and CP61 of the invention are effective in increasing in vivo transfection of DNA into many tissues.

Delivery Complex Coated with CP36 Dimer This delivery complex was prepared as follows.

1. Preparation of NBC28: DNA complex with charge ratio ± 4 and DNA concentration of 25 μg: 92.6 μg / ml solution of NBC28 and 50 μg / ml pCMVl
A solution of uc DNA was prepared in 10 mM HEPES pH 7.4. This peptide solution is added to the DNA solution at a ratio of 1: 1 (v / v), and
Left for hours. NBC28 has the following amino acid sequence: TKKKKKKKKKKKKKKKK.
It is a nucleic acid-enriched peptide having KKKKYCG.

2. Coating of the conjugate: The coating peptide is added to the conjugate, followed by some 10 mM H
EPES buffer was added to a volume of up to 97% of the required final volume. The resulting complex was vortexed for 10 seconds and then stored at 4 ° C. overnight.

[0079] 3. Salt spike: Next morning (after about 18 hours), 5M salt (3% of final volume to give 150 mM salt) is added to the complex, which is then vortexed for 10 seconds, Then, it was left at room temperature for another hour.

[0080] 4. Reconstitution of the complex: This complex in 1.5 ml eppendorf was transferred to MSE Microcent
The mixture was centrifuged at 13000 rpm for 30 seconds in an aur centrifuge. Supernatant (75
%) Was removed and an equal volume of fresh HBS was added while gently vortexing and sucking down with a Gilson pipette for 30 seconds.

[0081] 5. Complex sonication Complexes in 1.5 ml Eppendorf tubes were sonicated for 30 seconds in a sonication bath prior to transfection.

[0082] The complex was then transfected into HUVEC cells according to the method described above.

FIG. 10 shows that the complex coated with CP36 dimer gives good transfection of HUVEC cells. In particular, the level of transfection is higher than that obtained with CP36 or NBC28 alone.

[Brief description of the drawings]

 The present invention is now illustrated in the accompanying examples with reference to the following figures.

FIG. 1 shows the erythrocyte lytic activity of CP36 dimer (CP36D), CP36 monomer (CP36M) and melittin (CP1).

FIG. 2 shows the erythrocyte lytic activity of CP36 and CP48.

FIG. 3 shows CP12, 18, 36, 39, 41, 42, complexed at various charge ratios in the presence or absence of DTT and in the presence of 10% fetal calf serum.
HepG2 when transfected with 43, 44, 45 and 48
4 shows transfection of cells.

FIG. 4 shows CP12, 18, 36, 39, 41, 42, complexed at various charge ratios in the presence or absence of DTT and in the presence of 10% fetal calf serum.
HepG2 when transfected with 43, 44, 45 and 48
Shows the protein levels of the cells. Reduced protein levels indicate a toxic effect on the cells.

FIG. 5 shows CP36 / 1 (first batch), CP36 / 1.2 (second batch) in the presence or absence of DTT and in the presence or absence of 10% fetal calf serum. 2) shows transfection of HepG2 cells with complexed pCMVβ. The name Red means the presence of the reduced form, ie, 5 mM DTT; and NR, means the non-reduced form, ie, no addition of DTT.

FIG. 6 shows CP36 from different batches of CP36 peptide at four charge ratios.
4 shows data from experiments comparing luciferase expression from HUVECs transfected with complexed DNA. Batches of peptides refer to different syntheses and were named CP36 / 1, / 3, / 4, / 5, / 6.

FIG. 7 shows CP36-complexed DNA and PEI at various charge ratios or N: P ratios.
Shows a compilation of a number of experiments comparing average luciferase expression from HUVECs transfected with complexed DNA (experiment numbers are in parentheses). The PEI used was a 22 kDa linear PEI (Exgen-500).

FIG. 8 shows CP36-complexed DNA and PEI at various charge ratios or N: P ratios.
Shown is a compilation of a number of experiments comparing the average% of cells expressing GFP from HUVECs transfected with complexed DNA (experiment numbers are in parentheses). The PEI used was a 22 kDa linear PEI (Exgen-500).

FIG. 9 shows luciferase expression in vivo after administration with 75 μg DNA using various peptides in many tissues.

FIG. 10 shows average luciferase expression from HUVECs transfected with CP36 dimer-coated NBC28: DNA particles.

──────────────────────────────────────────────────続 き Continued on the front page (31) Priority claim number 99169912.0 (32) Priority date July 19, 1999 (July 19, 1999) (33) Priority claim country United Kingdom (GB) (31) Priority claim number 9928303.8 (32) Priority date November 30, 1999 (November 30, 1999) (33) Priority claim country United Kingdom (GB) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Welsh, John Hamilton, England Estee 5 5 Sp. Park ( 72) Inventor Hussein, Ronda Daffee, England Estee 5 5 Espy Staffordshire, Kiel, University of Kiel, The Science Park F Term (reference) 4B024 AA01 AA20 BA80 CA01 CA11 DA02 EA10 GA11 HA01 HA17 4C076 AA95 CC41 CC50 EE51 4C084 AA13 NA13 4H045 AA10 AA20 AA30 BA10 BA54 CA50 DA00 EA20 FA10 FA34

Claims (26)

[Claims] 1. A delivery complex for delivering a negatively charged polymer to a cell, comprising a modified membrane disrupting peptide, wherein the membrane disrupting peptide forms a covalently bound multimer. The delivery complex has been modified to: 2. The delivery conjugate of claim 1, wherein said negatively charged polymer is a nucleic acid. 3. The delivery complex according to claim 1, wherein the membrane disrupting peptide is an α-helix membrane disrupting peptide and is toxic to cells. 4. The method of claim 1, wherein said modified membrane disrupting peptide is melittin.
The delivery complex according to any one of claims 1 to 3. 5. The delivery conjugate of any one of claims 1-4, wherein said modified membrane disrupting peptide is further modified to form a substantially continuous α-helix. 6. The modified membrane-disrupting peptide forms a substantially continuous α-helix by replacing an amino acid that disrupts α-helix formation with an amino acid that does not disrupt α-helix formation. The delivery complex of claim 5, wherein the delivery complex is modified to: 7. The delivery conjugate of claim 6, wherein proline at position 14 of the melittin peptide is replaced with alanine or tryptophan. 8. The method of claim 5, wherein the modified membrane disrupting peptide is modified to form a substantially continuous α helix by removing amino acids that stop α helix formation. Delivery complex. 9. The delivery complex of any one of claims 1 to 8, wherein the modified membrane disrupting peptide has been modified to form a dimer. 10. The modified membrane-disrupting peptide has a covalent bond directly to an amino acid of another modified membrane-disrupting peptide via a disulfide bond or a linking group. 10. The delivery complex of claim 9, wherein the delivery complex is modified by substitution with a forming amino acid. 11. Adding an amino acid capable of forming a covalent bond directly with the amino acid of another modified membrane-disrupting peptide via a disulfide bond or via a linking group. Claim 1 which is modified by
The delivery complex according to any one of claims 8 to 13. 12. The delivery complex of any one of claims 1 to 8, wherein the modified membrane disrupting peptide has been modified to form a trimer. 13. The delivery complex of any one of claims 1 to 8, wherein said modified membrane disrupting peptide is modified to form a tetramer. 14. The modified membrane-disrupting peptide, wherein two or more amino acids form a disulfide bond, directly or via a linking group, with the amino acids of another modified membrane-disrupting peptide. 14. The delivery conjugate of claim 12 or 13, wherein the delivery conjugate is modified by substituting the amino acid to form a trimer or tetramer. 15. The membrane disrupting peptide is modified by adding two or more amino acids that form a covalent bond directly with amino acids of another membrane disrupting peptide via a disulfide bond or via a linking group. 14. The delivery complex of claim 12 or 13, forming a trimer or tetramer. 16. The modified membrane-disrupting peptide has an amino acid sequence of: CIG.
16. The delivery complex of any one of claims 1 to 15, having AVLKVLTTGLAALISWIKRKRQQ. 17. The method of claim 1, wherein the modified membrane disrupting peptide comprises a lipid.
17. The delivery complex according to any one of claims 16. 18. The delivery complex of claim 1, wherein the modified membrane disrupting peptide has an extended α helix. 19. The delivery conjugate of any one of claims 1 to 18, wherein said modified membrane disrupting peptide has a basic amino acid residue added to the C-terminus of said peptide. 20. The delivery complex of any one of claims 1 to 19, comprising a functional homolog of the modified membrane disrupting peptide. 21. The delivery complex according to any one of claims 1 to 20, further comprising a nucleic acid enriched peptide. 22. The method of claim 1 for delivering a negatively charged polymer to a cell.
22. Use of a delivery complex according to any one of claims 21 to 21. 23. A method for forming a delivery complex according to claim 21, comprising: 1. concentrating the nucleic acid using the nucleic acid enriched peptide to form an enriched nucleic acid complex; Coating the enriched nucleic acid complex with a modified membrane disrupting peptide. 24. Any of claims 1-21 for use in therapy.
The delivery complex according to paragraph. 25. The method of claim 1 in the manufacture of a composition for the treatment of a genetic disorder.
Use of the delivery complex according to any one of claims 21 to 21. 26. A method for treating a genetic disorder, the method comprising:
21. administering an effective dose of the delivery conjugate of any one of claims 21 to a patient in need of such treatment, wherein the negatively charged polymer is a therapeutic nucleic acid. Method.