JP2008509205A - Vector containing polymer-modified siRNA liposomes - Google Patents

Abstract

The present invention relates to non-viral delivery vectors comprising liposomes, wherein one or more lipids of the liposome are reversibly or irreversibly coupled to one or more polymers, and the liposome comprises siRNA.

Description

The present invention relates to non-viral delivery vectors containing siRNA. The present invention also relates to targeted non-viral delivery vectors, methods of preparing the vectors, methods of using the vectors, and uses thereof.

Background of the Invention Methods for viral DNA delivery address many problems, including immune responses, inability to deliver viral DNA vectors repeatedly, difficulties in generating high viral titers, and the potential of infectious viruses. Worried. Non-viral delivery methods have provided an alternative system that does not have these problems, thus facilitating the development of a less harmful non-viral approach to gene transfer.

  A large potential non-viral delivery system usually involves the use of cationic liposomes consisting of neutral phospholipids and cationic lipids. They have been used to introduce DNA, mRNA, antisense oligonucleotides, proteins, and drugs into cells. A large number of cationic liposomes are commercially available and many new cationic lipids have recently been synthesized. The efficiency of these liposomes has been explained both in vitro and in vivo.

  RNAi in animals and basic eukaryotes, querying in fungi, and post-transcriptional gene silencing in plants are examples of a broad family of phenomena collectively referred to as RNA silencing (12,13). The phenomenon of specific RNA inactivation was first discovered in plants (14) and later in C. elegans (15, 16) as a defense mechanism against viral infection. A common feature of RNA silencing is the production of small (21-23 nt) double stranded RNA called siRNA that acts as a specific determinant for downregulation of gene expression (13). The key enzyme in intracellular production of small double-stranded RNA is Dicer, a cytosolic ribonuclease III that digests long double-stranded RNA into 21-23 nt units (13,17,18). These short double-stranded RNAs are unwound and one of the two strands appears to associate with a complex of protein and target transcript, termed the RNA-induced silencing complex (RISC). This leads to the destruction of the target RNA (19). The discovery that synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides can replace this process and have the ability to specifically down-regulate gene function in cultured mammalian cells (20 ) Opened the door to the application of the RNAi concept in functional genomics programs and even in therapy. Early studies in vivo demonstrated the ability of synthetic and transgenic siRNA to down-regulate both exogenous and endogenous gene expression in adult mice (21, 22). Therefore, the potential side effects caused by siRNA appear as a stimulation of the interferon system, although only slightly (23, 24).

  Although the delivery of many different nucleic acids has been widely described, studies on siRNA delivery are at a preliminary level. Thus, despite the widespread use of cationic lipid / liposome systems to deliver plasmid DNA (pDNA) and oligodeoxynucleotides (ODN) to cells (7, 9, 25-28), either in vitro or in vivo There is very little reported in the literature regarding the formation of siRNA in cationic lipids / liposomes and their delivery to cells (siFection). Even basic studies on the formation of cationic lipid / liposome systems with siRNA have not yet been reported.

  The present invention aims to provide improved non-viral delivery of siRNA.

Summary of the Invention
One reason that studies on siRNA delivery are still at a preliminary level is the obvious confusion that all nucleic acids should be fairly similar and should be delivered to cells in a comparable manner using comparable delivery systems . For example, due to the similar chemistry of oligonucleotides (ODN), pDNA and siRNA, the formulation of siRNA and ODN / pDNA behaves similarly and mechanically predicts that the two species will exhibit similar characteristics .

  On the surface, this is true. Both pDNA and siRNA have an anionic phosphodiester backbone with the same negative charge / nucleotide (nt) ratio and thus interact electrostatically with the cationic liposome / lipid system to introduce nucleic acid into the cell It should form possible cationic lipid-nucleic acid (lipoplex) particles. However, pDNA and siRNA are otherwise very different in molecular weight and molecular topography, with potentially important consequences.

  All pDNA condenses into small nanoparticles of 60-100 nm (27-31) following neutralization of 70-90% of the charge of its phosphodiester backbone with cationic agents. Cationic drug condensed pDNA can then exist in a variety of different forms depending on the cationic condensing agent, such as spheres, toroids and rods (27, 28). Despite the drug, there is a minimum size for pDNA condensation corresponding to about 400 nucleotides (32). Such behavior ensures that the pDNA is almost completely encapsulated or encapsulated by the cationic drug and protected from enzymatic or physical degradation within the nanometer particle (26,33-40) .

  In contrast to pDNA, siRNA cannot aggregate into nanometer-sized particles that are already small sub-nanometer nucleic acids. Thus, electrostatic interactions between siRNA and cationic lipid / liposome systems cause two potential problems. First, a relatively uncontrolled interaction process that leads to oversized and poorly stable siRNA lipoplex (LsiR) particles. Second, incomplete encapsulation of the siRNA molecule, thereby potential enzymatic or physical degradation of the siRNA prior to delivery to the cell.

  Such considerations make it clear that pDNA and siRNA are completely different types of nucleic acids.

  Thus, the present invention is based in part on the fact that non-viral delivery vectors comprising liposomes coupled to siRNA and polymers dramatically reduce liposomes against aggregation without compromising the ability of siRNA to down-regulate target genes. Based on the surprising discovery of stabilization. In particular, liposomes containing siRNA can be generated at an average size of 30-60 nm, which can be incubated with various amounts of polymer, including polymers (e.g., polymer functional groups) and lipids. A bond such as a covalent bond can be formed between the two.

  Due to the similar chemistry of oligonucleotides (ODN), pDNA and siRNA, siRNA and ODN / pDNA formulations behave similarly and, mechanically, the two species are expected to exhibit similar characteristics. In other words, PEGylated siRNA complexes were predicted not to mediate down regulation of target genes even at low pegylation. Because the literature described herein does not allow these complexes to be internalized, escape the endosome, or find the target compartment / molecule necessary to mediate its biological action. This is because one of them is shown.

  The evidence presented herein surprisingly shows that such assumptions do not apply to siRNA.

  The invention is also based, in part, on the surprising discovery that the non-viral vectors described herein can be further incubated with an agent (eg, a target moiety such as an oxidized IgG antibody). While not intending to be bound by any particular theory, rather, a polymer that coats the excess free functional groups of lipids exposed on the surface of the liposome, the second of the ligand, such as a targeting moiety on a non-viral vector. It seems that a layer is formed.

Overview Aspects of the Invention In a first aspect, the invention relates to a non-viral delivery vector comprising a liposome, wherein one or more lipids of the liposome are reversibly or irreversibly coupled to one or more polymers. And the liposome contains siRNA.

  In a second aspect, the present invention relates to a targeted non-viral delivery vector comprising liposomes, wherein one or more lipids of the liposome are reversibly or one or more polymers and one or more drugs. It is irreversibly coupled and the liposome is contained in the siRNA.

  In a third aspect, the invention includes providing a non-viral delivery vector according to the first aspect of the invention or a targeted delivery vector according to the second aspect of the invention to the environment of a cell, tissue or organ. , Relates to a method of delivering siRNA to a cell.

  In a fourth aspect, the invention provides a non-viral delivery vector according to the first aspect of the invention or a targeted delivery according to the second aspect of the invention for use in delivering siRNA to a cell, tissue or organ. Regarding vectors.

  In a fifth aspect, the invention relates to a non-viral delivery vector according to the first aspect of the invention, or a second aspect of the invention in the manufacture of a composition for delivery of siRNA to a cell, tissue or organ. It relates to the use of targeted delivery vectors.

  In a sixth aspect, the invention relates to a non-viral comprising a liposome, wherein one or more lipids of a liposome are reversibly or irreversibly coupled to one or more polymers, and the liposome comprises siRNA. A process for preparing a delivery vector comprising: (i) contacting siRNA with a liposome; and (ii) reversibly or irreversibly coupling the liposome formed in step (i) to a polymer. It relates to a process including steps.

  In a seventh aspect, the present invention provides that one or more lipids of a liposome are reversibly or irreversibly coupled to one or more polymers and one or more drugs, and the liposome contains siRNA. A process for preparing a targeted non-viral delivery vector comprising a liposome, comprising: (i) contacting the siRNA with the liposome; (ii) reversibly converting the liposome formed in step (i) to a polymer Or irreversibly coupling; and (iii) reversibly or irreversibly coupling the liposome formed in step (i) or (ii) with one or more agents. .

  In an eighth aspect, the invention provides (i) providing a vector according to the first or second aspect of the invention; (ii) optionally contacting the vector with a cryoprotectant; and (iii) a vector Is freeze-dried.

  In a ninth aspect, the present invention relates to a lyophilized vector obtainable or obtained by the method according to the eighth aspect of the present invention.

  In a tenth aspect, the invention relates to a liposome comprising a lipid and a coupling moiety, wherein the distance between the lipid and the coupling moiety is at least 1.5 nm.

  In an eleventh aspect, the invention provides a non-viral delivery vector according to the first aspect of the invention, or a targeted delivery vector according to the second aspect of the invention, or a liposome according to the tenth aspect, and pharmaceutically It relates to a pharmaceutical composition comprising an acceptable carrier or diluent.

  In a twelfth aspect, the invention provides a non-viral delivery vector according to the first aspect of the invention, or a targeted delivery vector according to the second aspect of the invention, a liposome according to the tenth aspect, or a It relates to a method of treating a disease in a subject comprising administering to the subject a medically effective amount of a pharmaceutical composition according to the eleventh aspect.

  In a thirteenth aspect, the invention provides a non-viral delivery vector according to the first aspect of the invention, or a targeted delivery vector according to the second aspect of the invention, or a tenth aspect, for use in the treatment of a disease. It relates to a liposome according to an aspect.

  In a fourteenth aspect, the invention provides a non-viral delivery vector according to the first aspect of the invention, or a targeted delivery vector according to the second aspect of the invention, in the manufacture of a composition for the treatment of disease, or It relates to the use of liposomes according to the tenth aspect.

  In a fifteenth aspect, the present invention relates to the use of a polymer-coupled liposome in the preparation of a non-viral delivery vector comprising siRNA.

  In a sixteenth aspect, the present invention relates to the use of a liposome coupled to a polymer and one or more agents in the preparation of a targeted non-viral delivery vector comprising siRNA.

  In an seventeenth aspect, the present invention relates to a non-viral delivery vector or targeted non-viral delivery vector substantially as described herein and referred to in any one of the Examples or Figures.

  In an eighteenth aspect, the present invention relates to a method substantially as described herein and with reference to any one of the examples or drawings.

  In a nineteenth aspect, the present invention relates to a use substantially as described herein and with reference to any one of the examples or drawings.

  In a twentieth aspect, the present invention relates to a liposome substantially as described herein and with reference to any one of the Examples or Figures.

Preferred Embodiments Preferably, one or more lipids of a liposome that are reversibly or irreversibly coupled to one or more polymers are exposed on the surface of the liposome.

式中、Bは脂質であり；Xは自由選択の(optional)リンカー基であり、R₂はHまたはヒドロカルビル基である。 Preferably, the liposome comprises one or more aminoxy group-containing lipids of formula (I):

Where B is a lipid; X is an optional linker group and R ₂ is H or a hydrocarbyl group.

Preferably, the aminoxy group-containing lipid is cholesteryl- (dPEG ₄ ) ₂ -aminoxy lipid (CPA).

  Preferably, the liposome comprises one or more cationic lipids and / or one or more non-cationic co-lipids.

  Preferably the cationic lipid comprises at least one alicyclic group.

  Preferably, at least one alicyclic group is cholesterol.

Preferably, the cationic lipid is N ¹ -cholesteryloxycarbonyl-3,7-diazanononane-1,9-diamine (CDAN).

  Preferably, the non-cationic colipid is phosphatidylethanolamine. More preferably, the non-cationic colipid is dioleoylphosphatidylethanolamine (DOPE).

  Preferably the polymer comprises one or more aldehyde and / ketone groups. More preferably, the polymer is PEG.

  Preferably, the liposomes are coupled with about 0.1 to about 5% PEG.

  Preferably, the liposome comprises one or more drugs, or the liposome is reversibly or irreversibly coupled to one or more drugs.

  Preferably, the agent of the targeted non-viral delivery vector is selected from the group consisting of sugars, carbohydrates, and ligands.

  Preferably, the sugar is selected from the group consisting of glucose, mannose, lactose, fructose, maltotriose, maltoheptose.

  Preferably the ligand is an antibody.

  Preferably, the process according to the sixth aspect of the invention comprises the further step of reversibly or irreversibly coupling the liposomes formed in step (i) or step (ii) with one or more agents.

  Preferably, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and lactose.

  Preferably, the method according to the eighth aspect of the invention comprises the further step of (iv) rehydrating the vector prior to use.

式中、Bは脂質であり；Xはリンカー基であり、カップリングはカップリング部分であり、X骨格は少なくとも30個の原子を含む。 Preferably, the liposome according to the tenth aspect of the present invention is of the formula:

Where B is a lipid; X is a linker group, the coupling is a coupling moiety, and the X backbone contains at least 30 atoms.

  Preferably, the X skeleton contains at least 40 atoms.

式中、nおよびmは独立して0〜6、好ましくは1〜6、より好ましくは2、3、または4、より好ましくは2または4である。 Preferably, X is a group of the following formula or includes a group of the following formula:

In the formula, n and m are independently 0 to 6, preferably 1 to 6, more preferably 2, 3, or 4, more preferably 2 or 4.

式中、nおよびmは独立して0〜6、好ましくは1〜6、より好ましくは2、3、または4、より好ましくは2または4である。 Preferably, X is a group of the following formula or includes a group of the following formula:

In the formula, n and m are independently 0 to 6, preferably 1 to 6, more preferably 2, 3, or 4, more preferably 2 or 4.

式中、mは0〜6、好ましくは1〜6、より好ましくは1、2、または3、より好ましくは1であり、かつnは0〜20、好ましくは5〜15、より好ましくは10、11、または12、より好ましくは11である。 Preferably, X is a group of the following formula or includes a group of the following formula:

Wherein m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, and n is 0-20, preferably 5-15, more preferably 10, 11 or 12, more preferably 11.

式中、mは0〜6、好ましくは1〜6、より好ましくは1、2、または3、より好ましくは1であり、かつnは0〜20、好ましくは5〜15、より好ましくは10、11、または12、より好ましくは11である。 Preferably, X is a group of the following formula or includes a group of the following formula:

Wherein m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, and n is 0-20, preferably 5-15, more preferably 10, 11 or 12, more preferably 11.

  Preferably, the disease is liver disease and / or liver damage.

Advantages The present invention has a number of advantages. These advantages are evident in the following description.

  By way of example, the present invention is advantageous because it provides a method of delivering siRNA using non-viral mediated methods.

  As a further example, the present invention is advantageous because the non-viral delivery vectors described herein are serum resistant and less susceptible to degradation.

  As a further example, the present invention is advantageous because non-viral delivery vectors are dramatically stabilized against aggregation without compromising the ability of the siRNA to down-regulate the target gene.

  As a further example, since the non-viral delivery vector can be coated with an additional agent such as an antibody to generate a targeted non-viral delivery vector for delivery of siRNA to a specific site of interest, It is advantageous.

Detailed Description of the Invention
siRNA
As noted above, siRNA is the basis of the so-called “RNA-induced interference” (RNAi) concept, a method of post-transcriptional gene regulation that is conserved throughout many eukaryotes.

  RNAi is induced by short (typically less than 30 nucleotides) double-stranded RNA molecules present in cells (Fire A et al. (1998), Nature 391: 806-811). These short dsRNA molecules (or siRNAs) cause destruction of messenger RNA that shares sequence homology with siRNA up to one nucleotide degradation (Elbashir SM et al. (2001), Genes Dev, 15: 188-200) . siRNA and targeted mRNA bind to the RNA-induced silencing complex, which is thought to cleave the targeted mRNA. siRNA is clearly recycled, much like many turnover enzymes, and one siRNA molecule can induce the cleavage of about 1000 mRNA molecules. Thus, siRNA-mediated RNAi degradation of mRNA is very effective in inhibiting target gene expression.

  The siRNAs described herein include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as one or more nucleotide additions, deletions, substitutions And / or can include modified RNA that differs from the naturally occurring RNA by modification.

  Such alterations include modifications that make the siRNA resistant or even more resistant to nuclease digestion, e.g., to the end of the siRNA or to one or more internal nucleotides of the siRNA. Such addition of non-nucleotide material can be included.

  Many different types of modifications are known in the art. These include methylphosphonate and phosphorothioate backbones and / or the addition of acridine or polylysine chains at the 3 ′ and / or 5 ′ ends of the molecule. The nucleotide sequence can also be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifetime of the siRNA.

  One or both strands of the siRNA may contain a 3 ′ overhang.

  Thus, the siRNA can comprise, for example, at least one 3 ′ overhang of 1 to about 6 nucleotides in length (including ribonucleotides or deoxynucleotides). If both strands of the siRNA molecule contain 3 'overhangs, the length of the overhang can be the same or different for each strand.

  In order to enhance the stability of the siRNA, the 3 ′ overhang can be stabilized against degradation. Overhangs can be stabilized by including purine nucleotides such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogs can be tolerated and does not affect the efficiency of RNAi degradation.

  Typically, siRNAs are isolated comprising short double-stranded RNAs of about 17 nucleotides to about 29 nucleotides in length, such as about 19-25 contiguous nucleotides in length that target the target mRNA. There is a form of siRNA. The siRNA includes a sense RNA strand and a complementary antisense RNA strand that are annealed together by standard Watson-Crick base pairing interactions. The sense strand includes a nucleic acid sequence that is identical to the target sequence contained within the target mRNA.

  As used herein, the term “isolated siRNA” means that the siRNA has been modified or removed from the natural state through human intervention. An isolated siRNA can exist in a substantially pure form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered.

  The sense and antisense strands of siRNA can contain two complementary single-stranded RNA molecules, or a single molecule in which two complementary portions are base-paired and covalently joined by a single-stranded hairpin Can be included.

  Human mRNA can contain target sequences in common with their respective alternative splice forms, homologues or variants. Thus, a single siRNA containing such a common target sequence can induce RNAi-mediated degradation of different RNA types that contain the common target sequence.

  The target sequence in the target mRNA can be selected from a given sequence, such as a cDNA sequence, corresponding to the target mRNA using various methods in the art. For example, a rational design of siRNA is described in Nat Biotechnol. (2004) 22 (3): 326-30. siRNA can be designed based on the following guidelines. First, a sequence of about 21 nucleotides in the target mRNA is identified, which starts with an AA dinucleotide. Each AA is recorded and the 3 'adjacent nucleotide is identified as a potential siRNA target site. This is based on the observation by Elbashir et al. That siRNAs with 3 'protruding UU dinucleotides are most effective (EMBO J (2001) 20: 6877-6888, Nature (2001) 411: 494-498.2 and Genes & Dev). (2001) 15: 188-200). However, siRNAs with 3 ′ terminal dinucleotide overhangs have been shown to effectively induce RNAi. Preferably, then, the target site from within the sequence identified above is one or more of the following criteria: (i) selection of siRNA with 30-50% GC content; (ii) in the target sequence> Avoidance of 4 T or A stretches; (iii) selection of siRNA target sites at different positions along the length of the gene sequence; and (iv) more than 16-17 contiguous base pairs of homology to other coding sequences Are further selected using one or more of the following, excluding any target sequence having:

  siRNA sequences confirmed off-target down-regulation as described in Kumiko Ui-Tei et al., Nucl. Acids Res. 2004, Vol 32, No. 3, p.936-48 It can be exactly derived from the algorithm.

  If the selected siRNA sequence does not function for silencing, the following steps can be used. Searches can be made for sequencing errors and possible polymorphisms in the gene. Studies on the specificity of target recognition by siRNA indicate that a single point mutation located in the paired region of the siRNA duplex is sufficient to stop target mRNA degradation. Second and / or third targets can also be selected and corresponding siRNAs can be prepared and tested.

  Although siRNA silencing is highly effective by selecting a single target in mRNA, it is desirable to design two independent siRNA duplexes and use them to control the specificity of the silencing effect .

  A number of techniques known to those skilled in the art can be used to obtain siRNA. For example, siRNA can be chemically synthesized using an appropriately protected ribonucleotide phosphoramidite and a conventional DNA / RNA synthesizer. siRNAs can be synthesized as two separate complementary RNA molecules or as a single RNA molecule with two complementary regions.

  Additional methods for siRNA design can be found, for example, on the QIAGEN, Ambion and Ocimum Biosolutions websites.

  siRNA can also be purchased from several companies such as Dharmacon (USA) and Qiagen GmbH (Hilden, Germany).

  siRNA can even be labeled. As an example, siRNA can be labeled with 3′-FITC labeled anti-GFP.

  siRNA can be produced recombinantly using methods known in the art. For example, siRNA can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. The recombinant plasmids of the invention can also contain inducible or regulatable promoters for expression of siRNA in specific tissues or in specific intracellular environments.

  SiRNA expressed from recombinant plasmids can be isolated from cell expression systems cultured by standard techniques. siRNA can be expressed from recombinant plasmids as two separate complementary RNA molecules or as a single RNA molecule with two complementary regions.

  Selection of suitable plasmids for expressing the siRNA of the invention, methods for inserting nucleic acid sequences for expressing siRNA, and methods for obtaining siRNA are described, for example, in Science (2002) 296: 550-553; Nat Biothechnol. (2002) 20: 497-500; Genes Dev. (2002), 16: 948-958; Nat. Biothechnol. (2002) 20: 500-505; and Nat. Biothechnol. (2002) 20: 505- 508.

  siRNA sequences can be cytokines, chemokines, hormones, antibodies, engineered immunoglobulin-like molecules, single chain antibodies, fusion proteins, enzymes, immune co-stimulatory molecules, immune modulatory molecules, transdominant negative variants of target proteins, toxins, For therapeutic and / or diagnostic applications such as conditional toxins, antigens, tumor suppressor proteins, growth factors, membrane proteins, vasoactive proteins and peptides, antiviral proteins and / or ribozymes.

である。 The target mRNA can be or be derived from the anti-apoptotic protein libin-2 (U73857), which is used to stimulate caspase-3, resulting in the initiation of apoptosis in siRNA transfected cell lines Can do. One example of such an siRNA sequence targeting this mRNA is

It is.

  The target mRNA can be or can be derived from HBV, HCV and / or P-pg.

  The siRNA can be targeted to a target mRNA that is or is derived from HBV, HCV and / or P-glycoprotein.

  In particular, the sequence of hepatitis B virus is hepatitis B virus isolate 2-AII-BR large S protein (S) gene (accession number AY344099.1); hepatitis B virus isolate 6-AIII-BR large S protein (S) gene (accession number AY344104.1); hepatitis B virus isolate j13 small surface protein (S) gene (accession number AY639927.1); hepatitis B virus isolate j7 small surface protein (S) gene (Accession number AY639924.1); hepatitis B virus isolate 17993 (accession number AY217367.1); and / or hepatitis B virus isolate Q7-1 (accession number AY217365.1).

からなる群より選択される。配列は、HBVコア遺伝子の保存された配列に対して向けられたGPboostアルゴリズムを用いて得られた。これらの19nt RNA配列の全ては、双方の3'鎖において2つのDNA塩基対dTdT突出を設けて、Dharmacon(Colorado, USA)によって化学的に合成された。全ての配列をPAGEで精製した。配列IA3は、インビトロおよびインビボにおけるHBV表面抗原のダウンレギュレーションの潜在的パターンを明らかにした(結果は示さず)。 Preferably, the HBV siRNA sequence is directed against the conserved sequence of the HBV core gene. More preferably, the HBV siRNA sequence is

Selected from the group consisting of The sequence was obtained using the GPboost algorithm directed against the conserved sequence of the HBV core gene. All of these 19 nt RNA sequences were chemically synthesized by Dharmacon (Colorado, USA) with two DNA base pair dTdT overhangs on both 3 ′ strands. All sequences were purified by PAGE. The sequence IA3 revealed a potential pattern of down-regulation of HBV surface antigen in vitro and in vivo (results not shown).

  Hepatitis C virus is one of the main causes of liver-related morbidity and mortality. The virus establishes a persistent infection in the liver, leading to the development of, for example, chronic hepatitis, cirrhosis and hepatocellular carcinoma. A satisfactory treatment has not yet been developed and the current treatment, interferon combined with ribavirin, fails in nearly 50% of patients. HCV virus is a positive standard RNA virus that contains a single long open reading frame encoding structural and nonstructural proteins. Translation of the viral genome is degraded by an internal ribosome entry site (IRES) located in the untranslated region at the 5 'end (5'-UTR; accession number D31603), which is also conserved in 99.6% of all virus strains Is done. It therefore constitutes an ideal target for siRNA drugs. Similarly, 3'-UTR (accession number D63922) is highly conserved and has been shown to play an important role for viral replication in vivo.

  The sequence of hepatitis C virus is human hepatitis virus C capsid and envelope: x protein (accession number M55970.1); 5′-UTR region (341 nt) conserved across all HCV isolates (accession number M55970.1 And / or non-structural proteins such as NS3, NS4, NS5A, and NS5B.

  The sequence of hepatitis C virus includes a modified form of hepatitis C virus NS3 protease (accession number BD270935.1).

からなる群より選択される。 Preferably, the one or more siRNA sequences are directed to the untranslated region (5'-UTR accession number D31603) or 3'-UTR (accession number D63922) at the 5 'end of HCV. More preferably, the HCV sequence is

Selected from the group consisting of

  All of these 19 nt RNA sequences were chemically synthesized by Dharmacon (Colorado, USA) with two DNA base pair dTdT overhangs on both 3 ′ strands. All sequences were PAGE purified. The effectiveness of individual sequences was compared to siRNA331 (5'-GGUCUCGUAGACCGUGCAC) described by Yokota et al., EMBO Reports 4, 6, 2003, 602ff.

  The sequence of P-glycoprotein (MDR1 gene product) includes human P-glycoprotein (ABCB1) (accession number AF399931.1).

  Also included within the scope of the invention are variants, homologues, fragments and derivatives of the nucleotide sequences described herein.

  In preferred embodiments, the siRNA is, for example, about 19-25 contiguous nucleotides in length, such as from about 19-25 contiguous nucleotides, which are targeted to or derived from HBV, HCV and P-pg proteins. It is a form of isolated siRNA containing short double stranded RNA from about 29 nucleotides.

Liposomes Liposomes are typically completely closed structures comprising a lipid bilayer membrane containing an encapsulated aqueous volume. Liposomes can contain many concentric lipid bilayers separated by an aqueous phase (multilamellar vesicles or MLV) or they can contain a single membrane bilayer (monolayer vesicles). The lipid bilayer is composed of two lipid monolayers having a hydrophobic “tail” region and a hydrophilic “head” region. In the membrane bilayer, the hydrophobic (nonpolar) “tail” of the lipid monolayer is directed to the center of the bilayer, while the hydrophilic (polar) “head” is directed to the aqueous phase.

  Lipid components that can be used in the liposomes described herein are generally described in the literature. In general, these are phospholipids and / or sphingolipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylic acid, phosphatidylinositol. Additional components such as sterols such as cholesterol or other components such as fatty acids (eg stearic acid, palmitic acid), diethyl phosphate or cholesterol hemisuccinate can be used. In addition, the liposome membrane may contain a preservative. Liposome membranes can also contain components that modify their dispersion behavior. They include, for example, PEGylated derivatives of phosphatidylethanolamine, lipids such as GM1, or conjugates of sugars and hydrophobic components such as dextran palmitate or stearate.

  The basic structure of liposomes can be made by various techniques known in the art.

  For example, liposomes have typically been prepared using the process of Bangham et al. (1965 J. Mol. Biol., 13: 238-252), which allows lipids suspended in an organic solvent. Evaporate under reduced pressure in a reaction vessel to give a dry film. The appropriate amount of aqueous phase is then added to the vessel and the mixture is stirred. The mixture is then allowed to stand substantially undisturbed for a time sufficient to form a multilamellar vesicle.

  Liposomes can be reproducibly prepared using a number of currently available techniques. The types of liposomes that can be produced using a number of these techniques are small unilamellar vesicles (SUVs) [see Papahadjapoulous and Miller, Biochem. Biophys. Acta., 135, p.624-638 (1967)], and vice versa. Phase evaporation vesicles (REV) [see U.S. Pat. No. 4,235,871 issued Nov. 25, 1980], stable pull lamella vesicles (SPLV) [see U.S. Pat. No. 4,522,803 issued Jun. 11, 1985 And a large unit manufactured by the extrusion technique described in co-pending U.S. Patent Application No. 622,690, filed June 20, 1984, by the name of the invention "Extrusion Technique for Producing Unilamellar Vesicles". Includes layer vesicles.

  In a preferred embodiment, liposomes are prepared using the following method. Lipids are pipetted into a round bottom flask pretreated with nitric acid and dithymyl dichloride, for example, appropriate amounts of stock solutions of CDAN, DOPE and aminoxy lipid (CPA), respectively, the solvent is evaporated, and then vortexed Is prepared by hydrating a dry lipid film with water under vigorous stirring to yield multilamellar liposomes.

  Unilamellar liposomes can be produced by sonicating multilamellar liposomes for 30 minutes. Preferably this continues until a size of less than about 30 nm is reached.

  To add siRNA to the liposomes, a solution of siRNA in water is added dropwise to these liposomes under vigorous stirring by vortexing. Preferably, this is continued until a final siRNA concentration of about 0.1 mg / mL is reached.

  The resulting siRNA lipoplex is typically scaled by a diameter of about 30-50 nm as measured by PCS.

  The vectors described herein can be lyophilized. Preferably the vector is lyophilized. Accordingly, in a further aspect, a method comprising: (i) providing a vector described herein; (ii) optionally contacting the vector with a cryoprotectant; and (iii) lyophilizing the vector. Is provided.

  Advantageously, the lyophilized vector can be stored for a long time. Following storage, the vector can be rehydrated in water prior to use.

  Advantageously, lyophilization in the presence of cryogens such as sucrose, trehalose and lactose does not damage the vector in terms of size (PCS) and activity. Surprisingly, it has been found that vectors lyophilized in the presence of trehalose are even better than freshly prepared vectors.

  The polymer is added, for example, at 1 mg / mL using, for example, a solution of polyethylene glycol-α / γ-bisaldehyde (Mw 2000 or 3400, respectively; NEKTAR, USA). The appropriate amount of polymer is then added to the siRNA lipoplex under vortexing. Typically, this results in covalently pegylated surface siRNA lipoplexes with varying amounts of polymer (eg, 0.1-5%, m / m total lipid). Half of the volume can be evaporated at reduced pressure and can be supplemented by the addition of PBS prior to use, eg, injection into animals.

  It is also desirable to include other components in the liposome, such as radiolabels, dyes, chemiluminescent and fluorescent markers; contrast media; imaging aids; diagnostic markers including drugs and the like.

式中、Bは脂質であり；Xは自由選択のリンカー基であり、およびR₂はHまたはヒドロカルビル基である。好ましい脂質は、国際(PCT)特許出願番号PCT/GB01/05385のものである。 The liposome preferably comprises one or more aminoxy group-containing lipids of formula (I):

Where B is a lipid; X is an optional linker group and R ₂ is H or a hydrocarbyl group. Preferred lipids are those of International (PCT) patent application number PCT / GB01 / 05385.

  Here, in the context of formula (I), the term “hydrocarbyl group” means a group containing at least C and H, and may optionally contain one or more other suitable substituents. Examples of such substituents can include halo, alkoxy, nitro, alkyl groups, cyclic groups, and the like. In addition to the possibility of a substituent being a cyclic group, the combination of substituents can form a cyclic group. If the hydrocarbyl group contains more than one C, the carbons need not necessarily be linked to each other. For example, at least two of the carbons can be linked via a suitable element or group. Thus, hydrocarbyl groups can contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for example, sulfur, nitrogen and oxygen. A non-limiting example of a hydrocarbyl group is an acyl group.

  A typical hydrocarbyl group is a hydrocarbon group. In the present specification, the term “hydrocarbon” means any one of an alkyl group, an alkenyl group, and an alkynyl group, and the group may be a linear, branched or cyclic group, or an aryl group. The term “hydrocarbon” includes those groups except that they are optionally substituted. If the hydrocarbon is a branched structure having substituents thereon, the substitution may be either on the hydrocarbon skeleton or on the branch; or the substitution may be on the hydrocarbon skeleton and on the branch. possible.

  The hydrocarbyl / hydrocarbon / alkyl may be linear or branched and / or may be saturated or unsaturated.

  In one preferred aspect, the hydrocarbyl / hydrocarbon / alkyl can be selected from a straight or branched chain hydrocarbon group containing at least one heteroatom in the group.

  In one preferred aspect, the hydrocarbyl / hydrocarbon / alkyl may be a hydrocarbyl group containing at least 2 carbons, or the total number of carbon and heteroatoms is at least 2.

  In one preferred aspect, the hydrocarbyl / hydrocarbon / alkyl can be selected from hydrocarbyl groups that contain at least one heteroatom in the group. Preferably, the heteroatom is selected from sulfur, nitrogen and oxygen.

  In one preferred aspect, the hydrocarbyl / hydrocarbon / alkyl can be selected from a straight or branched chain hydrocarbon group containing at least one heteroatom in the group. Preferably, the heteroatom is selected from sulfur, nitrogen and oxygen.

In one preferred aspect, the hydrocarbyl / hydrocarbon / alkyl is a linear or branched alkyl group, preferably C _1-10 alkyl, more preferably C _1-5 alkyl, containing at least one heteroatom in the group. More can be selected. Preferably, the heteroatom is selected from sulfur, nitrogen and oxygen.

In one preferred aspect, the hydrocarbyl / hydrocarbon / alkyl is selected from linear alkyl groups, preferably C _1-10 alkyl, more preferably C _1-5 alkyl, containing at least one heteroatom in the group. be able to. Preferably, the heteroatom is selected from sulfur, nitrogen and oxygen.

Hydrocarbyl / hydrocarbon / alkyl is
・ C _1-10 hydrocarbyl ・ C ₁ -C ₅ hydrocarbyl ・ C ₁ -C ₃ hydrocarbyl ・ Hydrocarbon group ・ C ₁ -C ₁₀ hydrocarbon ・ C ₁ -C ₅ hydrocarbon ・ C ₁ -C ₃ hydrocarbon ・ alkyl The group can be selected from: C ₁ -C ₁₀ alkyl, C ₁ -C ₅ alkyl, C ₁ -C ₃ alkyl.

  The hydrocarbyl / hydrocarbon / alkyl may be linear or branched and / or may be saturated or unsaturated.

  The hydrocarbyl / hydrocarbon / alkyl can be a linear or branched hydrocarbon group containing at least one heteroatom in the group.

Preferably R ₂ is H or a hydrocarbyl group.

In a preferred aspect, the R ₂ hydrocarbyl group contains a freely selected heteroatom selected from O, N and halogen.

In a preferred aspect, R ₂ is H.

  Preferably, the lipid is a cholesterol group or is derived from or comprises a cholesterol group.

  The cholesterol group may be or may be derived from cholesterol or a derivative thereof.

Examples of cholesterol derivatives include substituted derivatives, wherein one or more of the cyclic CH ₂ or CH groups and / or one or more of the linear CH ₂ or CH groups are suitably substituted. Alternatively or in addition, one or more of the cyclic groups and / or one or more of the linear groups can be unsaturated.

  In a preferred embodiment, the cholesterol group is cholesterol.

  In a preferred aspect, there is an optional linker X.

  In a preferred aspect, X is a hydrocarbyl group.

  In a preferred aspect, the linker X contains a polyamine group or is linked to the lipid via the polyamine group.

  It is considered advantageous because the polyamine group increases the DNA binding capacity and the efficiency of gene transfer of the resulting liposomes.

  In one embodiment, preferably the polyamine group is a non-naturally occurring polyamine. Preferably, two or more of the amine groups of the polyamine group of the present invention are separated by one or more groups not found in nature that separate the amine groups of naturally occurring polyamine compounds (i.e., preferably The polyamine groups of the present invention have non-natural spacing).

Preferably, the polyamine group contains at least two amines of the polyamine group that are separated from each other (spaced from each other) by ethylene (—CH ₂ CH ₂ —) groups.

Preferably, each of the amines of the polyamine group is separated (spaced from each other) by an ethylene (—CH ₂ CH ₂ —) group.

式中、nおよびmは独立して0〜6、好ましくは1〜6、より好ましくは2、3、または4、より好ましくは2または4である。非常に好ましい局面において、mは2であり、かつnは4である。 In one preferred aspect, X is or comprises a group of the formula:

In the formula, n and m are independently 0 to 6, preferably 1 to 6, more preferably 2, 3, or 4, more preferably 2 or 4. In a highly preferred aspect, m is 2 and n is 4.

式中、nおよびmは独立して0〜6、好ましくは1〜6、より好ましくは2、3、または4、より好ましくは2または4である。非常に好ましい局面において、mは2であり、かつnは4である。 In one preferred aspect, X is or comprises a group of the formula:

In the formula, n and m are independently 0 to 6, preferably 1 to 6, more preferably 2, 3, or 4, more preferably 2 or 4. In a highly preferred aspect, m is 2 and n is 4.

式中、Bは脂質であり、nおよびmは独立して0〜6、好ましくは1〜6、より好ましくは2、3、または4、より好ましくは2または4である。非常に好ましい局面においては、mは2であり、かつnは4である。 In one preferred aspect, the liposome is of the formula:

Where B is a lipid and n and m are independently 0-6, preferably 1-6, more preferably 2, 3, or 4, more preferably 2 or 4. In a highly preferred aspect, m is 2 and n is 4.

式中、mは0〜6、好ましくは1〜6、より好ましくは1、2、または3、より好ましくは1であり、nは0〜20、好ましくは5〜15、より好ましくは10、11、または12、より好ましくは11である。 In one preferred aspect, X is or comprises a group of the formula:

In which m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, and n is 0-20, preferably 5-15, more preferably 10, 11 Or 12, more preferably 11.

式中、mは0〜6、好ましくは1〜6、より好ましくは1、2、または3、より好ましくは1であり、nは0〜20、好ましくは5〜15、より好ましくは10、11、または12、より好ましくは11である。 In one preferred aspect, X is or comprises a group of the formula:

In which m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, and n is 0-20, preferably 5-15, more preferably 10, 11 Or 12, more preferably 11.

式中、Bは脂質であり、mは0〜6、好ましくは1〜6、より好ましくは1、2、または3、より好ましくは1であり、nは0〜20、好ましくは5〜15、より好ましくは10、11、または12、より好ましくは11である。 In one preferred aspect, the liposome is of the formula:

Wherein B is a lipid, m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, n is 0-20, preferably 5-15, More preferably 10, 11, or 12, more preferably 11.

  Typical examples of suitable polyamines include spermidine, spermine, cardopentamine, norspermidine, and norspermine. Another preferred polyamine is cardopentamine.

  In a highly preferred embodiment, the aminoxy group-containing lipid is CPA.

  Preferably, the liposome comprises one or more cationic lipids.

  Various cationic lipids are known in the art. Exemplary structures for such cationic lipids are provided in Table 1 of WO 95/02698.

  In general, either monovalent or polyvalent cationic lipids can be used.

  Multivalent cationic lipids are generally preferred.

  Cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides or derivatives thereof. The linear and branched alkyl and alkene groups of the cationic lipid can contain from 1 to about 25 carbon atoms. Preferred straight or branched alkyl or alkene groups have 6 or more carbon atoms. The alicyclic group can contain from about 6 to 30 carbon atoms. Preferred alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, among others, chloride, bromide, iodide, fluoride, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.

  A well known cationic lipid is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) chloride.

  DOTMA and similar diester DOTAP (1,2-bis (oleoyloxy) -3 (trimethylammonium) propane) are commercially available.

  Additional cationic lipids structurally related to DOTMA are described in US Pat. No. 4,897,355.

  Another useful group of cationic lipids related to DOTMA and DOTAP are commonly referred to as DORI ethers or DORI esters. DORI lipids differ from DOTMA and DOTAP in that one of the methyl groups of the trimethylammonium group is replaced with a hydroxyethyl group. The oleoyl group of DORI lipids can be replaced with other alkyl or alkene groups such as palmitoyl or stearoyl groups. The hydroxyl group of the DORI-type lipid can be used as a site for further functionalization for esterification to an amine, such as carboxyspermine.

  Additional cationic lipids that can be used in the delivery vectors or complexes of the invention include those described in WO 91/15501 useful for transfection of cells.

  Cationic sterol derivatives such as 3β [N- (N ′, N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) in which cholesterol is linked to a trialkylammonium group can also be used in the present invention. . DC-Chol has been reported to provide more effective transfection and lower toxicity than DOTMA-containing liposomes for some cell lines. DC-Chol polyamine variants such as those described in WO 97/45442 can also be used.

  Polycationic lipids containing carboxyspermine are also useful in the delivery vectors or conjugates of the present invention. EP-A-304111 describes carboxyspermine-containing cationic lipids including 5-carboxyspermylglycine dioctadecyl-amide (DOGS) and dipalmitoylphosphatidylethanolamine 5-carboxyspermilamide (DPPES). Additional cationic lipids can be obtained by replacing the octadecyl and palmitoyl groups of DOGS and DPPES with other alkyl or alkene groups, respectively.

  In a preferred embodiment, the cationic lipid comprises at least one saturated or unsaturated alicyclic ether or ester of an amine, amide or derivative thereof. The alicyclic group can contain from about 6 to 30 carbon atoms. A highly preferred alicyclic group is cholesterol.

  Thus, in a highly preferred embodiment, the polycationic lipid is a cholesterol-based lipid.

式中、Cholは下記式の基を示す。

Preferably, the cholesterol-based lipid is N ¹ -cholesteryloxycarbonyl-3,7-diazanononane-1,9-diamine (CDAN):

In the formula, Chol represents a group of the following formula.

  Preferably, the liposomes described herein comprise non-cationic colipids, preferably neutral lipids, to form liposomes or lipid aggregates.

  Neutral lipids useful in the present invention include lecithin; phosphatidylethanolamines such as DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyl oleoylphosphatidylethanolamine) and DSPE (distearoylphosphatidylethanolamine); Phosphatidylcholines such as DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine) and DSPC (distearoylphosphatidylcholine); phosphatidylglycerol; DOPG (dioleoylphosphatidylglycerol), DPPG Phosphatidylglycerols such as palmitoylphosphatidylglycerol) and DSPG (distearoylphosphatidylglycerol) Including and mixtures thereof; diphosphatidylglycerol; fatty acid esters; - dioleoyl or dipalmitoyl phosphatidylserine, such as dipalmitoyl phosphatidylserine glycerol esters; sphingolipids; Karudoripin; cerebrosides; and ceramides. Neutral lipids include cholesterol and other 3DOH-steroyl.

  Preferably, the non-cationic colipid is phosphatidylethanolamine. More preferably, the non-cationic colipid is DOPE (dioleoylphosphatidylethanolamine).

  In one preferred aspect, the lipid is a lipid suitable for use in imaging applications. The imaging lipid can be a lipid selected from fluorescent lipids, magnetic resonance imaging lipids, nuclear magnetic resonance imaging lipids, electron microscopy and image processing lipids, electron spin resonance lipids and radioactive imaging lipids. Suitable and preferred lipids in each of these classes are presented below.

Fluorescent lipid:
For example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (5-dimethylamino-1-naphthalenesulfonyl, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- ( 1-pyrenesulfonyl), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (carboxyfluorescein), 1-oleoyl-2- [6-[(7-nitro-2-1,3 -Benzoxadiazol-4-yl) amino] hexanoyl] -sn-glycero-3-phospho-L-serine, 25- {N-[(7-nitrobenz-2-oxa-1,3-diazol-4- Yl) -methyl] amino} -27-norcholesterol, -oleoyl-2- [6-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] hexanoyl] -sn- Glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (risamine rhodamine B sulfonyl).

Lipids for magnetic resonance imaging and nuclear magnetic resonance imaging:
Gd-DTPA-bis (stearylamide) (Gd-BSA); Gd-DTPA-bis (myristylamide) (GdDTPA-BMA), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine diethylenetriaminepentaacetate : Gd ³⁺ (DMPEDTPA: Gd ³⁺ ); D ₃₅ -1,2-dihexanoyl-sn-glycero-3-phosphocholine.

Electron microscope and image processing:
1,2-dioleoyl-sn-glycero-3-{[N (5-amino-1-carboxypentyl) iminodiacetic acid] succinyl}-(nickel salt).

Electron spin resonance:
1,2-diacyl-sn-glycero-3-phosphotempocholine, 1-palmitoyl-2-stearoyl (n-DOXYL) -sn-glycero-3-phosphocholine.

Radioactive imaging:
(99m) Tc-DTPA-bis (stearylamide); (99m) Tc-DTPA-bis (myristylamide).

  Furthermore, one or more amphiphilic compounds can optionally be incorporated to modify their surface properties. Amphiphilic compounds useful in the present invention include, among others, neoglycolipids such as GLU4 and GLU7, N- (O-methoxy (polyoxyethylene) oxycarbonyl) phosphatidylethanolamine, N-monomethoxy (polyoxyethylene) Polyethylene glycol lipids such as succinylphosphatidylethanol-amine and polyoxyethylene cholesteryl ether; alkyl glycosides, alkyl methyl glucamide, sucrose esters, alkyl polyglycerol ethers, alkyl polyoxyethylene ethers, and alkyl sorbitan oxyethylene ethers and steroidal oxy Nonionic surfactants such as ethylene ether; block copolymers such as polyoxyethylene polyoxypropylene block copolymers.

  Thus, in a preferred embodiment of the invention, the liposome comprises one or more aminoxy group-containing lipids, one or more cationic lipids (more preferably one or more polycationic lipids), and one or more. Non-cationic colipids (more preferably, neutral lipids).

  More preferably, the liposome comprises one or more aminoxy group-containing lipids of formula (I) (preferably CPA) and / or mixtures thereof; DOTMA, DOTAP, DORI ether or DORI ester, (DC-Chol), DOGS, One or more polycationic lipids selected from the group consisting of DPPES, and / or CDAN, and / or mixtures thereof; and DOPE, POPE, DSPE, DOPC, DPPC, DSPC, DOPG, DPPG, DSPG, postfat One or more neutral lipids selected from the group consisting of dilserine, diphosphatidylglycerol, fatty acid esters, glycerol esters, sphingolipids, cardipine, cerebrosides; and / or ceramides; and / or mixtures thereof.

  More preferably, the liposome comprises one or more lipids selected from the group consisting of CPA, CDAN and DOPE.

  Most preferably, the liposome comprises CPA, CDAN and DOPE.

  The inventors have surprisingly found that certain liposomes used according to the invention have special advantages. These advantages are applicable to a wide range of systems as well as the present delivery vector containing siRNA. The inventors have determined that the present liposomes comprising a lipid linked to a coupling moiety via a linker with minimal length allows the preparation of targeted delivery vectors. In particular, using such liposomes, some liposomes are coupled to one or more polymers, and the coupling portion of the liposomes with a minimum length linker (`` long linker '') is created by the polymer. Vectors that protrude beyond the shell can be adjusted. The protruding coupling portion of the liposome containing the long linker can then be used to couple to additional groups, for example, the protruding coupling portion can be used to couple to a target moiety such as an antibody.

  Accordingly, in a further aspect, the present invention provides a liposome comprising a lipid and a coupling moiety, wherein the distance between the lipid and the coupling moiety is at least 1.5 nm. Preferably the distance between the lipid and the coupling moiety is at least 2 nm, such as at least 3 nm or at least 5 nm.

式中、Bは脂質であり；Xはリンカー基であり、カップリングはカップリング部分であり、X骨格は少なくとも30個の原子を含む。 In a further aspect, the present invention provides a liposome of the formula:

Where B is a lipid; X is a linker group, the coupling is a coupling moiety, and the X backbone contains at least 30 atoms.

  The term “X skeleton” means the shortest chain of directly bonded atoms within the X moiety between the X bond to the coupling and the X bond to B.

  Preferably, the X skeleton comprises at least 40 atoms, such as at least 60 atoms, such as at least 50 atoms.

  Preferably, the X skeleton comprises at least 40 carbon atoms, such as at least 60 carbon atoms, such as at least 50 carbon atoms.

  As an example, the cholesteryl aminoxy lipid 4 described herein has not been successful in conjugating oxidized IgG antibodies. This is because the spacer between the cholesteryl moiety and the aminoxy functional group is not long enough. However, it successfully couples polyethylene glycol-bisaldehyde (Mw 2000 and 3400).

  Lipid B may be any lipid described herein. Particularly preferred are lipids selected from phospholipids such as distearoylphosphatidylethanolamine (DSPE), steroid-based lipids such as cholesterol, and amine-based lipids such as dicyclohexylamine.

  Linker X may be any linker described herein. A linker is typically understood to mean a non-reactive linker. Preferably the linker is hydrophilic.

The linker is
・ Hydrocarbyl group such as octadecyl group ・ Peptide derived from polyglycine ・ Poly (ethylene carbonate), polyethylene glycol (PEG), N- (2-hydroxypropyl) methacrylamide (HMPA), poly (D, Polymer dextran, such as L-lactide-co-glycolide) (PLGA), poly (D, L-lactide) (PLA), and poly (glycolide) (PGA), polymannose, hyaluronic acid, oligosaccharides, dextran, It can be a carbohydrate such as pullulan.

  In some aspects, the linker may itself be chemically reactive, for example, the linker may be derived from a peptide such as poly (lysine) (PL) or poly (glutamic acid).

式中、nおよびmは独立して0〜6、好ましくは1〜6、より好ましくは2、3、または4、より好ましくは2または4である。非常に好ましい局面において、mは2であり、かつnは4である。 In one preferred aspect, X is or comprises a group of the formula:

In the formula, n and m are independently 0 to 6, preferably 1 to 6, more preferably 2, 3, or 4, more preferably 2 or 4. In a highly preferred aspect, m is 2 and n is 4.

式中、nおよびmは独立して0〜6、好ましくは1〜6、より好ましくは2、3、または4、より好ましくは2または4である。非常に好ましい局面において、mは2であり、かつnは4である。 In one preferred aspect, X is or comprises a group of the formula:

In the formula, n and m are independently 0 to 6, preferably 1 to 6, more preferably 2, 3, or 4, more preferably 2 or 4. In a highly preferred aspect, m is 2 and n is 4.

式中、mは0〜6、好ましくは1〜6、より好ましくは1、2、または3、より好ましくは1であり、nは0〜20、好ましくは5〜15、より好ましくは10、11、または12、より好ましくは11である。 In one preferred aspect, X is or comprises a group of the formula:

In which m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, and n is 0-20, preferably 5-15, more preferably 10, 11 Or 12, more preferably 11.

式中、mは0〜6、好ましくは1〜6、より好ましくは1、2、または3、より好ましくは1であり、nは0〜20、好ましくは5〜15、より好ましくは10、11、または12、より好ましくは11である。 In one preferred aspect, X is or comprises a group of the formula:

In which m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, and n is 0-20, preferably 5-15, more preferably 10, 11 Or 12, more preferably 11.

  The coupling may be any coupling moiety described herein. Particularly preferred are nucleophilic groups, especially amines, thiols, alcohols, aminoxys, hydrazines, hydrazides, azides, electrophilic groups, especially isothiocyanates, aldehydes, ketones, isocyanates, maleimides, Michael acceptors, in general, Halides, tosylates, chemical leaving groups, generally active esters, photoligative groups, especially azides.

Polymer As used herein, the term “polymer” includes one or more functional groups that interact with, bind to, or couple to one or more lipids contained in a liposome. Any polymer is also referred to.

  The polymer may be a naturally occurring polymer or a derivative thereof.

  The polymer may be a chemically modified polymer, where the polymer is modified to include one or more functional groups.

  In a more preferred embodiment, one or more lipids of the liposome are coupled to one or more polymers. Advantageously, the lipid coupled to the polymer is exposed on the surface of the liposome so that the polymer remains on the surface of the liposome. Without being bound by any particular theory, it is believed that the polymer effectively coats the surface of the liposome through multiple interactions between the lipid and polymer of the liposome.

  Coupling between lipids and polymers can be via any type of interaction, such as hydrogen bond interactions, charge interactions, hydrophobic interactions, covalent interactions, van der Waals interactions, or dipole interactions. It may be mediated.

  In preferred embodiments, the interaction is mediated through covalent interactions.

  Preferably, the covalent interaction occurs between one or more groups (eg, functional groups) of the polymer and one or more lipids of the liposome.

  One skilled in the art can select appropriate groups to achieve the desired interaction between one or more groups (e.g., functional groups) of the polymer and one or more lipids of the liposome. it can.

  In one preferred aspect, the covalent interaction is one or more groups of the polymer, and amines, thiols, alcohols, aminoxys, hydrazines, hydrazides, azides, isothiocyanates, aldehydes, ketones, isocyanates, maleimides, halides, Occurring between one or more functional groups of one or more lipids of a liposome selected from tosylate and ester. Particularly preferred are aminoxy group and hydrazine group.

  More preferably, the covalent interaction occurs between one or more aldehyde and / or ketone groups of the polymer and one or more aminoxy functional groups of one or more lipids of the liposome.

  Advantageously, the provision of a lipid containing an aminoxy group allows for simple attachment of the polymer to the lipid via the aminoxy group. When reacted with a polymer containing an aldehyde or ketone group, a compound in which the polymer and lipid are linked via an amide group is provided. Such a linkage can simply be prepared in a “one pot” reaction. This method avoids extensive purification procedures with simple dialysis or excess unreacted reagents.

  Preferably, the polymer is monofunctional or difunctional poly (ethylene glycol (“PEG”)), poly (vinyl alcohol) (“PVA”); other poly, such as poly (propylene glycol) (“PPG”). (Alkylene oxide); and poly (oxyethylated glycerol), poly (oxyethylated sorbitol), poly (oxyethylated polyol) such as poly (oxyethylated glucose), and the like.

  As discussed in the background of the invention teaching US-A-2001 / 0021763, the polymer is a monomer of the polymer, linear or branched, or substituted or unsubstituted, similar to mPEG, and to a linker Based on other capped monofunctional PEGs having a single active site available for coupling, can be homopolymers or random or block copolymers and terpolymers.

  Specific examples of suitable additional polymers include poly (oxazoline), poly (acryloylmorpholine) (“PAcM”), and poly (vinyl pyrrolidone) (“PVP”). PVP and poly (oxazoline) are well-known polymers in the art and their preparation and use in the syntheses described for mPEG should be readily apparent to those skilled in the art. PAcM, and its synthesis and use are described in US-A-5,629,384 and US-A-5,631,322.

  In a preferred embodiment, the polymer is polyethylene glycol (PEG) with functional aldehyde and / or ketone groups, or a chemical derivative thereof.

  In a preferred embodiment, the polymer has a molecular weight of 2000-1000. In a preferred embodiment, polyethylene glycol (PEG) has a molecular weight of 2000-1000.

  In preferred embodiments, the polymer has one or more functional groups that can be coupled to one or more lipids. In a preferred embodiment, the polymer has 1 or 2 and only 1 or 2 functional groups that can be coupled to one or more lipids.

  Different sizes of PEG can be used as described in FIG. 1, which can include mono- and bis-aldehyde PEG.

  Polyethylene glycol (PEG) has previously been used to modify the biophysical properties of drug delivery systems. For example, it can be used to confer serum stability to gene delivery vectors (2-4). This concept has been described in the literature and a common consensus is that the formulation of PEG into a gene delivery vehicle dramatically reduces the transfection efficiency of gene delivery vectors (5-8,42). Recent investigations of why this phenomenon occurs suggest that PEG-formulated non-viral gene delivery systems are equally internalized to cultured cells but exhibit dramatically altered intracellular transport (9). These PEG-formulated vectors are not released from endosomes, or PEG-induced stealth does not release pDNA into cells, or PEG as a molecule along the cell microtubule system towards the cell nucleus PDNA appears to inhibit intracellular transport. Similar observations have been made with lipoplexes loaded with oligonucleotides (ODN). Song and co-workers have shown that unilamellar DODAC / DOPE / PEG-lipid cationic liposome systems with different lengths of PEG undergo endocytosis like non-pegylated lipoplexes, but their endosomal release is severe (10). In part, according to these data, Hoekstra and co-workers included 10 mol% PEG-phosphatidylethanolamine in the ODN lipoplex, inhibiting its internalization in more than 70% Chinese hamster ovary cells, We report that the intracellular fraction remains trapped in the endosome / lysosome pathway. Hoekstra noted that the method of formulating PEG into the lipoplex is critical to the ability of the lipoplex to escape the endosome. When liposomes were formed in the presence of DSPE-PEG and then loaded with ODN, ODN uptake was strongly inhibited in a PEG / lipid-concentration dependent manner. Even at the lower dose of PEG (1%), dissociation of the complex was inhibited. In contrast, when DSPE-PEG is incorporated into preformed ODN lipoplexes, PEGylation is restricted to the outer perimeter of ODN lipoplexes, and only such PEGylated complexes exhibit useless delivery. However, PEG lipids provide an exchangeable one (11).

  Based on these results, non-viral delivery vectors formulated with siRNA are expected to exhibit the same trend as observed with ODN and pDNA.

  Surprisingly, this is not the case.

  In fact, non-viral delivery vectors containing liposomes, where one or more lipids of the liposome are reversibly or irreversibly coupled to one or more polymers such as PEG, and the liposome contains siRNA, the target gene Dramatically stabilizes liposomes against aggregation without compromising the ability of siRNA to down-regulate.

  Thus, in a preferred embodiment, the non-viral delivery vector is a lipid of formula (I) containing one or more aminoxy groups, preferably CPA and / or mixtures thereof; DOTMA, DOTAP, DORI ether or DORI ester, One or more polycationic lipids selected from the group consisting of: DC-Chol), DOGS, DPPES, and / or CDAN, and / or mixtures thereof; DOPE, POPE, DSPE, DOPC, DPPC, DSPC, DOPG, One or more neutral lipids selected from the group consisting of DPPG, DSPG, phosphatidylserine, diphosphatidylglycerol, fatty acid ester, glycerol ester, sphingolipid, cardolipine, cerebroside; and / or ceramide; and / or mixtures thereof One or more polymers coupled to the containing liposomes.

  More preferably, the non-viral delivery vector is a lipid of formula (I) containing one or more aminoxy groups, preferably CPA and / or mixtures thereof; DOTMA, DOTAP, DORI ether or DORI ester, (DC- Chol), DOGS, DPPES, and / or CDAN, and / or a mixture thereof, one or more polycationic lipids; DOPE, POPE, DSPE, DOPC, DPPC, DSPC, DOPG, DPPG, A liposome comprising one or more neutral lipids selected from the group consisting of DSPG, phosphatidylserine, diphosphatidylglycerol, fatty acid ester, glycerol ester, sphingolipid, cardolipine, cerebroside; and / or ceramide; and / or mixtures thereof And PEG coupled with.

  More preferably, the non-viral delivery vector comprises one or more polymers coupled with liposomes comprising CPA, CDAN and DOPE and / or mixtures thereof.

  Most preferably, the non-viral delivery vector comprises PEG coupled with a liposome comprising CPA, CDAN and DOPE and / or mixtures thereof.

Non-viral delivery vectors As described herein, delivery vectors are typically made by contacting one or more lipids, including siRNA, and any other component to be included in a liposome. The The lipid may be part of a preformed liposome comprising one or more, preferably two or more lipid components. This final complex can be stored at about −80 ° C. with 10% sucrose (w / v) added until use.

  As described herein, the complex can also be lyophilized for storage purposes in the presence or absence of a cryogen such as sucrose, trehalose or lactose without loss of activity and particle integrity. it can.

  The components can be combined in any order. If additional components are to be added, they can be added at any stage, but preferably together with the siRNA.

  Non-viral delivery vectors may be particularly well suited for pharmaceutical applications, but they are not limited to that application, for food applications, agricultural applications, for imaging applications, and others described herein. Can be designed.

Drug
In addition to siRNA, liposomes can also include one or more agents therein.

  Further, as described herein, liposomes can be reversibly or irreversibly coupled to one or more agents, such as one or more targeting moieties.

  The drug may be an organic compound or other chemical. The drug may be a compound that can be obtained from or produced by any suitable source, whether natural or artificial. The agent can be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof. The agent can be a sense or antisense molecule, or even a polynucleotide molecule that can be an antibody, eg, a polyclonal antibody, a monoclonal antibody, or a monoclonal humanized antibody.

  The drug may be just a known drug, or compound, or an analog thereof.

  Agents can be designed or derived from libraries of compounds that can include peptides, as well as other compounds such as small organic molecules.

  By way of example, the drug may be a natural substance, a biological macromolecule, or an extract made from a biological substance such as a bacterium, fungus, or animal (especially mammalian) cell or tissue, an organic or inorganic molecule, a synthetic drug , Semi-synthetic drugs, structural or functional mimetics, peptides, peptidomimetics, peptides cleaved from the whole protein, or (for example, using a peptide synthesizer or by recombinant techniques, or combinations thereof) It can be a synthetically synthesized peptide, a recombinant drug, an antibody, a natural or non-natural drug, a fusion protein or equivalent thereof, and variants, derivatives or combinations thereof.

  The agent can be an organic compound. In some examples, the organic compound includes two or more hydrocarbyl groups.

  The agent can contain halo groups such as fluoro, chloro, bromo, or iodo groups.

  The agent can include one or more of alkyl, alkoxy, alkenyl, alkylene, and alkenylene groups, which can be unbranched or branched.

  Agents can exist as stereoisomers and / or geometric isomers, for example, agents can have one or more asymmetric and / or geometric centers, and thus two or more stereoisomers It can exist in body and / or geometric form. The present invention contemplates the use of all individual stereoisomers and geometric isomers of these agents, and mixtures thereof.

  The drug may be any chemical or substance that is desired to be applied, administered or used in liposomes, including but not limited to insecticides, herbicides, cosmetics, and perfumes, vitamins and It can include mineral supplements, seasonings, and other food additives including minerals, contrast agents, dyes, fluorescent markers, radioactive labels, plasmids, vectors, virus particles, toxins, catalysts, and the like.

  The agent can include one or more biologically active agents and acts as a beneficial or therapeutic compound to prevent disease when administered to an animal, preferably a mammal, more preferably a human. And any molecule that alleviates or treats. This may prevent a disease from occurring in a subject who may be predisposed to the disease but has never been diagnosed with it; inhibit the disease, ie prevent its occurrence; or It can include alleviating the disease, i.e. causing remission of the disease.

  Examples of drugs include, but are not limited to, anti-inflammatory agents; anti-cancer and anti-tumor agents; anti-microbial and anti-viral agents including antibiotics; anti-parasitic agents; vasodilators; bronchodilators, anti-allergy and Antiasthma drugs; peptides, proteins, glycoproteins, and lipoproteins; carbohydrates; receptors; growth factors; hormones and steroids; neurotransmitters; analgesics and anesthetics; narcotics; Contains substances.

  The agent can be selected from the group consisting of PEG, sugar, carbohydrate and ligand.

  The sugar can be selected from the group consisting of glucose, mannose, lactose, fructose, maltotriose, and maltoheptose (as shown in FIG. 7).

Chemical Synthesis Methods Drugs and / or siRNA can be prepared by chemical synthesis techniques.

  It will be apparent to those skilled in the art that sensitive functional groups may be required to be protected and deprotected during synthesis. This is the case, for example, as described in Protective Groups in Organic Synthesis by TW Greene and PGM Wuts, John Wiley and Sons Inc. (1991), and by PJ Kocienski in Protecting Groups Georg Thieme Verlag (1994). Can be achieved by technology.

  For example, if a base is used in a reaction with a substrate having an optical center that contains a base-sensitive group, any stereocenter present may be racemized under certain conditions during some of the reactions. Is possible. This is possible, for example, during the guanylation step. As is well known in the art, it should be possible to avoid such potential problems by selection of reaction sequences, conditions, reagents, protection / deprotection methods, and the like.

  Drugs and / or siRNA can be isolated and purified by conventional methods.

  Separation of diastereomers can be achieved by conventional techniques, for example by fractional crystallization, chromatography or H.P.L.C. of a stereoisomer mixture of a compound of formula (I) or a suitable salt or derivative thereof. The individual enantiomers of the compounds of formula (I) can be obtained from the corresponding optically pure intermediate or by the corresponding racemic HPLC using an appropriate chiral support, or an appropriately optically active acid or base. Diastereomeric salts formed by reaction of the corresponding racemate with can also be prepared by decomposition, such as by fractional crystallization.

  Drugs and / or siRNAs can be produced using chemical methods for synthesizing drugs and / or siRNAs in whole or in part. For example, if the drug comprises a peptide, the peptide can be synthesized by solid phase techniques, cleaved from the resin, and then purified by preparative high performance liquid chromatography (eg, Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing (eg, Edman degradation techniques; Creighton, supra).

  Peptide inhibitors can be synthesized using various solid phase techniques (Roberge JY et al (1995) Science 269: 202-204), and automated synthesis can be performed, for example, according to instructions provided by the manufacturer, This can be achieved using an ABI 43 1 A peptide synthesizer (Perkin Elmer). In addition, the amino acid sequence comprising the drug can be altered during direct synthesis and / or combined with sequences from other subunits, or any portion thereof using chemical methods, Can produce the drug.

  As used herein, the term “derivative” or “derivatization” includes chemical modification of an agent. Examples of such chemical modifications are substitution of hydrogen with halo, alkyl, acyl or amino groups.

  The agent may be a modified agent such as, but not limited to, a chemically modified agent.

  Chemical modification of the drug can enhance or reduce hydrogen bond interactions, charge interactions, hydrophobic interactions, van der Waals interactions or dipole interactions.

Targeted non-viral delivery vectors In a further aspect, the present invention relates to targeted delivery of non-viral delivery vectors.

  Targeted delivery of non-viral delivery vectors can be achieved by the addition of one or more agents (eg, targeting moieties) such as peptides and / or other ligands to the surface of the liposome, preferably the liposome. . Advantageously, the drug is coupled to the surface of the liposome via an interaction between the drug and one or more lipids of the liposome exposed on the liposome surface.

  Advantageously, this may allow delivery of the siRNA to specific cells, organs, and tissues that can bind to an agent, such as a targeting moiety. Typically, binding between cells, organs, and tissues is through specific binding between cells, organs, and / or tissues and drugs.

  In preferred embodiments, the cells, organs, and tissues may be the liver or may be derived from the liver. As used herein, the term “liver cell” refers to a cell located in the liver. Liver cells include, but are not limited to, cancerous liver cells, hepatocytes, Kupffer cells, Ito cells, endothelial cells lining hepatic sinusoids, vascular endothelial cells lining hepatic vessels, and accidentally in the liver Any cell of any origin present (eg, an ectopic metastatic cancer cell) can be included.

  Preferably, the liver cell is a liver cell (eg, HepG2 cell).

  Advantageously, the non-viral delivery vectors described herein exhibit strong accumulation in such cells, organs, and tissues.

  In another preferred embodiment, the cells, organs, and tissues can be or can be derived from the spleen, lungs, and / or lymph nodes.

  As used herein, the term “specific binding” refers to an interaction between one or more cells, organs, and / or tissues and a drug. This interaction is typically recognized by the drug, thereby allowing the interaction (e.g., binding) to occur in cells, organs and / or tissues, such as antigenic determinants or epitopes. Depends on the presence of specific structural features.

  Preferably, the agent is selected from the group consisting of sugars, carbohydrates, and / or ligands.

  Preferably, the sugar is selected from the group consisting of glucose, mannose, lactose, fructose, maltotriose, maltoheptose.

  The drug may be just a ligand.

  Many different ligands can be used depending on the site targeted for liposome delivery.

  A ligand can be designed or obtained from a library of compounds that can include peptides, as well as other compounds such as small organic molecules.

  Examples thereof are natural substances, biological macromolecules, or extracts made from biological materials such as bacteria, fungi, or animal (especially mammalian) cells or tissues, organic or inorganic molecules, Synthetic ligands, semi-synthetic ligands, structural or functional mimetics, peptides, peptidomimetics, derivatized ligands, peptides cleaved from whole proteins (e.g., using peptide synthesizers, or recombinant techniques or combinations thereof Synthetically synthesized peptides (such as by recombinant ligands, antibodies, natural or non-natural ligands, fusion proteins or equivalents thereof and variants, derivatives or combinations thereof).

  Preferably the ligand is an antibody.

As used herein, the term “antibody” refers to intact antibodies, bispecific antibodies or antibody fragments, Fv, ScFv, Fab ′ and F (ab ′) ₂ , monoclonal and polyclonal antibodies, chimeric, Includes engineered antibodies, including CDR-grafted and humanized antibodies, and artificially selected antibodies produced using phage display or alternative techniques.

  A chimeric antibody refers to a fusion created by genetic engineering between a part of a mouse antibody and a part of a human antibody. In general, a chimeric antibody contains approximately 33% mouse protein and 67% human protein.

  Humanized antibodies can be obtained not only by replacing the constant region of a mouse antibody with a human protein, but also by replacing part of the variable region of the antibody with a human protein. Generally, humanized antibodies are 5-10% mice and 90-95% humans.

  More sophisticated approaches to humanized antibodies not only provide for human-derived constant regions, but also include variable region modifications. This allows the antibody to be reshaped as closely as possible to the human form. This approach is, for example, Cancer Res (1993) 53: 851-856, Nature (1988) 332: 323-327, Science (1988) 239: 1534-1536, Proc Natl Acad Sci USA (1991) 88: 4181-4185 And J Immunol (1992) 148: 1149-1154.

  Antibody preparation is performed using standard laboratory techniques. Antibodies can be obtained from animal serum or, in the case of monoclonal antibodies or fragments thereof, can be produced in cell culture. Recombinant DNA technology can be used to produce antibodies according to established techniques in bacteria, or preferably in mammalian cell culture. The selected cell culture system preferably secretes the antibody product.

  The foregoing and other techniques are discussed, for example, in Kohler and Milstein, (1975) Nature 256: 495-497; US Pat. No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor. . Techniques for the preparation of recombinant antibody molecules are described in the references cited above and in, for example, EP 0623679; EP 0368684 and EP 0436597.

  Antibodies can be selected and created using phage display technology. Methods for the construction of bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (see, eg, Kang et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 4363 and Clackson et al. (1991) Nature, 352: 624).

  Advantageously, when the ligand is an antibody, the lipoplex coupled antibody exhibits substantially all of its activity.

  Preferably, the ligand is an RGD peptide (integrin receptor), a folate receptor and / or a receptor such as a receptor derived from or derived from a transferrin receptor.

Pharmaceutical Salts Non-viral delivery vectors can be administered in the form of pharmaceutically acceptable salts.

  Pharmaceutically acceptable salts are well known to those skilled in the art and include, for example, those mentioned by Berge et al. In J. Pharm. Sci., 66, 1-19 (1977). Suitable acid addition salts are formed from acids that form non-toxic salts, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate. Tart, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate Contains salt.

  Where one or more acidic moieties are present, suitable pharmaceutically acceptable base addition salts can be formed from bases that form non-toxic salts such as aluminum, calcium, lithium, magnesium, potassium, sodium, Includes zinc and salts of pharmaceutically active amines such as diethanolamine.

Pharmaceutically active salts Non-viral delivery vectors can be administered as pharmaceutically acceptable salts. Typically, a pharmaceutically acceptable salt can be readily prepared suitably with the desired acid or base. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.

Pharmaceutical Compositions The pharmaceutical compositions of the present invention can comprise a therapeutically effective amount of a non-viral delivery vector.

  The pharmaceutical composition may be for human or veterinary use in human and veterinary medicine, typically one or more of any of pharmaceutically acceptable diluents, carriers or excipients. including. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable binder, lubricant, suspending agent, coating agent, solubilizer as, or in addition to, a carrier, excipient or diluent.

  Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

  There may be different composition / formulation requirements depending on the different delivery systems. By way of example, the pharmaceutical composition of the invention is formulated using a minipump or by the mucosal route, for example as a nasal spray or aerosol for inhalation or ingestion solutions, or by injection form for delivery. It can be administered parenterally, for example, by intravenous, intramuscular or subcutaneous routes. Alternatively, the formulation can be designed and administered by multiple routes.

  If the drug is to be administered mucosally through the gastrointestinal mucosa, it can remain stable during movement through the gastrointestinal tract; for example, it is resistant to proteolysis, stable at acidic pH, and bile detergency Should be resistant to the effect.

  Where appropriate, the pharmaceutical composition may be applied by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dust, by the use of a skin patch, such as starch or lactose. Administered orally in the form of tablets containing the form, or in capsules or cavity suppositories, alone or mixed with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents Alternatively, the pharmaceutical composition can be injected parenterally, eg, intravenously, intramuscularly, or subcutaneously. For parenteral administration, the composition can best be used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, the compositions can be administered in the form of tablets or lozenges that can be conventionally formulated.

  Non-viral delivery vectors can be used in combination with cyclodextrins. Cyclodextrins are known to form drug molecules, inclusion bodies, and non-inclusion body complexes. Formation of a drug-cyclodextrin complex can modify the solubility, dissolution rate, bioavailability and / or stability properties of the drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes. As an alternative to direct complexation with the drug, cyclodextrin can be used as an auxiliary additive, eg as a carrier, diluent or solubilizer. α-, β-, and γ-cyclodextrins are most commonly used and suitable examples include International Publication (WO-A) No. 91/11172, International Publication No. (WO-A) No. 94/02518 and It is described in International Publication (WO-A) No. 98/55148.

  Pharmaceutical compositions containing non-viral delivery vectors can also be used in combination with conventional treatments for the disease of interest.

Administration Non-viral delivery vectors can be administered alone, but in general, for example, the vectors are suitable pharmaceutical excipients, diluents, or carriers selected according to the intended route of administration and standard pharmaceutical practice. Would be administered as a pharmaceutical composition.

  For example, non-viral delivery vectors contain flavoring or coloring agents for immediate release, delayed release, modified release, sustained release, pulsed release, or controlled release applications. It can also be administered in the form of tablets, capsules, suppositories, elixirs, solutions or suspensions.

  If the medicament is a tablet, the tablet is an excipient such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, starch (preferably corn, potato or tapioca starch), starch Disintegrants such as sodium glycolate, croscarmellose sodium and certain complex silicates, and granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia Can be contained. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

  Similar types of solid compositions can also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactose or high molecular weight polyethylene glycols. In aqueous suspensions and / or elixirs, the drug can be a variety of sweetening or flavoring agents, coloring materials or pigments, emulsifiers and / or suspending agents, and water, ethanol, propylene glycol and glycerin, and combinations thereof. Can be combined with a diluent such as

  The route for administration (delivery) is, but not limited to, oral (e.g., as a tablet, capsule, or as a feeding solution), topical, (e.g., as a nasal spray or aerosol for inhalation), mucosa, Nasal, parenteral (e.g. by injection form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intraorgan, intravaginal, intraventricular, intracerebral, One or more of subcutaneous, (including intravitreal or intraocular), eye, transdermal, rectal, buccal, vagina, epidural, sublingual can be included.

Dosage levels Typically, the physician will determine the actual dosage that is most appropriate for an individual subject.

  The particular dose level and frequency of administration for any particular patient can vary, including the activity of the particular compound used, its metabolic stability and length of action, age, weight Depends on a number of factors, including general health, gender, diet, mode and time of administration, rate of elimination, drug combination, severity of specific symptoms and the individual being treated.

Formulation Non-viral delivery vectors can be formulated into pharmaceutical compositions by using techniques known in the art, such as by mixing with one or more suitable carriers, diluents or excipients.

Diseases Aspects of the invention can be used in the treatment and / or prevention of diseases such as those listed in WO-A-98 / 09985.

  For ease of reference, a portion of that list is provided here: macrophage inhibitory and / or T cell inhibitory activity, and thus anti-inflammatory activity; anti-immune activity, ie responses not related to inflammation Inhibitory effects on cellular and / or humoral immune responses including: diseases associated with viruses and / or other intracellular pathogens; ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, and up in T cells Inhibiting regulated fas receptor expression; inhibiting unwanted immune responses and inflammation, including: arthritis including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen Disease and other autoimmune diseases, inflammation associated with atherosclerosis, arterial stiffness Disease, atherosclerosis heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorder, respiratory distress syndrome, or other cardiopulmonary disease, inflammation associated with peptic ulcer, ulcerative colitis, and gastrointestinal tract Other diseases, liver fibrosis, cirrhosis or other liver disease, thyroiditis or other adenopathy, glomerulonephritis or other kidney and urological diseases, otitis or other otolaryngology, dermatitis or other skin Disease, periodontal disease or other dental diseases, testicular or epididymal testicularitis, infertility, testicular trauma or other immune-related testicular disease, placental dysfunction, placental failure, habitual abortion, eclampsia, preeclampsia and Other immune and / or inflammation related gynecological diseases, posterior uveitis, middle uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation (e.g. retinitis Or cystoid macular edema), cross Ophthalmitis, scleritis, retinitis pigmentosa, immune and inflammatory component of degenerative fondus disease, inflammatory component of ocular trauma, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemia Symptomatic optic neuropathy, excessive scarring (eg after glaucoma filtration surgery), immune and / or inflammatory response to ocular implants, and other immune and inflammation-related eye diseases, both the central nervous system (CNS) or any other organ Inflammation, Parkinson's disease, complications and / or side effects from treatment of Parkinson's disease, Parkinson's disease, HIV-related brain disorders associated with AIDS-related dementia, in which suppression of immunity and / or inflammation is beneficial in , Devic disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, symptoms or disorders of the CNS, seizure inflammatory component, post-polio syndrome, psychiatric disability And inflammatory components, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillain-Barre syndrome, Sydenham chorea, myasthenia gravis, pseudobrain tumor, Down syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory component of CNS compression or CNS trauma or CNS infection, muscular atrophy and dystrophic inflammatory component, and central and peripheral nervous system immune and inflammation related diseases, symptoms, Or disorders, post-traumatic inflammation, septic shock, infections, inflammatory complications or side effects of surgical procedures, bone marrow transplantation or other transplant complications and / or side effects, inflammation and / or immune complications and side effects of gene therapy (e.g. Of natural or artificial cells, tissues, and organs, such as cornea, bone marrow, organ, lens, pacemaker, natural or artificial skin tissue Monocyte or leukocyte proliferation by reducing the amount of monocytes or lymphocytes to suppress or inhibit humoral and / or cellular immune responses for prevention and / or treatment of transplant rejection Due to infection with a viral carrier or inflammation associated with AIDS to treat or reduce sexually transmitted diseases such as leukemia). Specific cancer-related disorders include, but are not limited to, solid tumors; tumors that occur in blood such as leukemia; tumor metastases; benign tumors such as hemangiomas, optic neuromas, neurofibrosis, trachoma, and suppuration Granulomas; rheumatoid arthritis, psoriasis; ocular angiogenesis diseases such as diabetic retinopathy, immature retinopathy, macular degeneration, corneal graft rejection, neovascular glaucoma, fibroproliferation, lebeosis; Osler-Webber syndrome Myocardial angiogenesis; plaque angiogenesis; telangiectasia; hemophilia joint; vascular fibrosis; wound granulation; coronary collateral branch; cerebral collateral branch; arterial venous malformation; ischemic limb angiogenesis; Neonatal glaucoma; fibroproliferation; diabetic angiogenesis; Helicobacter-related diseases, fractures, angiogenesis, hematopoiesis, ovulation, menstruation, and placenta formation.

  Aspects of the invention can be used in the treatment and / or prevention of diabetes, such as type I and type II diabetes.

  Aspects of the present invention include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical cancer, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, female reproductive cancer, male reproductive system Cancer, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon and rectal cancer, colon cancer, endometrial cancer, Endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, Leukemia, leukemia, liver cancer, lung cancer, malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal and sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblast Tumor, non-Hodgkin lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, Head cancer, pituitary tumor, plasma cell neoplasm, primary CNS lymphoma, prostate cancer, rectal cancer, respiratory system, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, stomach cancer, testicular cancer It can also be used in the treatment and / or prevention of cancers such as thyroid cancer, urinary cancer, uterine sarcoma, vaginal cancer, vascular system, Vardenstrom macroglobulinemia, and Wilms tumor.

  In preferred embodiments, the disease is a disease, disorder, or symptom of or associated with liver disease and / or liver damage.

  Liver damage can be associated with exposure to alcohol, hepatotoxic drugs, and combinations thereof. For example, damaging agents include anticonvulsants, phenytoin, carbamazepine and phenobarbital, recreational drugs such as ecstasy (3,4-methylenedioxymethamphetamine), antituberculosis drugs, and chemotherapeutic agents such as isoniazid and rifampicin. Can do.

  Liver damage can also be associated with infectious agents such as bacterial, parasite, fungal and viral infections. For example, liver damage may include Aspergillus fungal infection, Schistosoma parasite infection, and adenovirus, retrovirus, adeno-associated virus (AAV), hepatitis A virus, hepatitis B virus, hepatitis C virus May be derived from various viral infections such as hepatitis E virus, herpes simplex virus (HSV), Epstein-Barr virus (EBV) and paramyxovirus infection.

  Liver disease includes but is not limited to acute hepatitis, fulminant hepatitis, chronic hepatitis, cirrhosis, fatty liver, alcoholic liver injury, drug-induced liver injury (drug-addictive hepatitis), congestive hepatitis, autoimmune hepatitis, It can include primary biliary cirrhosis, hepatic porphyria, perichoilitis, sclerosing cholangitis, liver fibrosis and chronic active hepatitis.

Use The non-viral delivery vectors described herein can be used to effectively transfect siRNA into cells, such as eukaryotic cells, particularly mammalian cells.

  The non-viral delivery vectors described herein can be used to effectively transfect the liver.

  Non-viral delivery vectors can be used for various siRNA delivery applications such as gene therapy, DNA vaccine delivery and in vitro transfection experiments.

  Non-viral delivery vectors can be used for various siRNA delivery applications such as liver, gene therapy, DNA vaccine delivery and in vitro transfection experiments.

  Non-viral delivery vectors can also be used to administer therapeutic agents to patients suffering from disease.

Variants / homologues / derivatives The present invention also includes the use of homologues and fragments.

  Here, the term “homolog” means an entity having a certain homology with the nucleotide sequences described herein. Here, the term “homology” may be equivalent to “identity”.

  In this context, homologous sequences are employed to include nucleotide sequences that may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, a homolog contains the subject sequence and the same sequence encoding the active site. Homology can also be considered in terms of similarities (i.e., amino acid residues having similar chemical properties / functions), but in the context of the present invention, it is preferred to represent homology in terms of sequence identity. .

  Homology comparisons can be performed by eye or more usually with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate% homology between two or more sequences.

  % Homology can be calculated over contiguous sequences, i.e., aligning one sequence with another sequence and replacing each amino acid in one sequence with the corresponding amino acid in the other sequence, one residue at a time. Compare directly with the group. This is called “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

  This is a very simple and consistent method, for example, that in one otherwise identical pair of sequences, one insertion or deletion will remove the next amino acid residue from the alignment and thus overall It does not take into account that a significant reduction in% homology would potentially result when a correct alignment is performed. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without improperly penalizing the overall homology score. This is accomplished by inserting “gaps” in the sequence alignment to try to maximize local homology.

  However, these more complex methods often result in “gap penalties” for sequences with as few gaps as possible, reflecting the higher association between the two compared sequences for the same number of identical amino acids. Assign each gap occurring in the alignment to achieve a higher score of more than 1 in the gap. “Affine gap costs” are typically used, which impose a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimal alignment with fewer gaps. Most alignment programs allow gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

  Thus, the calculation of maximum% homology first requires creating an optimal alignment taking into account gap penalties. A suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A .; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid-Chapter 18), FASTA (Atschul et al., 1990, J Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for some applications it is preferred to use the GCG Bestfit program. A tool called BLAST2 Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol Lett 1999 174 (2): 247-50; FEMS Microbiol Lett 1999 177 (1): 187-8).

  Although the final% homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. In fact, a scaled similarity score matrix is commonly used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. Examples of such matrices commonly used are the BLOSUM62 matrix, the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either public default values or custom symbol comparison tables when supplied (see user manual for further details). For some applications, it is preferable to use public default values for the GCG package, or in the case of other software, a default matrix such as BLOSUM62.

  Once the software has produced an optimal alignment, it is possible to calculate% homology, preferably% sequence identity. The software typically does this as part of the sequence comparison and produces a numerical result.

  Nucleotide sequences for use in the present invention can include within them synthetic or modified nucleotides. Modifications to many different types of oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and / or acridine or polylysine chain loadings at the 3 ′ and / or 5 ′ ends of the molecule. For the purposes of the present invention, it should be understood that the nucleotide sequences described herein can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifetime of the nucleotide sequences useful in the present invention.

  The present invention can include the use of nucleotide sequences complementary to the sequences described herein, or any derivative, fragment or derivative thereof.

  Sequence alleles are also included. An “allele” or “allelic sequence” is an alternative form of a sequence encoding a protein. Alleles result from mutations, ie changes in the nucleic acid sequence, generally resulting in a modified mRNA or polypeptide whose structure or function may or may not be altered. Preferably, the function is not altered. Any given gene has no allelic form and can have one or more allelic forms. Common mutational changes that give rise to alleles are generally attributed to amino acid deletions, additions or substitutions. Each of these types of changes can occur one or more times in a given sequence, either alone or in combination with others.

  The term “allele” also includes genetic polymorphisms such as SNPs (single nucleotide polymorphisms).

  The term “fragment” in relation to a nucleotide sequence includes any substitution, mutation, modification, substitution, deletion or addition of one (or more) nucleotide sequence from or to the sequence, provided that the resulting protein Is biologically active and preferably has at least as much biological activity as the protein encoded by the full-length nucleotide sequence. A fragment may comprise 50, 60, 70, 80, 90, 95, 96, 97, 98 or 99% of the full length nucleotide sequence.

  In a further aspect, the siRNA is delivered to the cell or cellular environment, comprising providing a non-viral or targeted delivery vector described herein to the environment of the cell, tissue or organ (eg, liver). A method is also provided.

General Recombinant DNA and Method Techniques The present invention uses conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology that are within the ability of one skilled in the art unless otherwise indicated. Such techniques are explained in the literature. For example, J. Sambrook, EF Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons; MJ Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and DMJ Lilley and JE Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press reference.

  The present invention will now be further described by way of example, which is meant to assist one of ordinary skill in the practice of the present invention, but is not intended to limit the scope of the invention in any way.

Examples Materials and Methods Thin Layer Chromatography Thin layer chromatography (TLC) performed on Merck-Kieselgel 60 F ₂₅₄ aluminum backing plates pre-coated, suitably UV light, iodine, ammonium ammonium molybdate (IV), acidic Visible with ethanolic vanillin or other drugs. Flash column chromatography was achieved with Merck-Kieselgel 60 (230-400 mesh). Mass spectra were recorded using a Brucker Esquire 3000, VG-7070B or JEOL SX-102 instrument. ¹ H and ¹³ C NMR resonances were recorded on an Advance Brucker 400 UltrashieldTM machine using residual isotope solvent as internal reference (s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, br = Broad singlet).

はDharmacon(USA)から購入し、製造業者によって示されるように20μM溶液として貯蔵した。非特異的siRNA配列

は、製造業者によって示されるように20μMで貯蔵されたDharmacon(USA)から入手した。3'-FITC標識抗GFP siRNA配列

は、Qiagen GmbH(Hilden,Germany)から入手した。配列は、オフターゲットダウンレギュレーションを確認するアルゴリズムに由来するものでよい(Kumiko Ui-Tei et al., Nucl. Acids Res. 2004, Vol 32, No.3, p.936-48)。このアルゴリズムに由来する1つのそのような配列は、ATG開始コドンの上流のlacZ遺伝子648〜670位を標的とする抗β-Gal配列

である。
この配列は、例えば、ヒト遺伝子TIMM8A(内部ミトコンドリア膜8ホモログAのトランスロカーゼ)およびNM＿01132.1(AFG3 ATPaseファミリー遺伝子3)その他の潜在的オフターゲットダウンレギュレーションを欠くことにおいてDharmacon配列とは異なる。 siRNA
Anti-β-gal siRNA-1 used throughout this experiment to evaluate the down-regulation of the lacZ gene product

Was purchased from Dharmacon (USA) and stored as a 20 μM solution as indicated by the manufacturer. Non-specific siRNA sequence

Was obtained from Dharmacon (USA) stored at 20 μM as indicated by the manufacturer. 3'-FITC labeled anti-GFP siRNA sequence

Was obtained from Qiagen GmbH (Hilden, Germany). The sequence may be derived from an algorithm that confirms off-target down regulation (Kumiko Ui-Tei et al., Nucl. Acids Res. 2004, Vol 32, No. 3, p. 936-48). One such sequence derived from this algorithm is an anti-β-Gal sequence that targets positions 648-670 upstream of the ATG start codon lacZ gene

It is.
This sequence differs from the Dharmacon sequence, for example, in the lack of human genes TIMM8A (translocase of internal mitochondrial membrane 8 homolog A) and NM — 01132.1 (AFG3 ATPase family gene 3) and other potential off-target down-regulations.

単層リポソームは、多重膜リポソームをSonomatic水浴(Longford Ultrasonics, UK)において30分間音波処理することによって生じさせた。リポソームを十分に音波処理することによって、溶液は完全に透明となり、リポソームのサイズはPCSによってもはや測定することができず、これは、リポソームのサイズが30nmよりも小さいことを示す。水中のsiRNAの溶液(0.28mg/mL)を、0.1mg/mLの最終siRNA濃度にて、ボルテックスによる激しい撹拌下でこれらのCDAN/DOPE/CPAリポソーム(3mg/mL)に滴下した。得られたsiRNAリポプレックスは、典型的には、PCSによって決定して30〜50nm直径と測定された。1mg/mLのポリエチレングリコール-α/γ-ビスアルデヒド(各々、Mw 2000または3400；NEKTAR, USA)を調製して、適当な量をボルテックスによる撹拌下でリポプレックスに加え、共有結合により表面がペグ化された、異なる量のPEG(0.1〜5%、m/m合計脂質)を有するsiRNAリポプレックスを得た。容量の半分を減圧下で蒸発させ、動物への注射に先立ってPBSの添加によって補った。 Liposome / Lipoplex Preparation All CDAN / DOPE / aminoxylipids are prepared by adding appropriate amounts of CDAN, DOPE and aminoxylipid (CPA) stock solutions to nitric acid (HNO ₃ , pure; 10 min) and dimethylsilyl dichloride, respectively. Pipet into a round bottom flask pretreated with (Sigma, UK10; several minutes), evaporate the solvent, and then hydrate the dry lipid film with water (milliQ, 18Ω) under vigorous stirring by vortexing

Unilamellar liposomes were generated by sonicating multilamellar liposomes for 30 minutes in a Sonomatic water bath (Longford Ultrasonics, UK). By fully sonicating the liposomes, the solution becomes completely transparent and the size of the liposomes can no longer be measured by PCS, indicating that the liposome size is smaller than 30 nm. A solution of siRNA in water (0.28 mg / mL) was added dropwise to these CDAN / DOPE / CPA liposomes (3 mg / mL) under vigorous stirring by vortexing at a final siRNA concentration of 0.1 mg / mL. The resulting siRNA lipoplexes were typically measured at 30-50 nm diameter as determined by PCS. Prepare 1 mg / mL polyethylene glycol-α / γ-bisaldehyde (Mw 2000 or 3400, respectively; NEKTAR, USA) and add the appropriate amount to the lipoplex under vortexing and the surface is PEGylated by covalent bonding. SiRNA lipoplexes with different amounts of PEG (0.1-5%, m / m total lipid) were obtained. Half of the volume was evaporated under reduced pressure and supplemented by the addition of PBS prior to injection into the animals.

Cell culture 24 hours prior to experiments in growth medium (DMEM / 10% FCS / penicillin / streptomycin), inoculate HeLa or IGROV-1 cells at 40000 cells / well in 48-well plates at 37 ° C. Cultured (10% CO ₂ ). Prior to transfection, the medium was replaced with fresh growth medium. OptiMEM-I / DMEM was purchased from Invitrogen (UK).

In vivo transfection
A. Fluid dynamic model
Balb / C mice weighing 20-25 g are injected with siRNA-containing lipoplexes (200 uL, 0.1 mg / mL siRNA) via tail vein injection or via intraperitoneal injection, 8 hours or 24 hours, respectively. I left it alone. After each resting phase, the animals were treated within 1 second with 1 μg of pUMVC1 (encoding the lacZ gene under the CMV promoter, 7528Bp, University of Michigan Vector Core; http: //www.med.umich. edu / vcore / Plasmids /) was injected and left for 24 hours, after which the liver was dissected and assayed for β-galactosidase activity using a standard ELISA test (ROCHE). The relative light units obtained were normalized by dividing through the total cellular protein content measured by BCA assay (Pierce).

B. Recombinant adenovirus (lacZ) model
AdRSVβGal was purchased from Transgene (Strasbourg, France). It is a type-5 adenovirus in which most of the E3 region has been deleted. Virus was titrated by injecting different amounts of stock into Balb / C mice and the titer that resulted in liver expression of approximately 10,000 RLU / mg protein was selected. Typically, 15 μL of viral tok was diluted in PBS (200 μL) and injected into the tail vein or intraperitoneal cavity of Blab / C mice, respectively. The two protocols followed: 1. AdRNAVβGal injection 2 hours prior to siRNA lipoplex injection (FIG. 5A) and 2. AdRSVβGal injection (intraperitoneal) 200 μL siRNA per animal 8 hours prior to injection Tail vein-injection of lipoplex (0.1 mg / mL siRNA) (FIG. 5B). All β-galactosidase ELISA assays were performed 24 hours after virus injection.

Transfection The β-Gal reporter gene (pUMVC1-β-Gal, 7528Bp) was transfected with PRIMOfect ™ (IC-Vec Ltd., UK) according to the manufacturer's instructions. Typically, 0.1 μg (HeLa) or 0.25 μg (IGROV-1) pDNA was transfected per 48-well plate. The total nucleic acid: lipid ratio was 1:12 (w / w) as recommended in the directive manual. After 3 hours of pDNA transfection, the transfection medium was replaced with fresh growth medium (150 μL), followed by LsiR siFection experiments using LsiR particles prepared in fresh OptiMEM (final volume 100 μL) immediately before siFection. Went.

All siFections described in this experiment were performed in 48-well plates. For this purpose, siRNA (0.1 μg) was diluted with fresh OptiMEM to a final volume of 100 μL. Under vortexing, 4.35 μL of CDAN / DOPE (0.3 mg / mL) was added (a lipid / siRNA ratio of 13: 1 [w / w] was obtained) and the LsiR lipoplex was left for 5 minutes. Finally, the LsiR mixture was then introduced into the appropriate wells of a given 48-well plate containing cells in complete growth medium (150 μL) (containing FCS / antibiotics) at 37 ° C., 10% at CO ₂ and incubated for 3 hours. The medium was then replaced with fresh growth medium and the cells were incubated for 16-72 hours prior to the β-Gal reporter assay (Roche, UK).

Example 1
Referring to pegylated siRNA lipoplexes Figure 1A, DOPE (140μL, CHCl ₃ in 10.68mg / mL), CDAN · 3HCl (271μL, CHCl 3 in 4 mg / mL) and CPA (100 [mu] L, in CHCl ₃ 4.4mg / mL) Was pipetted into a round bottom flask (5 mL) pretreated with nitric acid and dimethyldichlorosilane and the solvent was removed under reduced pressure to form a lipid film, which was washed with water (milliQ, 1 mL) under vortexing. Hydrated by addition. Subsequently, the multilamellar liposome formulation was sonicated in a sonomatic® water bath (Longford Ultrasonics) for 30 minutes to obtain small unilamellar vesicles (SUVs). 250 μL of these liposomes were pipetted into a 5 mL falcon tube and a solution of siRNA (0.28 mg / mL) was added dropwise with vigorous stirring by vortexing, followed by addition of polyethylene glycol-bisaldehyde (Mw 3400, 7.8 μL, 10 mg / mL; 5% PEG / total lipid). Samples were left at room temperature for 15 minutes, after which PBS (483 μL) was added. The sample was left at room temperature for 16 hours, and then the volume was reduced to 750 μL to obtain a siRNA lipoplex (LsiR) of 0.1 mg / mL siRNA.

  Referring to FIG. 1B, pUMVC1-β-Gal was transfected with PRIMOfect ™ (IC-Vec Ltd., UK) according to the manufacturer's instructions. Typically, 0.1 μg (HeLa) or 0.25 μg (IGROV-1) pDNA was transfected per 48-well plate. After a 3 hour pDNA transfection time, the transfection medium was replaced with fresh growth medium (150 μL). LsiR conjugates with different amounts of PEG (0-5%) were made as described in A. In each experiment, 3 doses of siRNA were selected (0.02 / 0.1 / 0.5 μg siRNA / well; 5/30/150 nM) and diluted with OptiMEM to give a final volume of 100 μL, which was then reconstituted with 150 μL of fresh growth medium. In addition to each containing well (48-well plate), cells were incubated for 16-72 hours prior to β-Gal reporter assay (Roche, UK).

Example 2
Stability of PEGylated siRNA lipoplexes
The stability of PEGylated (Mw 2000) siRNA lipoplex in 80% serum (FCS) was investigated. Surface pegylated LsiR complexes were made as described in Example 1 and FIG. 1 and incubated with FCS for different times before measuring particle size by photon correlation (PCS). The result is shown in figure 2. With increasing degree of PEG, the particle size does not increase over the time scale investigated.

  Advantageously, PEGylated siRNA lipoplexes made by post-coupling PEG to siRNA-loaded lipoplexes are serum stable with increasing amounts of PEG coupled to the surface. Presents.

Example 3
Tissue distribution experiments Liposome formulations were labeled with lipid [4- ¹⁴ C] cholesterol (Amersham Biosciences) at a final molar ratio of CDAN / DOPE / CPA700 / [4- ¹⁴ C] cholesterol 39.95: 50: 10: 0.05. SiRNA lipoplexes were made in the ratio liposome / siRNA 13: 1 (w / w) by adding siRNA to the liposomes under vortexing. Samples were concentrated to half of the total volume and made up to the original volume by adding PBS. 200 μl (0.1 mg / mL siRNA) of these complexes were injected into the lateral tail vein of each mouse weighing approximately 30 g. Radioactivity was adjusted to approximately 0.035 μCI per animal.

  One hour later, the mice were anesthetized and blood was obtained by cardiac puncture and mixed immediately with 15 U heparin. Assuming that the total blood weight was 6% of the body weight, the blood concentration of liposomes was calculated. After cervical dislocation, the liver, spleen, kidney, lung and heart were dissected and weighed. Organs were homogenized in PBS at a concentration of 5 mL PBS / g organ. An aliquot of 200 μL of each organ and 100 μL of blood was solubilized with Solvable at 60 ° C. for 1 hour. The sample was then treated with 0.1 mL EDTA (0.1 M) followed by treatment with 0.3 mL to 0.5 mL 30% hydrogen peroxide in a 0.1 mL aliquot. After 15-30 minutes at room temperature, the samples were further incubated at 60 ° C. for 1 hour. Then 10 mL of Ultima Gold ™ was added to each vial and radioactivity was measured using a liquid scintillation counter. The results shown in FIG. 3 are expressed as a percentage of the injected dose per organ (% ID).

  Pegylated siRNA lipoplexes generated by post-coupling PEG to siRNA-loaded lipoplexes exhibit a different pharmacokinetic profile than non-pegylated analogs, and the amount detected in the liver with increasing amounts of PEG is It gradually decreased.

Example 4
Down-regulation of lacZ gene in liver
200 μL of LsiR PEGylated with PEG ²⁰⁰⁰ (CHO) ₂ (0.1%) was injected intravenously into Balb / C mice and the animals rested for 8 hours, after which 1 μg pUMVC1 in 2.5 mL PBS within 10 seconds. -β-Gal was injected (hypermuscular injection). Animals were sacrificed 24 hours after pDNA injection and assayed for β-galactosidase expression by ELISA. pDNA alone and pDNA + LsiR (GFP control) give the same expression level of β-Gal, whereas LsiR (anti-β-Gal) mediates more than 80% of the β-Gal protein.

  The results are shown in FIG.

  Surprisingly, the down-regulation of the lacZ gene introduced into the liver of female Balb / C mice by hydrodynamic injection of 1 μg pDNA (in 2 ml PBS) was 20 μg after 8 or 24 hours of rheological injection. More than 80% was reached after systemic delivery of siRNA lipoplex (PEG 0.1%).

Example 5
Down-regulation of adenovirus-expressed β-Gal protein in pegylated LsiR lipoplex To examine the effect on the level of PEGylation on the down-regulation of adenovirus-expressed β-Gal protein, tail vein injection ( AdRSVβGal was injected via a total volume of 200 μL, and the animals were left for 2 hours before each injection of LsiR (anti-β-Gal) PEGylated with 0 / 0.1 / 5% PEG ²⁰⁰⁰ (CHO) ₂ . The results are shown in FIG. 5A.

To examine the effect of the adenoviral injection route, AdRSVβGal was injected intraperitoneally 8 hours after intravenous injection of LsiR lipoplex PEGylated with PEG ³⁴⁰⁰ (CHO) ₂ (5%) (200 μL total volume). ). Due to the different routes of AV injection, the total level of β-Gal protein was reduced to 10% of the level obtained by the intravenous route.

  The results are shown in FIG. 5B.

  Surprisingly, it was infected with a given dose of lacZ-adenovirus and 20 μg siRNA lipoplex (PEG 0% / 0.1% / 5%) via tail vein injection obtained with viral infection after 2 hours. Male Balb / C mice showed maximal down-regulation (> 70%) of the highest pegylated siRNA lipoplex (5% PEG).

Example 6
(A)
HPLC analysis
HPLC analysis of CDAN / CPA / DOPE (20:30:50, m / m / m) liposomes was performed. 30 μL of liposomes (3 mg / mL) were diluted with 70 μL of water and 90 μL of this solution was injected into the HPLC. Peaks were analyzed by an evaporative light scattering detector (ELS). The results are shown in FIG. 6A.

Antibody coupling to the surface of CDAN / CPA / DOPE (20:30:50, m / m / m) was also investigated. Dissolve 2 mg rabbit IgG (Sigma) in 1 mL NaOAc (20 mM), NaCl (0.15 M) pH 5.9; in a separate tube, prepare 1 mL fresh H ₅ IO ₆ and combine the two tubes And left at room temperature for 1.5 hours. Prior to introducing the entire reaction mixture into dialysis tubing (Spectrum Labs, USA; MWCO 12000-14000), the reaction was quenched by the addition of 0.5 mL ethylene glycol and against 0.1 MK ₂ HPO ₄ /0.1% Triton X. Dialyzed for 16 hours. The solution was collected and IgG ^OX concentration was measured by BCA assay. 80 μL CDAN / CPA / DOPE (20:30:50, m / m / m; 2 mg / mL) and 100 μL oxidized IgG ^OX (0.94 mg / mL) were incubated at 37 ° C./16 hours for analysis For this, 90 μL was injected into the HPLC. The results are shown in FIG. 6A. Compared to a control in which non-oxidized IgG was incubated with liposomes, the CPA peak at rt = 27 minutes was reduced by 48%. A new peak at rt = 36 minutes was observed with the strong absorbance at λ = 280 nm shown for the protein, which was isolated and analyzed on the SDS page and covalently bonded through the oxidized Fc-carbohydrate unit via an oxime bond It was found to be IgG with about 20 copies of CPA made.

Double incubation of liposomes with (i) PEG ²⁰⁰⁰ (CHO) ₂ followed by (ii) IgG ^OX was also performed. The results are shown in FIG. 6C. Surprisingly, the high reactivity of the aminoxy functional group of CPA lipids is due to incubation of CDAN / CPA / DOPE (20:30:50, m / m / m) liposomes with PEG ²⁰⁰⁰ (CHO) ₂ (1%). First, it is possible to further react with oxidized IgG ^OX to produce serum-protected liposomes that allow the antibody to be covalently conjugated to the surface of the PEGylated liposome (FIG. 6C (B)). . For this purpose, 30 μL of CDAN / CPA / DOPE (20:30:50, m / m / m) before the addition of 40, 60, 80 or 100 μL of oxidized IgG ^OX (0.94 mg / mL), respectively. ) Liposomes were incubated with ₂ μL of PEG ²⁰⁰⁰ (CHO) ₂ (10 ⁻⁸ mol) for 15 min / RT (FIG. 6C (B), 1-4). The reaction mixture was incubated for 16 hours and 90 μL was injected into the HPLC for analysis (FIG. 6C (A and B)). The CPA signal at rt = 27 min gradually decreased with increasing amounts of IgG ^OX (bottom panel), despite the prior incubation with PEG (CHO) ₂ , indicating the remaining free aminoxy of CPA lipids. The group is still reactive, indicating that the antibody is covalently co-ligated to the surface of the PEGylated liposome. Note that the HPLC signal at rt = 35 min shows a strong absorption at λ = 280 nm which represents the protein.

  LsiR lipoplexes made from siRNA and CDAN / DOPE / CPA (40/50/10; m / m / m) liposomes PEGylated with 0.1-1% total lipid (molar ratio in lipoplex) are Aminoxy liposomes can be incubated with oxidized IgG antibodies at acidic pH, so that before incubation with oxidized IgG (Figure 6a) and after incubation with oxidized IgG (Figure 6b) As shown by HPLC analysis, the antibody was covalently coupled to CPA lipids through its partially oxidized carbohydrate units.

(B)
(i) Preparation of liposomes and IgG
(CDAN / DOPE / CPA):
164 μL DOPE (9.05 mg / ml, 744 g / mol), 279 μL CDAN3HCl (3.88 mg / mL, 680 g / mol) and 107 μL CPA (4 mg / mL, 1075 g / mol) in a 5 mL round bottom flask Mix and evaporate the solvent at about 30 ° C. to form a dry lipid film. Multilamellar liposomes were made by adding 1 mL of water and vortexing for 1 minute. Liposome samples were sonicated for 20 minutes to make small <100 nm sized monolayer vesicles.

CDAN / DOPE / CPA: DSPE ^rhod :
159 μL DOPE (9.05 mg / ml, 744 g / mol), 277 μL CDAN3HCl (3.88 mg / mL, 680 g / mol), 45 μL CPA (8 mg / mL, 1075 g / mol) and 25.8 μL DSPE-rhodamine ( 2 mg / mL, 1301 g / mol) were mixed in a 5 mL round bottom flask. The solvent was evaporated at about 30 ° C. to form a dried lipid film. Multilamellar liposomes were made by adding 1 mL of water and vortexing for 1 minute. Liposome samples were sonicated for 20 minutes to obtain small monolayer vesicles <100 nm size.

IgG oxidation:
(a) 260 μL of IgG stock (0.38 mg / mL) and 260 μL of periodic acid (20 mM in water) were combined and left at room temperature in the dark for 30 minutes. The sample was then desalted in a NAP-5-column. After oxidation, different IgG dilutions were prepared (0 μL to 170 μL oxidized IgG filled with water to a total volume of 170 μL) and mixed with 30 μL liposomes (3 mg / mL). Samples of 40 μg protein were analyzed on SDS page gel (FIG. 6d, A1, lane 2).

  (b) 200 μL of IgG stock (0.38 mg / mL) and 200 μL of periodic acid (20 mM in water) were combined and left at room temperature in the dark for 1 hour. The sample was then dialyzed against sodium acetate buffer (20 mM sodium acetate and 150 mM NaCl, pH 5.5) for 2 hours. After oxidation, different IgG dilutions were prepared (0 μL to 170 μL oxidized IgG filled with water to a total volume of 170 μL) and mixed with 30 μL liposomes. Samples of 40 μg protein were analyzed on SDS page gel (FIG. 6d, A1, lane 3).

  (c) 250 μL of IgG stock (0.38 mg / mL) and 250 μL of periodic acid (20 mM in water) were combined and left at room temperature in the dark for 2 hours. The sample was then dialyzed against sodium acetate buffer (20 mM sodium acetate and 150 mM NaCl, pH 5.5) for 2 hours. After oxidation, different IgG dilutions were prepared (0 μL to 70 μL oxidized IgG filled with water to a total volume of 70 μL) and mixed with 30 μL liposomes. A sample of 40 μg protein was analyzed on SDS page gel (FIG. 6d, A1, lane 4).

IgG ^OX fluorescent label:
70 μL (0.2 mg / mL) antibody was fluorescently labeled by incubating with 50 μL NHS-FITC (10 mg / mL, DMSO). Purification was achieved with a slide-A-Lyzer minidialysis unit, 10000 MWCO. The protein concentration was measured by the BCA test and found to be 0.11 mg / mL.

ELISA:
To each well of the NUNC-Immuno plate, 5 μL of HFN (1 mg / mL) and 40 μL of Tris buffer (NaCl 0.25M, Tris 0.02M, pH 7.6) were added and the plate was incubated for 30 minutes at room temperature. The plate was then washed 3 times with wash buffer (Tris 0.02M, NaCl 0.5M, Triton 0.5%, pH 7.6). The wells were blocked by adding 3 μL BSA (1 mg / mL) and 40 μL Tris buffer to each well. The plates were then incubated for 1 hour at 37 ° C. Following this, plates were added with 75 μL of different dilutions of oxidized IgG (0.18 mg / mL) between 0-60 pmol, or IgG ^OX -coupled lipoplex (0.11 mg / mL, protein). Plates were incubated at 37 ° C. for 1 hour and then washed. Next, 50 μL of sheep anti-mouse antibody coupled to horseradish peroxidase (1: 5000 dilution) was added to each well. Plates were incubated for an additional hour at 37 ° C and washed. Finally, 50 μL of SIGMA FAST ™ OPD substrate was added to each well and the absorbance at 405 nm was measured with a plate reader.

(ii) Lipoplex formation Lipoplex 1; CDAN / DOPE / CPA and Cy3-GFP labeled siRNA
100 μL of CDAN / DOPE / CPA liposome stock (3 mg / mL) was mixed with 125 μL of water. 75 μL of siRNA Cy3-GFP (0.4 mg / mL) was slowly added to the solution while the mixture was vortexed. 53 μL of PEG ²⁰⁰⁰ (CHO) ₂ (0.135 mg / mL) was added to the lipoplex and incubated for 1 hour. 100 μL of IgG ^OX- (FITC) sample (0.11 mg / mL) was added to the mixture and incubated for 3 hours.

^Lipoplex 2; Liposome ^rhod and unlabeled siRNA
30 μL of liposomal ^rhod stock (3 mg / mL) was pipetted into a plastic Eppendorf tube and 23 μL of siRNA (0.4 μg / μL) was added to the liposomes with gentle vortexing. 53 μL of PEG ²⁰⁰⁰ (CHO) ₂ (0.135 mg / mL) was then added to the lipoplex. 100 μL of IgG ^OX- (FITC) sample (0.11 mg / mL) was added to the mixture and incubated for 3 hours.

  The lipoplexes were purified in a SORVALL RC M150 GX centrifuge with reverse sucrose gradients (20%, 10%, 5% and 0%) at 45000 rpm for 1.5 hours. (Figure 6d, B). A pink layer was removed from each sample, dialyzed in a slide-A-Lyzer minidialysis unit (10000 MWCO, Pierce) and concentrated under reduced pressure to a final volume of 200 μL. The protein concentration in the sample was measured using the BCA assay. 30 μL of each sample (0.11 mg / mL protein) was lyophilized and redissolved in 15 μL of loaded dye. The sample was then run on a 12% Tris-Glycine SDS page gel (Figure 6D, A2, lane 3/4). Lipoplexes were also purified by FPLC on Sepharose CL 6B, 20, using a 0.6 cm column (0.2 MPa, flow rate 0.4 mL / min, sensitivity 0.05 / 0.1). Sodium acetate buffer (20 mM sodium acetate and 150 mM NaCl, pH 5.5) was used for separation and the detector was set at 280 nm. The collected fractions were then concentrated with rotavap and the PCS was measured. Typically, two fractions are collected, the first giving a 200 nm lipoplex size, the second giving a smaller fraction lipoplex size of 10000 nm, which are aggregated LsiR-IgG lipoproteins. Plex (FIG. 6D, lanes 2 and 3, respectively).

(iii) Results The integrity and activity of the antibody after oxidation were confirmed by SDS page and ELISA, respectively (FIG. 6D). The number of oxidized antibodies can be controlled by the incubation time in periodic acid and the concentration of periodic acid. Typically, a 1 hour incubation in 10 mM periodic acid provides a sufficient number of oxidized carbohydrates to effectively couple to CPA lipids in both liposomes and lipoplexes without compromising activity. It is clear from FIG. 6D / A1 that the antibody is not damaged even with increasing incubation time. After coupling of IgG ^OX to lipoplexes and FPLC, or sucrose gradient purification, SDS is slightly above the 50 kD molecular weight ladder that can be attributed to IgG Fc fragments coupled with different amounts of CPA lipids. Two different bands are revealed (Figure 6D, lane 2/3). The LsiR-IgG lipoplex ELISA shows a sufficient number of lipoplex-coupled antibodies.

Example 7
Cholesterylamine 2
Cholesteryl chloroformate 1 (7.5 g, 0.0167 mol) was dissolved in ethylene-1,2-diamine (180 ml) and the mixture was stirred for 18 hours. The reaction was quenched with water and extracted with dichloromethane. The organic extract was dried (MgSO ₄ ) and the solvent removed in vacuo to give a residue which was flash column chromatographed [CH ₂ Cl ₂ / MeOH / NH ₃ = 192: 7: 1 followed by CH ₂ Cl ₂ / MeOH / NH ₃ = 92: 7: 1 (v / v)] to give pure product 2 (5.5 g, 73%) as a white solid (mp 175-177 ° C.): FTIR (Nujol mull), ν _max [cm ⁻¹ ] 3338 (amine), 2977 (alkane), 2830 (alkane), 1692 (carbamate).

Example 8
Boc-aminoxycholesteryl lipid 3
Boc-amine-oxyacetic acid (145 mg, 0.758 mmol) in anhydrous dichloromethane was treated sequentially with DMAP (292 mg, 2.39 mmol), HBTU (373 mg, 0.987 mmol) and amine 2 (272 mg, 0.576 mmol), and the mixture The mixture was stirred at room temperature for 15 hours under a nitrogen atmosphere. The reaction was quenched with 7% aqueous citric acid and extracted with dichloromethane. The dried (MgSO ₄ ) extract was concentrated in vacuo to give a residue that was purified by flash column chromatography (gradient 20% ethyl acetate / hexanes to 65% ethyl acetate / hexanes) to give pure Boc- Aminoxycholesteryl lipid 3 (302 mg, 81%) was obtained.

Example 9
Cholesteryl aminoxy lipid 4
Boc-aminoxycholesteryl lipid 3 (86 mg, 0.067 mmol) in propan-2-ol (3 ml) was then treated with 4M HCl in dioxane (3 ml) and the mixture was stirred at room temperature for 3 hours. The solvent was removed in vacuo to give aminoxy lipid 4 (37 mg, 98%).

Example 10
Synthesis of CPA Compound The synthesis of cholesteryl- (dPEG ₄ ) ₂ -aminoxylipid (CPA) 6 was completed in two steps.
1) Solid phase synthesis of short protected PEG-aminoxy linker 3 (PABoc, Scheme 1) and
2) Solution phase coupling of cholesteryl-amine (C) to PABoc3 (Scheme 2).

PABoc3 was synthesized with 2-chlorotrityl polystyrene resin [PS-chlorotrityl-Cl] (Argonaut, USA) using the standard peptide Fmoc solid phase method. First, a short PEG linker, N-Fmoc-amide-dPEG ₄ ™ -acid (Quanta BioDesign, Inc., USA) was loaded onto the resin under basic conditions, the Fmoc protecting group was subsequently removed with piperidine, Amine 1 was obtained (Scheme 1). The N-Fmoc-amido-dPEG ₄ ™ -acid unit was then coupled to 1 using HBTU coupling reagent (Novabiochem, UK) and the Fmoc group was subsequently deprotected again. The resulting amine was coupled to N-Boc-aminooxyacetic acid (Novabiochem, UK) under HBTU conditions to give resin-bound PABoc2, which was then cleaved from the resin under mild acidic conditions to give the composition PABoc3 Which was considered pure enough to continue in the next step without further purification (TLC).

スキーム1：Boc-アミノキシ-(dPEG₄)₂-CO₂Hの合成
試薬および条件：a)N-Fmoc-アミド-dPEG₄(商標)-酸(3当量)、DMF中のヒューニッヒ(Hunig)塩基(5当量)、2時間、室温；b)DMF中の20%ピペリジン(3×5分)、室温；c)N-Fmoc-アミド-dPEG₄(商標)-酸(3当量)、HBTU(5当量)、DMF中のヒューニッヒ塩基(5当量)、1時間、室温；d)DMF中の20%ピペリジン(3×5分)、室温；e)Boc-アミノ-オキシ酢酸(3当量)、HBTU(5当量)、DMF中のヒューニッヒ塩基(5当量)、1時間、室温；およびf)DCM中の50%1,1,1-トリフルオロエタノール、1時間、室温。

Scheme 1: Boc-aminoxy- (dPEG ₄ ) ₂ —CO ₂ H synthesis reagents and conditions: a) N-Fmoc-amide-dPEG ₄ ™ -acid (3 eq), Hunig base in DMF (5 eq), 2 h, room temperature; b) 20% piperidine in DMF (3 × 5 min), room temperature; c) N-Fmoc-amido-dPEG ₄ ™ -acid (3 eq), HBTU (5 Eq.), Hunig's base in DMF (5 eq), 1 h, room temperature; d) 20% piperidine in DMF (3 × 5 min), rt; e) Boc-amino-oxyacetic acid (3 eq), HBTU ( 5 equivalents), Hunig's base in DMF (5 equivalents), 1 hour, room temperature; and f) 50% 1,1,1-trifluoroethanol in DCM, 1 hour, room temperature.

Completion of the synthesis of CPA is shown in Scheme 2. Briefly, commercially available cholesteryl chloroformate (Aldrich, UK) was treated with excess ethylenediamine in pure form to give cholesteryl-amine 4. PABoc3 was then coupled to amine 4 using HBTU as a coupling reagent to give Boc-protected cholesteryl- (dPEG ₄ ) ₂ -aminoxy 3 (71% yield). Deprotection of the Boc group with 4M HCl in dioxane yielded CPA [cholesteryl- (dPEG ₄ ) ₂ -aminoxylipid, 6] (> 97% yield according to analytical HPLC) without further purification. Used in physics experiments.

スキーム2：CPA脂質の合成
試薬および条件：a)エチレンジアミン(大過剰)、室温、18時間、75%；b)Boc-アミノ-オキシ酢酸、HBTU、DMAP、塩化メチレン、室温、18時間、81%；およびc)4M HCl/ジオキサン、プロパン-2-オール、3時間、99%。

Scheme 2: CPA lipid synthesis reagents and conditions: a) ethylenediamine (large excess), room temperature, 18 hours, 75%; b) Boc-amino-oxyacetic acid, HBTU, DMAP, methylene chloride, room temperature, 18 hours, 81% And c) 4M HCl / dioxane, propan-2-ol, 3 hours, 99%.

Synthesis of Cholesteryl-Gly-PEG (n = 11) -hydrazide (CP ₁₁ Hyd) The synthesis of cholesteryl-Gly-PEG (n = 11) -hydrazide (CP ₁₁ Hyd) is shown in Scheme 3. Treatment of cholesterol chloroformate 7 with glycine 8 gives cholesteryl glycine 9 in good yield. Next, in the presence of DMAP, O- (2-aminoethyl) -O- [2- (Boc-amino) ethyl] decaethylene glycol was coupled to 9 using the peptide coupling agent HBTU, and Boc -Protected cholesteryl-glycine-PEG _{n = 11} -amine 10 is obtained. Removal of the Boc group with TFA yielded the free amine 11, which this time used polystyrene coupled DCC-derived resin PS-carbodiimide (Argonaut, UK) as the coupling reagent and N ^h -tert-butyloxycarbonyl-succinic acid. The acid monohydrazide 14 was pure enough to be used immediately in coupling. Therefore, Boc-protected hydrazide 12 was obtained in good 68% yield in two steps. Treatment of 4 with 4M HCl in dioxane or TFA provided the desired hydrazide 13 smoothly in 54% yield.

スキーム3：CP₁₁Hydの合成
試薬および条件：a)Et₃N(1.2当量)、ジオキサン/水、12時間、室温、63%；b)O-(2-アミノエチル)-O-[2-(Boc-アミノ)エチル]デカエチレングリコール、HBTU、DMAP、DCM、2日、室温、97%；c)TFA/DCM(1:1)、1時間、室温；d)N^h-tert-ブチルオキシカルボニル-コハク酸モノヒドラジド14、Et₃N、PS-カルボジイミド、DCM、24時間、室温、2工程で68%；e)ジオキサン、2-プロパノール中4M HCl、室温、3時間、54%。

Scheme 3: CP ₁₁ Hyd synthesis reagents and conditions: a) Et ₃ N (1.2 eq), dioxane / water, 12 h, room temperature, 63%; b) O- (2-aminoethyl) -O- [2- (Boc-amino) ethyl] decaethylene glycol, HBTU, DMAP, DCM, 2 days, room temperature, 97%; c) TFA / DCM (1: 1), 1 hour, room temperature; d) N ^h -tert-butyloxy Carbonyl-succinic acid monohydrazide 14, Et ₃ N, PS-carbodiimide, DCM, 24 hours, room temperature, 68% over 2 steps; e) Dioxane, 4M HCl in 2-propanol, room temperature, 3 hours, 54%.

Experimental Method Materials and Methods
Boc-amino-oxyacetic acid and HBTU were obtained from Novabiochem (CN Biosciences, UK). N-Fmoc-amide-dPEG ₄ ™ -acid was purchased from Quanta BioDesign Ltd. (Powell, OH, USA). PS-carbodiimide and PS-chlorotrityl-Cl resin were obtained from Argonaut Technologies, Inc. (Foster City, CA, USA). All other chemicals were purchased from Sigma Aldrich (Dorset, UK) unless otherwise noted. Dried dichloromethane was distilled over phosphorus pentoxide; other solvents were pre-dried from Sigma-Aldrich (Dorset, UK) or BDH Laboratory Supplies (Poole, UK) or purchased if necessary. HPLC-grade acetonitrile was purchased from Fisher Chemicals (Leicester, UK) and other HPLC-grade solvents were purchased from BDH Laboratory Supplies (Poole, UK). Thin layer chromatography (TLC) is performed on pre-coated Merck-Kieselgel 60 F ₂₅₄ aluminum-backed plates and, where appropriate, ultraviolet light, iodine, acidic ammonium molybdate (IV), acidic ethanolic vanillin, or other reagents. And made it visible. Flash column chromatography was achieved with Merck-Kieselgel 60 (230-400 mesh). Weight spectra were recorded using a Bruker Esquire 3000, VG-7070B or JEOL SX-102 instrument. ¹ H and ¹³ C NMR were recorded on an Advance Brucker 400 UltrashieldTM machine with residual isotope solvent as internal reference (s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, br = Broad singlet). Analytical HPLC (Hitachi-LaChrom L-7150 pump system with Polymer Laboratories PL-ELS 1000 Evaporative Light Scattering Detector) has a gradient of 0.1% aqueous TFA to 100% acetonitrile (0.1% TFA) [0-15 min], then , 100% acetonitrile (0.1% TFA) [15-25 min] followed by 100% methanol [25-45 min] on a Vydac C4 peptide column.

Boc-アミノキシ-(dPEG₄)₂-CO₂H 3は標準的なペプチド固相合成戦略を用いて合成した：塩化クロロトリチル樹脂(1.27mmol/g負荷、55mg、0.070mmol)をDcm中で16時間膨潤させた。第一の酸を、樹脂を、DMF(15ml)中のN-Fmoc-アミド-dPEG₄(商標)-酸(102mg、0.209mmol)およびヒューニッヒ塩基(60μl、0.349mmol)で1時間処理することによって樹脂に負荷した。Fmoc脱ブロッキングは、DMF中のピペリジン(20%)(2×5分)を用い、続いて、DMFで十分に洗浄することによって達成した。次に、得られた樹脂結合遊離アミンを、DMF(15ml)中のヒューニッヒ塩基(60μl、0.349mmol)中のHBTU(132.5mg、0.209mmol)で1時間活性化したN-Fmoc-アミド-dPEG₄(商標)-酸(102mg、0.209mmol)と反応させた。(各カップリング工程では、3当量のアミノ酸、5当量のDIEAおよび3当量のHBTUを用いた。各カップリングは1時間行い、続いて、3当量のDIEAの存在下で、DMF中の無水酢酸(10%)でキャッピングした。最後に、Boc-アミノ-オキシ酢酸(40mg)をカップリングさせて樹脂結合生成物を得た。DCM中の50%トリフルオロエタノールからなる3mLの溶液を用い、化合物を4時間にわたって切断して、粗製残渣(40mg、0.058mmol)を得た。

Boc-aminoxy- (dPEG ₄ ) ₂ -CO ₂ H 3

Boc-aminoxy- (dPEG ₄ ) ₂ —CO ₂ H 3 was synthesized using a standard peptide solid-phase synthesis strategy: chlorotrityl chloride resin (1.27 mmol / g loading, 55 mg, 0.070 mmol) in 16 cm in Dcm. Swelled for hours. A first acid, the resin, DMF (15 ml) N-Fmoc-amide-dPEG ₄ in (TM) - acid (102 mg, 0.209 mmol) and Hunig's base (60 [mu] l, 0.349 mmol) by 1 hour at The resin was loaded. Fmoc deblocking was achieved by using piperidine (20%) in DMF (2 × 5 min) followed by extensive washing with DMF. The resulting resin bound free amine was then activated with N-Fmoc-amide-dPEG ₄ activated with HBTU (132.5 mg, 0.209 mmol) in Hunig's base (60 μl, 0.349 mmol) in DMF (15 ml) for 1 hour. Reacted with (trademark) -acid (102 mg, 0.209 mmol). (In each coupling step, 3 equivalents of amino acid, 5 equivalents of DIEA and 3 equivalents of HBTU were used. Each coupling was performed for 1 hour followed by acetic anhydride in DMF in the presence of 3 equivalents of DIEA. (10%) Finally, Boc-amino-oxyacetic acid (40 mg) was coupled to give the resin bound product, using 3 mL solution consisting of 50% trifluoroethanol in DCM, Was cleaved over 4 hours to give a crude residue (40 mg, 0.058 mmol).

クロロギ酸コレステリル1(7.5g、0.0167mol)をエチレン-1,2-ジアミン(180ml)に溶解し、混合物を18時間攪拌した。反応を水でクエンチし、ジクロロメタンで抽出した。有機抽出物を乾燥し(MgSO₄)、溶媒を真空中で除去して残渣を得、これをフラッシュカラムクロマトグラフィー[CH₂Cl₂:MeOH:NH₃ 192:7:1→CH₂Cl₂:MeOH:NH₃ 92:7:1(v/v)]によって精製して、純粋な生成物2(5.5g、0.0116、73%)を白色固体(mp 175〜177℃)として得た：FTIR(ヌジョールマル)ν_max 3338(アミン)、2977(アルカン)、2830(アルカン)、1692(カルバマート)cm^-1。

Cholesteryl-amine 4

Cholesteryl chloroformate 1 (7.5 g, 0.0167 mol) was dissolved in ethylene-1,2-diamine (180 ml) and the mixture was stirred for 18 hours. The reaction was quenched with water and extracted with dichloromethane. The organic extract was dried (MgSO ₄ ) and the solvent removed in vacuo to give a residue that was flash column chromatographed [CH ₂ Cl ₂ : MeOH: NH ₃ 192: 7: 1 → CH ₂ Cl ₂ : MeOH: NH ₃ 92: 7: 1 (v / v)] to give pure product 2 (5.5 g, 0.0116, 73%) as a white solid (mp 175-177 ° C.): FTIR ( Nujolmar) ν _max 3338 (amine), 2977 (alkane), 2830 (alkane), 1692 (carbamate) cm ⁻¹ .

無水ジクロロメタン中のBoc-アミノキシ-(dPEG₄)₂-CO₂H(40mg、0.058mmol)を、順次、DMAP(22mg、0.18mmol)、HBTU(24mg、0.063mmol)およびコレステリルアミン2(28mg、0.0.06mmol)で処理し、混合物を窒素雰囲気下で室温にて15時間攪拌した。反応を7%クエン酸水溶液でクエンチし、ジクロロメタンで抽出した。乾燥した(MgSO₄)抽出物を真空中で濃縮して残渣を得、これをフラッシュカラムクロマトグラフィー(勾配DCM:MeOH:H₂O)によって精製し、純粋なBoc-アミノキシ-(dPEG₄)₂-コレステリル脂質3(47mg、0.0411mmol、71%)を得た。

Boc-aminoxy- (dPEG ₄ ) ₂ -cholesteryl lipid (BocCPA) 5

In anhydrous dichloromethane Boc- aminoxy _{- (dPEG 4) 2 -CO 2} H (40mg, 0.058mmol) and, sequentially, DMAP (22mg, 0.18mmol), HBTU (24mg, 0.063mmol) and cholesteryl amine 2 (28 mg, 0.0 .06 mmol) and the mixture was stirred at room temperature for 15 hours under a nitrogen atmosphere. The reaction was quenched with 7% aqueous citric acid and extracted with dichloromethane. The dried (MgSO ₄ ) extract was concentrated in vacuo to give a residue, which was purified by flash column chromatography (gradient DCM: MeOH: H ₂ O) to give pure Boc-aminoxy- (dPEG ₄ ) ₂ -Cholesteryl lipid 3 (47 mg, 0.0411 mmol, 71%) was obtained.

次いで、プロパン-2-オール(2ml)中のBoc-アミノキシ-(dPEG₄)₂-コレステリル脂質3(40mg、0.035mmol)をジオキサン(2ml)中の4M HClで処理し、混合物を室温にて3時間攪拌した。溶媒を真空中で除去して、CPA脂質4(37mg、98%)を得た。

分析HPLC：1ピーク、RT31分。 Cholesteryl- (dPEG ₄ ) ₂ -aminoxylipid 6 (CPA)

Boc-aminoxy- (dPEG ₄ ) ₂ -cholesteryl lipid 3 (40 mg, 0.035 mmol) in propan-2-ol (2 ml) was then treated with 4M HCl in dioxane (2 ml) and the mixture was stirred at room temperature for 3 minutes. Stir for hours. The solvent was removed in vacuo to give CPA lipid 4 (37 mg, 98%).

Analytical HPLC: 1 peak, RT 31 minutes.

0℃のジオキサン(35ml)中のクロロギ酸コレステロール7(1g、2.23mmol)およびトリエチルアミン(424μl、2.9mmol)に、水(15ml)中に溶解したグリシン8を加えた。混合物を室温にて12時間攪拌し、次いで、反応を7%クエン酸水溶液でクエンチし、水性層をジクロロメタンで抽出し、有機抽出物を乾燥し(MgSO₄)、真空中で濃縮した。得られた粗製残渣をフラッシュカラムクロマトグラフィー(EtOAc/ヘキサン)によって精製して、生成物を白色粉末(680mg、1.39mmol、63%)として得た。

Cholesteryl-glycine 9

Glycine 8 dissolved in water (15 ml) was added to cholesterol chloroformate 7 (1 g, 2.23 mmol) and triethylamine (424 μl, 2.9 mmol) in dioxane (35 ml) at 0 ° C. The mixture was stirred at room temperature for 12 hours, then the reaction was quenched with 7% aqueous citric acid, the aqueous layer was extracted with dichloromethane, the organic extract was dried (MgSO ₄ ) and concentrated in vacuo. The resulting crude residue was purified by flash column chromatography (EtOAc / hexane) to give the product as a white powder (680 mg, 1.39 mmol, 63%).

コレステリル-グリシン9(150mg、0.309mmol)、O-(2-アミノエチル)-O-[2-(Boc-アミノ)エチル]デカエチレングリコール(Fluka, UK)(198mg、0.307mmol)、HBTU(117mg、0.309mmol)およびDMAP(114mg、0.927mmol)を無水ジクロロメタン(50ml)に溶解し、窒素雰囲気下で2日間攪拌した。反応を7%クエン酸水溶液でクエンチし、水性層をジクロロメタン/MeOH混合物で抽出し、有機抽出物を乾燥し(MgSO₄)、真空中で濃縮した。得られた粗製残渣をフラッシュカラムクロマトグラフィー(CHCL3/MeOH/H2O)によって精製して、生成物(335mg；0301mmol、97%)を得た。

HPLC R_T=27分(C-4カラム)；ES-MS 1136.4[M+Na]^＋。 Cholesteryl-gly-PEG ₁₁ -NHBoc 10

Cholesteryl-glycine 9 (150 mg, 0.309 mmol), O- (2-aminoethyl) -O- [2- (Boc-amino) ethyl] decaethylene glycol (Fluka, UK) (198 mg, 0.307 mmol), HBTU (117 mg , 0.309 mmol) and DMAP (114 mg, 0.927 mmol) were dissolved in anhydrous dichloromethane (50 ml) and stirred for 2 days under nitrogen atmosphere. The reaction was quenched with 7% aqueous citric acid, the aqueous layer was extracted with a dichloromethane / MeOH mixture, the organic extract was dried (MgSO ₄ ) and concentrated in vacuo. The resulting crude residue was purified by flash column chromatography (CHCL3 / MeOH / H2O) to give the product (335 mg; 0301 mmol, 97%).

HPLC R _T = 27 min (C-4 column); ES-MS 1136.4 [M + Na] ⁺ .

TFA；DCM(5ml:5ml)中のコレステリル-gly-PEG₁₁-NHBoc10(300mg)を室温にて1時間攪拌した。溶媒を真空中で除去してアミン11を得、これをさらに精製することなく用いた(スキーム3参照)。従って、アミン11(60mg、0.059mmol)、N^h-tert-ブチルオキシカルボニル-コハク酸モノヒドラジド14(232mg、0.118mmol)(Dietzgen et al.；Z. Naturforsch. B；42, 4, (1987), pp.441〜453に従って合成)およびトリエチルアミン(16μl)およびポリスチレン結合DCC樹脂(負荷1.27mmol/g；140mg；0.1777mmol)、(Argonaut Technologies, UK)をジクロロメタン(10ml)中で24時間攪拌した。樹脂を濾過によって除去し、濾液を真空中で濃縮して残渣を得、これをフラッシュカラムクロマトグラフィー(クロロホルム/メタノール/水)によって精製して、Boc-保護ヒドラジド12(49mg、0.040mmol、68%)を得た。

HPLC R_T=27分(C-4カラム)；ES-MS 1250.3[M+Na]⁺。 Cholesteryl -gly-PEG ₁₁ - (Boc- hydrazide) 12

TFA: Cholesteryl-gly-PEG ₁₁ -NHBoc10 (300 mg) in DCM (5 ml: 5 ml) was stirred at room temperature for 1 hour. The solvent was removed in vacuo to give amine 11, which was used without further purification (see Scheme 3). Thus, amine 11 (60 mg, 0.059 mmol), N ^h -tert-butyloxycarbonyl-succinic acid monohydrazide 14 (232 mg, 0.118 mmol) (Dietzgen et al .; Z. Naturforsch. B; 42, 4, (1987) , pp.441-453) and triethylamine (16 μl) and polystyrene-bound DCC resin (load 1.27 mmol / g; 140 mg; 0.1777 mmol), (Argonaut Technologies, UK) were stirred in dichloromethane (10 ml) for 24 hours. The resin was removed by filtration and the filtrate was concentrated in vacuo to give a residue that was purified by flash column chromatography (chloroform / methanol / water) to give Boc-protected hydrazide 12 (49 mg, 0.040 mmol, 68% )

HPLC R _T = 27 min (C-4 column); ES-MS 1250.3 [M + Na] ⁺ .

コレステリル-gly-PEG₁₁-(Boc-ヒドラジド)12(40mg、0.0326mmol)を2-プロパノール(3ml)に溶解し、次いで、ジオキサン(3ml)中の4M HClで処理した。混合物を3時間攪拌し、真空中で濃縮した。残渣をMeOHに対するエーテル沈殿によって精製して、灰色がかった白色ガム(20mg、0.0177mmol、54%)を得た。

HPLC R_T=27分(C-4カラム)；ES-MS 1150.3[M+Na]⁺。 Cholesteryl-gly-PEG ₁₁ -hydrazide 13 (CP ₁₁ hyd)

Cholesteryl -gly-PEG ₁₁ - (Boc- hydrazide) 12 (40mg, 0.0326mmol) was dissolved in 2-propanol (3 ml), then treated with 4M HCl in dioxane (3 ml). The mixture was stirred for 3 hours and concentrated in vacuo. The residue was purified by ether precipitation against MeOH to give an off-white gum (20 mg, 0.0177 mmol, 54%).

HPLC R _T = 27 min (C-4 column); ES-MS 1150.3 [M + Na] ⁺ .

Example 11
LsiR lipoplex lyophilized and rehydrated
Multilamellar liposomes were prepared in deionized water by hydrating lyophilized powder of 3 mg / mL lipid CDAN / DOPE (50:50, m / m). The siRNA was diluted in water and slowly added to the liposomes with vortexing to obtain a LsiR complex with a final siRNA concentration of 20 μg / mL (siRNA) at a lipid / siRNA ratio of 12: 1 (w / w) . Prepare 30% (w / v) sucrose, trehalose and lactose (Sigma, UK) stock solutions and add appropriate volumes of these to the LsiR lipoplexes, respectively, 5, 10 and 20% (w / v) A final concentration of was obtained. The LsiR concentration in complex formation varied depending on the amount of cryoprotectant used. For example, LsiR without cryoprotectant was prepared at 20 μg / mL, whereas 20% cryoprotectant containing LsiR was prepared at 60 μg / mL. Lyophilize 25 μL of lipoplex (0.5 μg siRNA) and hydrate the resulting powder in water at 20 μg / mL (25 μL) or 5 μg / mL (100 μL) and vortex And left at room temperature for 15 minutes. Sizing these LsiR particles by PCS showed that the particles had the same size as before the lyophilization / rehydration process.

pDNA transfection was performed using 0.2 μg / well of pDNA / well as described. These lyophilized / rehydrated 0.1 μg / well of LsiR were incubated for 3 hours at 37 ° C./10% CO ₂ 3 hours after pDNA transfection. Briefly, for 20 μg / mL (siRNA) fresh LsiR and lyophilized / rehydrated LsiR, 5 μL of each complex is added to 250 μL of complete growth medium in each well of the growing cells. added. For 5 μg / mL (siRNA) thawed / rehydrated LsiR, 20 μL of complex was added to 250 μL of complete growth medium in each well of growing cells. After 3 hours of incubation, siFECTion medium was removed and replaced with 400 μL of fresh complete growth medium.

  The results are shown in FIG.

  Surprisingly, LsiR lipoplexes lyophilized in the presence of trehalose and rehydrated in either 25 or 100 μL water down-regulates the lacZ reporter gene over freshly prepared LsiR lipoplexes Just good to do.

  All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology and molecular biology or related fields are intended to be within the scope of the following claims. Is done.

References

(a) PEGylated siRNA lipoplexes resulting from post-coupling of PEG to siRNA-loaded lipoplexes are produced by the oxime bond formed between the aldehyde group of PEG and the aminoxy functional group of aminoxylipid CPA. Irreversible coupling to the surface of siRNA lipoplexes mediates specific down-regulation of specifically targeted genes in vitro in a dose-response dependent manner. (b) This specific down-regulation of specific genes depends on the amount of PEG coupled to the surface of the siRNA lipoplex. Pegylated siRNA lipoplexes generated by post-coupling PEG to siRNA-loaded lipoplexes exhibit serum stability with increasing amounts of PEG coupled to the surface. PEGylated siRNA lipoplexes generated by post-coupling PEG to siRNA-loaded lipoplexes exhibit a different pharmacokinetic profile than non-pegylated analogs, and amounts detected in the liver with increasing amounts of PEG Reduce gradually. Surprisingly, the down-regulation of the lacZ gene introduced into the liver of female Balb / C mice by hydrodynamic injection of 1 μg pDNA (in 2 ml of PBS) is 8 or 24 hours after hydrodynamic injection. More than 80% was reached after systemic delivery of 20 μg siRNA lipoplex (PEG 0.1%). Even more unexpectedly, infected with a given dose of lacZ-adenovirus, 2 hours after virus infection, 20 μg siRNA lipoplex (PEG 0% / 0.1% / 5% via tail vein injection) ) Obtained male Balb / C mice showed the greatest down-regulation (> 70%) of the highest PEGylated siRNA lipoplex (5% PEG). LsiR lipoplexes made from siRNA PEGylated with 0.1-1% total lipid (molar ratio in lipoplex) and CDAN / DOPE / CPA (40/50/10; m / m / m) liposomes have an acidic pH By means of HPLC analysis of aminoxy liposomes before incubation with oxidized IgG (Figure 6a) and after incubation with oxidized IgG (Figure 6b). As shown, covalent coupling of the antibody to its CPA lipid via its partially oxidized carbohydrate unit was effected. Double incubation of liposomes with (i) PEG ²⁰⁰⁰ (CHO) ₂ followed by (ii) IgG ^OX was also performed (FIG. 6c). Referring to FIG. 6d, FIG. 6d (A1) shows the result of SDS PAGE gel (12.5% Tris / Glycine). Mw, molecular weight BenchMark ™ Protein Ladder (Invitrogen); lane 1, native non-oxidized human fibronectin (HFN) IgG; lane 2, oxidation of HFN-IgG to 30 mM / 10 mM periodic acid; lane 3, 60 min Oxidation of HFN-IgG for 10 mM periodic acid; Lane 4, 120 min / 10 HFN-IgG oxidation for 10 mM periodic acid; gel was stained with Coomassie blue. FIG. 6d (A2) shows the results of the SDS page gel (12.5 Tris / Glycine). Lane 1, natural non-oxidized HFN-IgG; lane 2, HFN-IgG ^OX and LsiR lipoplex covalently coupled after FPLC purification, fraction 1; lane 3, HFN-IgG ^OX covalently coupled after FPLC purification LsiR lipoplex, fraction 2. Both FPLC fractions contain antibodies whose Fc fragment migrates above the 50 kD molecular weight band, which is indicative of CPA-lipid coupled to the oxidized carbohydrate of the Fc unit. Due to the different size of the lipoplex in the second fraction, which exhibits a much higher particle size (10000 nm) compared to the first fraction (200 nm), indicating the state of aggregation, two bands occur in FPLC . The gel was silver stained using standard techniques to visualize protein bands. FIG. 6d (B) shows the LsiR lipoplex results with HFN-IgG ^OX covalently coupled after a sucrose gradient. The fluorescent band (indicated by the arrow) is derived from fluorescently labeled (Cy3) -siRNA. FIG. 6d (C) shows the results of an HFN-IgG ^OX ELISA showing specific binding of IgG to human fibronectin (HFN). Figure 6d (D) shows the ELISA results of covalently coupled HFN-IgG ^OX and LsiR lipoplexes after purification on a sucrose gradient, which was observed with native and oxidized HFN-IgG. Shows similar binding features. Natural carbohydrates such as glucose, mannose, lactose, fructose, maltotriose, and maltoheptaose are siRNA and CDAN / DOPE / CPA (40%) PEGylated with 0.1-1% total lipids (molar ratio in lipoplex). / 50/10; m / m / m) can be incubated with LsiR lipoplexes made from liposomes to form covalent conjugation of CPA lipid aminoxy functional groups with C1-carbohydrate atoms. Down-regulation of β-galactosidase on lyophilized LsiR with HeLa. LsiR fresh, freshly prepared LsiR; LsiR 12 FD 25, lyophilized, LsiR complex hydrated again in 25 μl water, no cryoprotectant; LsiR 12 FD 100, lyophilized, again in 100 μl water Hydrated LsiR complex, no cryoprotectant. The graph shows three cryoprotectants (sucrose, 5% / 10% or 20% (w / v) used at 5% / 10% or 20% (w / v) rehydrated in either 25 μl (FD25) or 100 μl (FD100) water, respectively. Comparison of trehalose or lactose).

Claims (46)

  A delivery vector comprising a liposome, wherein one or more lipids of the liposome are reversibly or irreversibly coupled to one or more polymers, and the liposome comprises siRNA.   2. The delivery vector of claim 1, wherein the one or more lipids of the liposome that are reversibly or irreversibly coupled to one or more polymers are exposed at the surface of the liposome. The delivery vector of claim 1 or claim 2, wherein the liposome comprises one or more aminoxy group-containing lipids of formula (I):

Where B is a lipid; X is an optional linker group and R ₂ is H or a hydrocarbyl group.   4. The delivery vector according to claim 3, wherein the aminoxy group-containing lipid is CPA.   The delivery vector according to any one of claims 1 to 4, wherein the liposome comprises one or more cationic lipids and / or one or more non-cationic co-lipids.   6. A delivery vector according to claim 5, wherein the cationic lipid comprises at least one alicyclic group.   7. The delivery vector according to claim 6, wherein the at least one alicyclic group is cholesterol. 8. The delivery vector according to claim 6 or claim 7, wherein the cationic lipid is N ¹ -cholesteryloxycarbonyl-3,7-diazanononane-1,9-diamine (CDAN).   6. The delivery vector according to claim 5, wherein the non-cationic colipid is phosphatidylethanolamine.   10. The delivery vector according to claim 9, wherein the non-cationic colipid is dioleoylphosphatidylethanolamine (DOPE).   The delivery vector according to any one of the preceding claims, wherein the polymer comprises one or more aldehyde and / or ketone groups.   The delivery vector according to any one of the preceding claims, wherein the polymer is PEG.   13. The delivery vector of claim 12, wherein the liposome is coupled with about 0.1 to about 5% PEG.   The delivery vector according to any one of the preceding claims, wherein the liposome comprises or is reversibly or irreversibly coupled to one or more drugs.   A targeted delivery vector comprising a liposome, wherein one or more lipids of the liposome are reversibly or irreversibly coupled to one or more polymers and one or more agents, and the liposome comprises siRNA.   16. The targeted delivery vector of claim 15, wherein the agent is selected from the group consisting of sugars, carbohydrates, and ligands.   17. The targeted delivery vector of claim 16, wherein the sugar is selected from the group consisting of glucose, mannose, lactose, fructose, maltotriose, maltoheptose.   18. A targeted delivery vector according to claim 16 or claim 17, wherein the ligand is an antibody.   An siRNA comprising the step of providing a delivery vector according to any one of claims 1-14 or a targeted delivery vector according to any one of claims 15-18 to a cell, tissue or organ environment. A method for delivery.   19. A delivery vector according to any one of claims 1-14 or a targeted delivery vector according to any one of claims 15-18 for use in delivering siRNA to a cell, tissue or organ.   19. A delivery vector according to any one of claims 1-14 or a targeted delivery vector according to any one of claims 15-18 in the manufacture of a composition for delivery of siRNA to a cell, tissue or organ. Use of. (i) contacting siRNA with liposomes; and
(ii) comprising reversibly or irreversibly coupling the liposome formed in step (i) to a polymer,
A process for preparing a delivery vector comprising a liposome, wherein one or more lipids of the liposome are reversibly or irreversibly coupled to one or more polymers, and the liposome comprises siRNA.   23. The process of claim 22, comprising the further step of (iii) reversibly or irreversibly coupling the liposomes formed in step (i) or step (ii) to one or more agents. (i) contacting siRNA with liposomes;
(ii) reversibly or irreversibly coupling the liposomes formed in step (i) to a polymer; and
(iv) reversibly or irreversibly coupling the liposomes formed in step (i) or step (ii) with one or more agents,
A targeted delivery vector comprising a liposome, wherein one or more lipids of the liposome are reversibly or irreversibly coupled to one or more polymers and one or more agents, and the liposome comprises siRNA. Process for preparing. (i) providing the vector according to any one of claims 1 to 18;
(ii) optionally contacting the vector with a cryoprotectant; and
(iii) A method comprising a step of freeze-drying a vector.   26. The method of claim 25, wherein the cryoprotectant is selected from the group consisting of sucrose, trehalose, and lactose.   27. The method of claim 25 or claim 26, comprising the further step of (iv) rehydrating the vector prior to use.   27. A lyophilized vector obtainable or obtained by the method of claim 25 or claim 26.   A liposome comprising a lipid and a coupling moiety, wherein the distance between the lipid and the coupling moiety is at least 1.5 nm. The liposome of claim 29 of the following formula:

Wherein B is a lipid; X is a linker group, the coupling is a coupling site, and the X backbone contains at least 30 atoms.   32. The liposome of claim 30, wherein the X skeleton comprises at least 40 atoms. 32. The liposome according to claim 30 or 31, wherein X is a group of the following formula or contains a group of the following formula:

In the formula, n and m are independently 0 to 6, preferably 1 to 6, more preferably 2, 3, or 4, more preferably 2 or 4. The liposome according to claim 30 or 31, wherein X is a group of the following formula or contains a group of the following formula:

In the formula, n and m are independently 0 to 6, preferably 1 to 6, more preferably 2, 3, or 4, more preferably 2 or 4. 32. The liposome according to claim 30 or 31, wherein X is a group of the following formula or contains a group of the following formula:

In which m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, and n is 0-20, preferably 5-15, more preferably 10, 11 Or 12, more preferably 11. The liposome according to claim 30 or 31, wherein X is a group of the following formula or contains a group of the following formula:

Wherein m is 0-6, preferably 1-6, more preferably 1, 2, or 3, more preferably 1, n is 0-20, more preferably 5-15, more preferably 10, 11 or 12, more preferably 11.   A delivery vector according to any one of claims 1 to 14, a targeted delivery vector according to any one of claims 15 to 18, or a liposome according to any one of claims 29 to 35, and a pharmaceutical A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.   The delivery vector according to any one of claims 1 to 14, the targeted delivery vector according to any one of claims 15 to 18, or the liposome according to any one of claims 29 to 35, or the claim 38. A method for treating a disease in a subject, comprising administering to the subject a medically effective amount of the pharmaceutical composition according to item 36.   The delivery vector according to any one of claims 1 to 14, or the targeted delivery vector according to any one of claims 15 to 18, or any of claims 29 to 35, for use in the treatment of a disease. The liposome according to one item.   A delivery vector according to any one of claims 1 to 14, or a targeted delivery vector according to any one of claims 15 to 18, or claims 29 to 35 in the manufacture of a composition for the treatment of a disease. Use of the liposome according to any one of the above.   40. The method of claim 37, the vector of claim 39, or the use of claim 39, wherein the disease is liver disease and / or liver damage.   Use of liposomes coupled to polymers in the preparation of delivery vectors containing siRNA.   Use of a liposome coupled to a polymer and one or more agents in the preparation of a targeted delivery vector comprising siRNA.   A delivery vector substantially as described herein and with reference to any one of the Examples or Figures.   A method substantially as described herein and with reference to any one of the Examples or Figures.   Use substantially as described herein and with reference to any one of the Examples or Figures.   Liposomes substantially as described herein and with reference to any one of the Examples or Figures.

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