WO2016197264A1 - 一种结合异核苷修饰、末端肽缀合及阳离子脂质体的小干扰rna修饰方法及制剂 - Google Patents

一种结合异核苷修饰、末端肽缀合及阳离子脂质体的小干扰rna修饰方法及制剂 Download PDF

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WO2016197264A1
WO2016197264A1 PCT/CN2015/000402 CN2015000402W WO2016197264A1 WO 2016197264 A1 WO2016197264 A1 WO 2016197264A1 CN 2015000402 W CN2015000402 W CN 2015000402W WO 2016197264 A1 WO2016197264 A1 WO 2016197264A1
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sirna
cld
dipeptide
cationic lipid
modification
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杨振军
孙晶
范鑫萌
王晓锋
黄野
王坚成
邱崇
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Peking University
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Definitions

  • the invention relates to a comprehensive chemical modification method of siRNA, which is a combination of heteronucleoside modification, terminal peptide conjugation and cationic lipid carrier encapsulation, and the product obtained by the chemical modification method has stable physical and chemical properties. , biological behavior is good and controllable, biological activity and high efficiency, etc., can be widely used in anti-virus, anti-tumor drug research.
  • the invention belongs to the field of biomedical technology.
  • RNA interfering was first discovered by Nobel Prize winner Fire and its collaborators in C. elegans in 1998, and RNAi was first discovered in mammalian cells in 2001.
  • the RNAi phenomenon is currently considered to be a conservative body defense mechanism produced by living organisms during evolution, and is widely found in all animals.
  • Chemically synthesized siRNAs usually consist of two complementary 19-22 nt single-stranded RNAs, typically two nucleosides at the 3'-end of each strand do not participate in the pairing, called the 3'-overhang (3'-overhang) .
  • the RNA sequence that targets mRNA is designed to be completely complementary to the mRNA, called the guide strand or the antisense strand, and the other strand is identical to the mRNA sequence, called the passenger strand or Sense strand.
  • the synthetic 21-nt siRNA is first phosphorylated at the 5'-end of the C1p1 enzyme after it enters the cell. Thereafter, after being recognized by the TRBP protein and undergoing a series of processes, the Dicer, Ago 2 and TRBP proteins are combined to form an unactivated RISC complex. The activated RISC complex is formed as the sense strand is cleaved by Ago 2 cleavage. After the mRNA that is finally complementary to the guide strand is loaded into the RISC, it undergoes degradation by an endonuclease similar to the follower strand, thereby generating a gene silencing effect.
  • siRNAs composed of natural bases cannot satisfy these requirements, and it is necessary to use chemical modification means such as covalent or non-covalent to enhance the prospect of siRNA.
  • covalent chemical modification means include: sugar ring modification, base modification, phosphate skeleton modification, terminal modification, etc.; non-covalent modification means mainly adopting cation separation
  • the carrier of the sub-characteristics enables the encapsulation and delivery of siRNA.
  • a single chemical modification method often has certain limitations. For example, covalent conjugation and base modification of siRNA itself can improve the stability of siRNA and its selectivity to target mRNA to reduce off-target.
  • siRNA/carrier electrostatic complex cannot achieve the high efficiency of siRNA silencing effect due to the randomness of its inoculation mode.
  • too many cationic materials may bring certain cytotoxic side effects. Therefore, in order to thoroughly and systematically solve various problems in the presence of natural siRNA, the present invention attempts to achieve the safety and high efficiency of siRNA transfection by adopting a combination of various chemical modification methods.
  • the present invention provides a novel chemical modification method for small interfering RNA (siRNA), and the chemical modification method provided by the present invention is heteronucleoside modification, terminal peptide conjugate A combination of two or all of the modification methods of the three modification methods of the cationic lipid carrier is combined.
  • the product obtained by the chemical modification method has the advantages of stable physical and chemical properties, good biological behavior and controllability, high biological activity, and the like, and can be widely used in antiviral and antitumor drug research.
  • the invention combines two strategies (structural modification and carrier transport) which are commonly used in the current siRNA application process, and realizes the biological behavior of siRNA by consciously assembling and forming a controlled supramolecular complex system. Regulation of the cellular pathway and intracellular metabolism, which in turn enables it to exert silent activity efficiently. Therefore, the research of the present invention intends to guide the biological behavior by regulating the assembly structure of the siRNA/vector complex, and finally realize the high efficiency of the siRNA to exert its efficacy. This also helps to reduce the dosage of siRNA and its related carriers, further avoiding or reducing the side effects such as toxicity or immunogenicity.
  • a method for comprehensive chemical modification of a small interfering RNA (siRNA) of the present invention comprising incorporating a D-configuration or an L-configuration at one or more sites of the sense strand and/or the antisense strand of a small interfering RNA Isonucleosides, dipeptide-conjugated at the 3'-end of the sense strand and/or antisense strand of a small interfering RNA, and entrapped with a cationic lipid carrier of the Gemini type, by two or all of the above modifications
  • the method can effectively improve the serum stability of small interfering RNA, reduce the off-target effect, and achieve the regulation of its transmembrane pathway, so that small interfering RNA can enter the cell according to the desired pathway or proportion (cavernolin-mediated Swallowing the giant cell drinking route), thereby reducing the degree of intracellular destruction and degradation of small interfering RNA, further improving the silencing activity of siRNA, and finally achieving high efficiency and safety of si
  • a heteronucleoside is a nucleoside analog of a nucleobase position shifted from 1' to the 2' position of a glycosyl group.
  • the base is displaced from the 1' position of the sugar ring to the 2' position of the sugar ring, increasing the glycosidic bond. stability.
  • the laboratory has synthesized different configurations of D- (chemical formula I) and L- (chemical formula II) hetero-nucleosides using different raw materials, the general formulas are as follows:
  • the heteronuclear acid shown is a hetero-nucleoside in the D-configuration, and the heteronucleoside shown in Formula II is an iso-nucleoside in the L-configuration.
  • D-/L-isonucleosides of different configurations can have different effects on the local conformation of the oligonucleotide, thereby affecting the physicochemical properties and silencing activity of the oligonucleotide. .
  • the D-/L-isonucleoside since the D-/L-isonucleoside is structurally similar to the natural nucleoside, it can retain or approximate the original properties of the oligonucleotide to the greatest extent and can be applied to the nucleic acid as a pair of molecular probes. Areas of discussion of mechanisms such as interactions with proteins.
  • the peptide conjugation modification is carried out at the 3' end of the sense strand and/or the antisense strand of the small interfering RNA, and the conjugated peptide fragment may be a peptidomimetic, the peptide conjugation
  • the general formula for the fragment to be linked to the RNA moiety is:
  • X is a polypeptide sequence
  • A is a substituted or unsubstituted benzene ring structure or a carbon atom
  • n is 0, 1, 2, 3, 4 or 5.
  • the polypeptide sequence represented by X in Formula III is KALLAL or a sequence of similar peptides thereof.
  • Gemini cationic lipid carriers typically consist of a cationic head, an aliphatic tail, and a tether.
  • the head structure is mostly a cationic group such as a polypeptide or a glycolipid.
  • the tail is usually an aliphatic saturated or unsaturated long chain and a hydrophobic molecule such as cholesterol, and the connecting arm is mainly a disulfide bond or an amide bond which can be degraded in the body.
  • the carrier is electrostatically compressed by a positively charged cationic water-soluble head and a negative charge of a gene drug (DNA/PNA/siRNA) phosphate backbone, and the siRNA is effectively compressed and aggregated on the surface of the carrier lipid particle to form a nanocomposite. .
  • the structure of the complex changes from lamellar to hexahedral due to changes in the environment inside and outside the cell membrane, causing the carrier complex to disassemble, thereby escaping from the lysosome into the cytoplasm and releasing it. DNA/RNA, which in turn produces a silencing effect.
  • the cationic lipid carrier may be a commercial cationic lipid carrier such as RNAiMax, Oligofectamine or the like, or a Gemini cationic lipid carrier of the general formula IV:
  • X is a sulfur atom (S) or a carbon atom (C)
  • Y is a nitrogen-containing group or a targeting group having a positive charge
  • R is a saturated or unsaturated aliphatic chain or a hydrophobic molecule.
  • the unsaturated aliphatic chain represented by R in Chemical Formula IV is an oleyl alcohol structure.
  • one or more positions of the sense strand and/or the antisense strand of the small interfering RNA is achieved by solid phase synthesis, that is, the phosphoramidite monomer in the doping position using the heteronucleoside phosphoramidite monomer instead of the natural nucleoside is in the corresponding position. Coupling was carried out.
  • the heteronucleoside compound represented by the chemical formula I and/or the formula II is separately prepared into a heteronucleoside phosphoramidite monomer represented by the chemical formula V and/or the chemical formula VI, and each nucleoside is coupled.
  • the condition for synthesizing the DNA oligonucleotide chain is to increase the number of injections of the heteronucleoside phosphoramidated monomer to 3 times; the coupling time after each injection is 300 seconds /
  • the conditions for synthesizing the heteronucleoside modified RNA oligonucleotide strand were such that the coupling time after each cycle of injection was increased to 900 sec/time, coupled 3 times.
  • the comprehensive chemical modification method further includes co-use with other chemical modification strategies, including 2'-O-methoxy (2'-OMe), 2'-fluoro (2' -F), locked nucleotide (LNA), phosphorous-sulfur skeleton modification and other terminal conjugation methods.
  • other chemical modification strategies including 2'-O-methoxy (2'-OMe), 2'-fluoro (2' -F), locked nucleotide (LNA), phosphorous-sulfur skeleton modification and other terminal conjugation methods.
  • the small interfering RNA sequence to be modified is a siMek1 sequence that targets mRNA of the MEK1 protein in the ERK pathway and a siMB3 sequence that targets mRNA of the variant B-Raf kinase protein in the ERK pathway.
  • the sequences before the above siMek1 and siMB3 modification are as follows:
  • siMek1 Justice Chain: 5’-GCAACUCAUGGUUCAUGCUdtdt-3’;
  • Antisense strand 5’-AGCAUGAACCAUGAGUUGCdtdt-3’
  • siMB3 Justice Chain: 5’-GCUACAGAGAAAUCUCGAUdtdt-3’
  • Antisense strand 5'-AUCGAGAUUUCUCUGUAGCdtdt-3’
  • the peptide fragment of the formula III (PA/PS-siMB3) is conjugated to the 3' end of the positive and antisense strands of the siMB3 sequence described above, and only the 3' end conjugation formula of the sense strand
  • the peptide fragment of III (PS-siMB3) has a peptide fragment of the formula III (PA-siMB3) and an unmodified siMB3 sequence, and the PA/PS-siMB3 and PS-siMB3 sequences are compared to the 3' end of the antisense strand only. More High serum stability.
  • the first nucleotide is introduced into the 5' end of the sense strand of the above siMek1 sequence and the siMB3 sequence, and the heteronucleoside represented by the chemical formula I or the formula II is incorporated, and in the positive and negative
  • the 3' end of the sense strand is conjugated to the peptide fragment of Formula III, and is encapsulated by the cationic carrier RNAiMax, or the cationic lipid carrier Oligofectamine, or the cationic liposome formed by Formula IV, and the unmodified siMek1 is encapsulated compared to the above cationic liposome.
  • the sequence and the siMB3 sequence have higher silencing activity.
  • the peptide fragment of the formula III is conjugated to the 3' end of the antisense strand of the siMB3 sequence, and the cationic liposome material formed by the formula IV is used as a delivery carrier.
  • the preparation and investigation of related parameters such as preparation process conditions finally determined the preparation methods of two different siRNA/carrier complexes. The results showed that the obtained siRNA/carrier complexes had low cytotoxicity and high stability.
  • the peptide fragment of the formula III when the peptide fragment of the formula III is conjugated to the 3' end of the positive and antisense strands of the siMek1 sequence and the siMB3 sequence, and is encapsulated by the cationic liposome formed by the chemical formula IV, Uniform and stable distribution of specific spherical vesicles, which can form stronger complexes by forming unmodified siMek1 sequence and siMB3 sequence than cationic liposome formed by chemical formula IV, resulting in complex formation.
  • a nano-assembled structure with a lower surface potential, a larger particle size, and a denser interior.
  • the peptide complex of the formula III is conjugated to the 3' end of the positive and antisense strands of the siMB3 sequence, and the nanocomposite formed by the cationic liposome formed by the formula IV is It can enable small interfering RNA to enter cells (cavernolin-mediated endocytosis and macrocytoplasmic pathway) according to the desired pathway or proportion, so as to avoid the degradation process of intracellular lysosomes to a certain extent, to achieve more For the efficient use of silent activity.
  • the peptide complex of the formula III is conjugated to the 3' end of the positive and antisense strands of the siMB3 sequence, and the nanocomposite formed by the cationic liposome formed by the formula IV is Compared with the above cationic liposome, the unmodified siMB3 sequence has higher silencing activity.
  • the invention realizes the controllability of the assembly by optimizing the prescription process conditions when forming the preparation of the 3', 3"-dipeptide-siRNA conjugate and the cationic lipid carrier (formula: Formula IV), including Optimization of particle morphology and internal structure, particle size and potential.
  • the integrated chemical modification method most preferably, comprises the following steps:
  • the formed nanocomposite particles can regulate their pathways or proportions into cells by means of caveolin-mediated endocytosis and macrophage pathway.
  • the chemical modification strategy of the combination of heteronucleoside modification, terminal peptide conjugation and cationic lipid carrier encapsulation provided by the invention can exert the advantages of each of the three chemical modification methods used, and complement each other, and the obtained siRNA has higher serum. Stability and biological activity, and showing a good controllable transmembrane transport capacity and silencing effect of target mRNA, laid a good foundation for the clinical application of siRNA technology.
  • 2.3', 3"-dipeptide-siRNA conjugates have dual binding to cationic lipid carriers, achieving similar biological activity to commercial carriers with less carrier usage for more efficient delivery siRNA and reduce the biological toxicity caused by the vector itself.
  • siRNA and cationic lipid carrier Through the exploration and optimization of the preparation conditions and parameters of the nano-composites formed by siRNA and cationic lipid carrier, a uniform and stable assembly system can be obtained, and the regulation of its transmembrane pathway can be realized, thereby affecting its intracellular metabolism. Behavior, and ultimately, the silent activity is effectively utilized to further advance the clinical application of siRNA.
  • Figure 1 shows the results of serum stability (50% FBS) of a heteronucleoside-conjugated 3',3"-dipeptide-siMB3 conjugate.
  • Figure 2 shows the results of silencing activity of the heteronucleoside-conjugated 3',3"-dipeptide-siMek1 conjugate (30 nM, 24 h), the transfection reagent is cationic liposome RNAiMax, the upper panel shows the results of Western Blotting, the lower panel is Real-time PCR results.
  • Figure 3 is a Real-time PCR result (30 nM, 24 h) of a heteronucleoside-conjugated 3',3"-dipeptide-siMB3 conjugate, and the transfection reagent was a cationic liposome RNAiMax.
  • Figure 4 is a Real-time PCR result (30 nM) of a heteronucleoside-conjugated 3',3"-dipeptide-siMB3 conjugate,
  • the transfection reagent is the cationic liposome Oligofectamine.
  • Figure 5 shows the results of Western blotting of the heteronucleoside-conjugated 3',3"-dipeptide-siMB3 conjugate (30 nM), and the transfection reagent was a cationic liposome Oligofectamine.
  • Figure 7 is a graph showing the effect of various parameters of the two-phase mixing process (MT method) on the formation of natural and 3',3"-dipeptide-siRNA conjugates and cationic lipid carrier CLD formulations.
  • Figure 8 is a formulation formed by combining the optimal conditions of the MT preparation process.
  • Figure 9 is an in vitro property evaluation of four formulations of natural and 3',3"-dipeptide-siRNA conjugates and cationic lipid carrier CLD (serum stability assay, erythrocyte hemolysis assay, and dilution stability assay).
  • Figure 10 is a dynamic light micrograph (DLS) of four formulations of natural and 3', 3'-dipeptide-siRNA conjugates with cationic lipid carrier CLD.
  • DLS dynamic light micrograph
  • Figure 11 is a transmission electron microscope (TEM) image of a cationic lipid carrier combined with a natural siRNA and a 3',3"-dipeptide-siRNA conjugate to form a formulation (AT method preparation), wherein A is a cationic lipid carrier CLD form.
  • Figure B shows the ion pattern of cationic lipid carrier CLD and natural siRNA;
  • A is a single cationic lipid carrier CLD. Morphology
  • Figure B is a combination of cationic lipid carrier CLD and natural siRNA
  • Figure 13 shows the cellular uptake of the cationic lipid carrier CLD in combination with native siRNA and 3', 3'-dipeptide-siRNA conjugates to form four formulations.
  • Figure 14 shows the selection of transmembrane pathways and inhibitors of small molecule cellular pathways in each channel.
  • Figure 15 is a graph showing the cellular uptake of four formulations formed by binding of a cationic lipid carrier CLD to a natural siRNA and a 3',3"-dipeptide-siRNA conjugate.
  • Figure 16 is a graph showing the ratio of the inoculation pathway of the four preparations formed by the combination of the cationic lipid carrier CLD and the natural siRNA and the 3', 3"-dipeptide-siRNA conjugate.
  • Figure 17 is a graph showing the results of silencing activity RT-PCR of cationic lipid carrier CLD in combination with natural siRNA and 3', 3'-dipeptide-siRNA conjugates to form four formulations at different times and concentrations.
  • Figure 18 shows the results of silencing activity of a cationic lipid carrier CLD transfected with a natural and heteronucleoside in combination with a 3',3"-dipeptide-siRNA conjugate.
  • Blank is a natural siRNA
  • Na is a cationic lipid carrier CLD transfected with natural siRNA
  • PP is a cationic lipid carrier CLD transfected 3', 3"-dipeptide-siRNA conjugate
  • D1PP is a cationic lipid carrier CLD transfected with D-configuration heteronucleoside modified 3', 3"-double
  • the peptide-siRNA conjugate, L1PP was a cationic lipid carrier CLD transfected with an L-configuration heteronucleoside modified 3',3"-dipeptide-siRNA conjugate.
  • Fig. 19 is a graph showing the results of formulation stability of a nanocomposite formed by a cationic lipid carrier CLD combined with a natural siRNA and a heteronucleoside to modify a 3',3"-dipeptide-siRNA conjugate.
  • Example 1 Isonucleoside-binding terminal peptide-conjugated modified siRNA and its serum stability evaluation
  • the small interfering RNA to be modified is a siMB3 sequence that targets mRNA of the variant BRaf kinase protein in the ERK pathway, and the siMB3 sequence before modification is as follows:
  • siMB3 Justice Chain: 5’-GCUACAGAGAAAUCUCGAUdtdt-3’
  • Antisense strand 5'-AUCGAGAUUUCUCUGUAGCdtdt-3’
  • the modification strategy selects any of the following: 1) Incorporation of the first nucleotide at the 5' end of the siMB3 sequence sense strand described above into the heteronucleoside represented by Formula I or Formula II.
  • X is the 6-peptide sequence H-Leu-Ala-Leu-Leu-Ala-Lys-OH (KALLAL), A is a carbon atom, and n is 1.
  • siMB3 sequences were evaluated separately. Take natural siMB3, different modified siMB3 sequence 4 ⁇ L (20 ⁇ M) + 20 ⁇ L FBS+16 ⁇ L PBS into 200 ⁇ L microcentrifuge tube, remove 10 ⁇ L to 3 microcentrifuge tubes, and incubate in 37°C water bath. Samples were then taken at the same time point and immediately placed in a -80 ° C freezer or liquid nitrogen. After quenching on ice, the cells were analyzed by 20% denaturing polyacrylamide gel electrophoresis, and the nucleic acid dye was stained for 15 min. Finally, the electrophoresis results were imaged by a chemiluminescent gel imaging system.
  • the heteronucleoside modification is carried out at the 5' end of the sense strand, regardless of the D-/L-isonucleoside modification, the stability of the serum conjugated to the antisense strand monopeptide is increased, and the sense strand monopeptide is conjugated. Reduced serum stability. It indicates that the selectivity of the end group of ribozyme is related to thermodynamics, and the heteronucleoside can change the balance. Peptide conjugation modification compensates for the effect of heteronucleoside modification on serum stability, and a modification strategy that greatly improves stability can be obtained.
  • RNAiMAX modified siRNA at a final concentration of 30 nM, incubated at room temperature for 15 min, added to the cell culture plate, and incubated for 24 h.
  • Total RNA was extracted by TRizol, and total RNA was reverse transcribed into cDNA, and then Real-time experiment was performed using Gotaq Green Mix to investigate the gene silencing effect of siRNA at the mRNA level.
  • the total protein was extracted, and the total protein was quantified by BSA.
  • the protein was separated by polyacrylamide gel (10% separation gel and 5% concentrated gel). After transfection, the primary and secondary antibodies were immunoreactive and finally passed. Bio-rad's chemiluminescent gel detection system detects protein knockout effects.
  • siMB3 targeting Braf-mutant mRNA When the modification and activity evaluation of siMB3 targeting Braf-mutant mRNA was performed, according to the results of Real-time PCR (Fig. 3), the L-isonucleoside modified binding of the 5' end of the sense strand was still found in all siMB3 leader structures.
  • the ',3'-dipeptide-siRNA conjugate has the best silencing activity, but unlike the modified siMek1 sequence, the silence of the 3',3"-dipeptide-siRNA conjugate was found in the siMB3 modification results. The activity was significantly increased, even higher than the silencing activity results of the L-isonucleoside modified siRNA bound to the 5' end of the sense strand.
  • siMek1 is significantly more stable in serum than siMB3. Due to the lower serum stability of siMB3, the improvement in serum stability of dipeptide conjugation in siMB3 is also more pronounced.
  • the increased serum stability of siRNA means that more intact siRNA is present in the body, and the siRNA that exerts silencing activity also becomes more. Therefore, since the increase in serum stability is more pronounced for siMB3, the dipeptide conjugation is also more pronounced for the improvement of siMB3 silencing activity.
  • the effect of the complex is not obvious; when the thermodynamic difference at the 5' end of the antisense strand is small, the ability of the positive and negative strands to enter the RISC complex is similar when forming the RISC complex, so when the 5' end of the sense strand When the heteronucleoside modification hinders the ability of the sense strand to enter the RISC complex, the ability of the antisense strand to enter the RISC complex will increase more, and thus the silencing activity will be more pronounced.
  • silencing activity of the lead structures PA/PS-siMB3-S01D and PA/PS-siMB3-S01L was slightly decreased, the silencing activity was still better than that of natural siMB3, but 3', 3"-dipeptide-
  • the silencing activity of the siRNA conjugate was significantly higher than that at 24 h of incubation.
  • the incubation time was extended to 72 h, the mRNA level was significantly restored, which should be due to the fact that as the incubation time prolonged, the mRNA degraded by the siRNA was recovered by continuous transcription.
  • the bis-peptide conjugation and the double-peptide conjugation combined with the 5'-end L-isonucleoside modification of the sense strand had the best silencing activity, indicating that the dipeptide conjugation can be to some extent Prolong the time that siRNA exerts silencing activity.
  • the extension of silencing time should be related to the double peptide conjugation significantly increased the intracellular stability of siRNA.
  • the conjugated peptide is mostly degraded in the serum before the siRNA double-strand itself, that is, the peptide conjugation can hinder the ribozyme attack on the siRNA end.
  • the conjugated peptide can delay the attack of ribozyme on siRNA.
  • siMek1 The sequence of siMek1 before modification is as follows:
  • siMek1 Justice Chain: 5’-GCAACUCAUGGUUCAUGCUdtdt-3’;
  • Antisense strand 5’-AGCAUGAACCAUGAGUUGCdtdt-3’
  • the peptide fragment of formula III (PA/PS-siMek1) is conjugated to the 3' end of the positive and negative strands of the siMek1 sequence described above:
  • X is the 6-peptide sequence H-Leu-Ala-Leu-Leu-Ala-Lys-OH (KALLAL), A is a carbon atom, and n is 1.
  • the specific structure of the CLD molecule is as follows: lysine is used as the cation head, the intermediate linker is composed of cystine residues, and the tail is composed of oleyl alcohol (18,10-cis double bond, 18-carbon saturated aliphatic hydrocarbon) ) constitute a lipophilic tail chain.
  • the free double 3',3"-dipeptide- The siRNA conjugate band brightness has become very dark relative to the control siRNA brightness, at which point the cationic lipid carrier compound has been able to fully bind to the 3',3"-dipeptide-siRNA conjugate (see Figure 6).
  • the ability to bind to the 3',3"-dipeptide-siRNA conjugate demonstrates again that the presence of dual action enhances the ability of the siRNA to bind to the cationic lipid carrier cationic lipid carrier.
  • Example 5 Preparation and determination of process parameters for preparation of natural and 3',3"-dipeptide-siRNA conjugates and cationic lipid carrier CLD nanocomposites by two-phase mixing method (MT method)
  • a certain concentration of CLD ethanol solution was slowly added dropwise to a certain concentration of siRNA aqueous solution, and after vortexing for a certain time, the nanoparticles were prepared by ultrasonication.
  • B. material concentration ratio (N/P) N means CLD Contains protonated N numbers, P refers to the amount of phosphoric acid contained in the siRNA
  • Example 6 Evaluation of in vitro properties of four formulations of natural and 3',3"-dipeptide-siRNA conjugates with cationic lipid carrier CLD (serum stability assay, erythrocyte hemolysis assay, and dilution stability assay)
  • Serum stability test 100 ⁇ L of different preparations and 5% glucose solution were added to 96-well plates, mixed with 100 ⁇ L fetal bovine serum, and incubated at 37 ° C for 0 min, 5 min, 10 min, 30 min, 1 h, 3 h, respectively.
  • the absorbance (OD value) at 630 nm was measured by Bio-Rad microplate reader at 5h, 10h, 24h, 33h, 48h, and 3 replicate wells were set for each sample.
  • Red blood cell hemolysis test blood was taken from the venous plexus of SD rats, centrifuged at 1500 g for 10 min at 4 ° C, serum was discarded, and the separated red blood cells were washed with 0.9% physiological saline, and then suspended with PBS phosphate buffer at pH 7.38. 2% (v/v) red blood cell suspension. 100 ⁇ L of the blood cell suspension was added to the 96-well plate, and then 100 ⁇ L of PBS (negative control), 1% Triton X-100 (positive control) or serial concentration samples were added, and incubated at 37 ° C for 1 h. The intact red blood cells were removed by centrifugation, and the absorbance at 540 nm of the supernatant was measured using a Bio-Rad microplate reader. The relative hemolysis rate is calculated using the following formula:
  • Dilution stability test Four preparations prepared according to siRNA (200 ⁇ L, 2 ⁇ M) / CLD (40 ⁇ L, 50 ⁇ M), and then the preparations were diluted 5 times, 10 times, 20 times, 40 times, respectively, and the change of the characteristics was observed (measured Dilute 5 times when the potential is set.
  • the natural siRNA/CLD complex preparation prepared by the MT method has the highest hemolysis rate, which is related to the highest surface potential.
  • the hemolysis rate of the MT method is relatively higher than that of the AT method, which is related to the assembly method, that is, the siRNA in the MT method is mainly in the inner layer, and the siRNA in the AT method is more in the outer layer, and thus the potential is somewhat Differences, hemolysis rate will also vary.
  • the hemolysis rate of the four preparations is relatively low, which is relatively safe; in terms of the two preparation methods, the potential and particle size of the AT method are greatly changed during the dilution process, which indicates that the dilution of the preparation prepared by the MT method is stable.
  • the effect is significantly greater than the AT method, which also reflects from the side that in the MT method, whether the natural siRNA or the 3', 3"-dipeptide-siRNA conjugate interacts with the CLD is stronger than the AT method.
  • the surface formed by the 3',3"-dipeptide-siRNA conjugate was diluted to have a lower surface potential than the native siRNA.
  • Example 7 Dynamic Light Scan (DLS) of four formulations of natural and 3',3"-dipeptide-siRNA conjugates with cationic lipid carrier CLD
  • Example 4 Four formulations of natural and 3',3"-dipeptide-siRNA conjugates with cationic lipid carrier CLD were prepared as in Example 4 and Example 5. Each dose of aqueous solution was placed in an EP tube. In the dynamic light scattering instrument (Dynamic Light Scattering Instrument, model: Zetasizer Nano ZSP), the corresponding parameters such as hydrated particle size, surface potential and polydispersity coefficient were determined.
  • Dynamic Light Scattering Instrument, model: Zetasizer Nano ZSP the corresponding parameters such as hydrated particle size, surface potential and polydispersity coefficient were determined.
  • the experimental results (as shown in Figure 10 and Table 2): Through the exploration and investigation of the preparation process conditions, relatively uniform and stable spherical-like nanoparticle composites can be obtained by both preparation processes.
  • the polydispersity index (PDI) of the four formulations was less than 0.3, indicating that the formed formulation had a good dynamic distribution.
  • the surface potentials of the four preparations can be controlled in the range of +20 to 30 mV, and the surface potential of this range is favorable for the effective uptake of the preparation by the cells, while minimizing the cytotoxicity caused by the positive charge.
  • the surface potential of the 3',3"-dipeptide-siRNA conjugate/CLD forming preparation is lower than that of the natural siRNA/CLD preparation, which is based on ensuring effective cell uptake, 3
  • the ',3'-dipeptide-siRNA conjugate has lower cytotoxic side effects and is safer to exert its silencing activity.
  • the particle size can be controlled to be between 100 nm and 150 nm by this formulation process. Particles of this particle size have better passive targeting effects in the body, that is, enhanced permeability and retention. Effect, EPR).
  • 3', 3"-dipeptide-siRNA conjugate and cationic lipid carrier CLD can form nanocomposites with larger particle size and lower surface potential. This feature is significantly different from the natural siRNA assembly system, and thus will produce the specificity of later biological behavior.
  • Example 8 Characterization of supramolecular structure of cationic lipid carrier CLD in combination with natural siRNA and 3',3"-dipeptide-siRNA conjugate (AT method preparation)
  • Example 4 CLD liposome and its natural siRNA (siMek1) and 3',3"-dipeptide-siRNA conjugate (PA/PS-siMek1) were examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM).
  • TEM transmission electron microscopy
  • AFM atomic force microscopy
  • the method of Example 4 was prepared to form the internal structure and apparent morphology of the composite particles. The operation was as follows: firstly, separate liposomes were prepared according to the operation of Example 4, natural siRNA and CLD nanocomposites, 3' , a nanocomplex formed by a 3"-dipeptide-siRNA conjugate and CLD.
  • Figure 11 shows the morphology of three groups of cationic lipid carrier particles, cationic lipid carrier CLD and natural siRNA complex, cationic lipid carrier CLD and 3',3"-dipeptide-siRNA conjugate complex.
  • Transmission electron microscopy results In the cationic lipid carrier microparticle group alone, the cationic lipid carrier exhibited a spherical shape and was a typical spherical lipid vesicle.
  • the cationic lipid carrier CLD was combined with natural siRNA and 3', 3"-dipeptide, respectively.
  • the structural morphology of the lipid microparticles showed a significant change: as for Fig.
  • Atomic force microscopy results of complexes formed by the interaction of cationic lipid microparticles with native siRNA (siMek1) and 3',3"-dipeptide-siRNA conjugate (siMek1) ( Figure 12), cationic lipid carrier
  • siMek1 native siRNA
  • siMek1 3',3"-dipeptide-siRNA conjugate
  • Figure 12 cationic lipid carrier
  • the complex formed with the natural siRNA and the 3',3"-dipeptide-siRNA conjugate exhibited a uniform circular shape and uniform distribution. This result is consistent with the TEM scan results.
  • the binding morphology of cationic lipid carrier to 3',3"-dipeptide-siRNA conjugate was different from that of cationic lipid carrier and natural siRNA.
  • the horizontal distance (ie diameter) of the cationic lipid carrier CLD is about 120 nm and the vertical distance is about 6 nm.
  • the reason for the large difference between the vertical distance and the horizontal distance is due to the use of the dry solvent method in the preparation of samples. A certain concentration of the material solution is about to be added to the mica sheet, and the solvent is volatilized and then detected, so that the measured particles will collapse downward by the influence of gravity during the drying process, thereby lowering the vertical distance.
  • Figure B shows the cationic lipid carrier.
  • the composite of CLD and natural siRNA has a diameter of about 100 nm and a vertical distance of about 5 nm.
  • the cationic lipid carrier CLD Due to the electrostatic adsorption of the cationic lipid carrier CLD and the natural siRNA, the cationic lipid carrier CLD is further compressed, so that the particle size of the complex is smaller than that of the cation.
  • the lipid carrier CLD is slightly smaller.
  • Figure 3 shows that the complex formed by the cationic lipid carrier CLD and the 3',3"-dipeptide-siRNA conjugate has a particle size of about 130 nm and a vertical distance of about 6.6 nm. . Its particle size and vertical distance are larger than the complex formed by natural siRNA and cationic lipid carrier CLD.
  • the lipid carrier CLD binds, and this phenomenon is caused by the covalently conjugated peptide sequence at the 3'-end.
  • the 3', 3"-dipeptide-siRNA conjugate and the cationic lipid carrier CLD form a supramolecular complex.
  • the mode of action of the compound is significantly different from the mode of action of natural siRNA and cationic lipid carrier CLD.
  • the chemical and biological significance of this difference will be combined with the subsequent cationic lipid carrier/natural siRNA and cationic lipid carrier/
  • the bioactivity evaluation results of the 3',3"-dipeptide-siRNA conjugate were further analyzed.
  • Example 9 Cationic lipid carrier CLD combined with native siRNA and 3',3"-dipeptide-siRNA conjugate to form cellular uptake of four formulations
  • Example 4 Four formulations of natural and 3',3"-dipeptide-siRNA conjugates with cationic lipid carrier CLD were prepared as in Example 4 and Example 5.
  • Melanoma A375 cells were used at 300,000/well. The cells were inoculated in a 6-well plate, cultured for 24 hours, and the cells were adhered to the cells for 15 min at 37 ° C. The cells were diluted 10 times with OPTI-MEM, and the final concentration of the dipeptide siRNA and the natural siRNA was 100 nM.
  • the medium was discarded and washed with 1 mL of PBS 2 2 mL of the above-described Cy3-labeled 3',3"-dipeptide-siRNA conjugate and natural siRNA preparation were added to each well and cultured for 4 hours. The uptake of the formulation was then tested by flow cytometry according to flow cytometry.
  • Example 10 Cellular uptake of four formulations of cationic lipid carrier CLD in combination with native siRNA and 3',3"-dipeptide-siRNA conjugates treated with inhibitors
  • the pathways for the uptake of exogenous substances by cells are mainly divided into the following types: cell phagocytosis, specific receptor-mediated introduction into cells, giant cell drink, clathrin-mediated endocytosis, and caveolin-mediated endocytosis.
  • cell phagocytosis Most of the exogenous substances that enter the cells through these pathways undergo acidification of the lysosomes and are eventually degraded or excreted.
  • Recent studies have shown that in these cellular pathways, when exogenous substances enter the cell in caveolin-mediated endocytosis, they can be directly released into the cytoplasm, to a certain extent, avoiding the degradation process of lysosomes.
  • the giant cell drinking pathway passes through the lysosome process, its escape rate is relatively fast.
  • the proportion of the preparation prepared by the two-phase mixing method is proportional to the two preparations prepared by the film hydration method (AT method).
  • the 3', 3"-dipeptide-siRNA conjugate prepared by the MT method has the largest proportion.
  • the membrane hydration method (AT method)
  • the natural siRNA obtained has the largest proportion of amantadine channel; in addition, after chlorpromazine inhibits cell channel, its cellular uptake increases, which indicates that after inhibition of this pathway, other pathways are activated to increase cell-to-siRNA. Ingestion.
  • the proportion of 3',3"-dipeptide-siRNA conjugates is the same under the same formulation process conditions (either AT or MT). Greater than native siRNA.
  • the four formulations were heavily dependent on the pathway and there was no significant difference between them.
  • the silencing activity of 3', 3"-dipeptide-siRNA conjugate and natural siRNA can also be speculated: 1) Whether it is MT method or AT method, 3', 3" - The dipeptide-siRNA conjugate has higher silencing activity than native siRNA (because the ratio of 3', 3"-dipeptide-siRNA conjugates in caveolin and giant cytosol is greater than that of natural siRNA); Among the four preparations, the 3',3"-dipeptide-siRNA conjugate/CLD complex prepared by the MT method has the best silencing activity (because the caveolin and giant cell drinking pathways occupy 99%); 3) The natural siRNA/CLD complex prepared by the AT method has the worst silencing activity (because clathrin has the largest proportion, which means that there are more siRNAs that are proteolytically degraded).
  • Example 11 Cationic Lipid Carrier CLD was combined with native siRNA and 3',3"-dipeptide-siRNA conjugate to form silent activity RT-PCR results for four formulations at different times and concentrations.
  • siMek1 The sequence of siMek1 before modification is as follows:
  • siMek1 Justice Chain: 5’-GCAACUCAUGGUUCAUGCUdtdt-3’;
  • Antisense strand 5’-AGCAUGAACCAUGAGUUGCdtdt-3’
  • the peptide fragment of the formula III (PA/PS-siMek1) is conjugated to the 3' end of the antisense strand of the siMek1 sequence described above;
  • X is the 6-peptide sequence H-Leu-Ala-Leu-Leu-Ala-Lys-OH (KALLAL), A is a carbon atom, and n is 1.
  • the first nucleotide is incorporated into the heteronucleoside (D1PP) of Formula I or the first nucleotide is added at the 5' end of the sense strand of the siMek1 sequence. Enter the heteronucleoside (L1PP) shown in formula II.
  • the 3',3"-dipeptide-siRNA conjugate was equivalent to the silencing effect of the native siRNA, indicating that the dipeptide was conjugated under conditions that ensured that the silencing activity was not affected.
  • siRNA is more effective in improving serum stability and reducing ribozyme degradation.
  • 3', 3"-dipeptide-siRNA conjugates modified with isonucleosides are more potent than 3', 3"-dipeptide-siRNAs.
  • the conjugate has better silencing activity, indicating that the modification strategy of heteronucleoside combined with dipeptide conjugate can significantly improve the silencing ability of the target on the basis of improving stability.
  • the nucleoside-conjugated 3',3"-dipeptide-siRNA conjugate has the best silencing activity when transfected with the cationic lipid carrier CLD. This indicates that the heteronucleoside-binding peptide conjugation modification and the small interfering RNA encased by the cationic lipid carrier have good application prospects and the value of continued development.
  • Example 4 Preparation of a heteronucleoside-modified 3',3"-dipeptide-siRNA conjugate/CLD preparation is described in Example 4.
  • the polyanion replacement assay was used to examine the 3', 3"- Stability of dipeptide-siRNA conjugate/cationic liposome CLD.
  • the polyanion of this experiment was selected from heparin.
  • Heparin is a sulfonated mucopolysaccharide.
  • There are many acidic proteins in the human body whether in the intracellular environment or in the extracellular environment. These proteins are negatively charged and can compete with siRNA to disrupt the stability of the siRNA/vector system. If the rate and extent of siRNA replacement by the heparin in the vector complex is poor, this indicates that the siRNA/carrier complex is stable, which increases the stability of the complex in vivo and facilitates better silencing activity.
  • CLD liposomes were prepared as in Example 4.
  • the hetero-nucleoside combined modified 3',3"-dipeptide-siRNA conjugate/CLD complex and the natural siRNA/CLD nanocomposite were prepared in a certain ratio to form a stable preparation.
  • the different preparations were respectively 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.5, 3.0 IU heparin/ ⁇ g siRNA mixed, incubate at 37 ° C for 30 min, add 5 ⁇ loading buffer, 80 V voltage on a 1% agarose gel containing 0.5 ⁇ g / mL EB After electrophoresis for 3 min and electrophoresis at 100 V for 15 min, the gel imaging system was used to observe EB/siRNA fluorescence. Free siRNA was used as a control group.

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Abstract

提供了一种新型的小干扰RNA(siRNA)的化学修饰方法，其是异核苷修饰、末端肽缀合及阳离子脂质载体包裹三种方法中的至少两种方法的联合使用。

Description

一种结合异核苷修饰、末端肽缀合及阳离子脂质体的小干扰RNA修饰方法及制剂 技术领域
本发明涉及一种综合的siRNA的化学修饰方法，该化学修饰方法是异核苷修饰、末端肽缀合及阳离子脂质载体包裹的联合使用，经该化学修饰方法所获得的产品具有理化性质稳定、生物学行为良好可控、生物活性高效等优点，能够广泛用于抗病毒、抗肿瘤药物研究中。本发明属于生物医药技术领域。
背景技术
RNA干扰现象(RNA interfering，RNAi)是由诺贝尔奖获得者Fire及其合作者于1998年在秀丽隐杆线虫中首次发现的，并在2001年在哺乳动物细胞中也首次发现了RNAi现象。RNAi现象目前被认为是生命体在进化过程中产生的一种保守的机体防御机制，广泛存在于所有的动物中。化学合成的siRNA通常由2条互补的19-22nt的单链RNA构成，一般在每条链3’-末端的2个核苷不参与配对，称为3’-悬垂端(3’-overhang)。靶向mRNA的RNA序列被设计成与mRNA完全互补的序列，称为引导链(guide strand)或反义链(antisense strand)，另外一条链与mRNA序列相同，称为随从链(passenger strand)或正义链(sense strand)。通常情况下，合成的21-nt的siRNA进入细胞后首先由C1p1酶在其5’-端进行磷酸化修饰。之后，被TRBP蛋白识别并经过一系列过程后，与Dicer，Ago 2和TRBP蛋白共同形成未活化的RISC复合体。随着正义链被Ago 2切割断裂离去后，能形成活化的RISC复合体。最终与引导链互补的mRNA被载入RISC后，发生与随从链类似的内切酶作用降解，从而产生基因沉默效应。
然而，要将siRNA技术发展成为理想的药物必须满足以下要求：(1)对靶序列具有高度的特异性和亲和性，以减少脱靶效应带来的免疫原性；(2)在体内具有高度的稳定性，能抵抗血浆中核酸酶的降解；(3)具有良好的细胞膜透过能力以实现细胞对其有效的摄取；(4)高效的抵达细胞质中(RNAi发挥作用的场所)，以减少溶酶体等细胞内物质对其的降解破坏。但天然碱基组成的siRNA无法满足上述这些要求，必须利用共价或非共价等化学修饰的手段，以提高siRNA的成药前景。目前，常用的共价化学修饰手段包括：糖环修饰，碱基修饰，磷酸骨架修饰，末端修饰等等；而非共价的修饰手段主要是采用含有阳离 子特性的载体来实现对siRNA的包载及递送。但是，单一的化学修饰方法往往具有一定的局限性，比如：对siRNA本身进行共价缀合和碱基修饰，在一定程度上能够提高siRNA的稳定性及其对靶mRNA的选择性从而降低脱靶效应，但是仍无法实现有效地细胞摄取；以非共价结合的方式采用载体对siRNA进行包裹，虽然在一定程度上解决了细胞摄取的难题，提升了siRNA的跨膜转运能力，但是这种单一的siRNA/载体静电复合物因其入胞方式的随机性并不能实现siRNA沉默效果的高效性，另外，过多的阳离子材料也会带来一定的细胞毒副作用。因此，为能够彻底而又系统性地解决天然siRNA存在的各种问题，本发明尝试采用多种化学修饰方式相结合的策略来实现siRNA转染的安全性和高效性。
发明内容
为了克服小干扰RNA在临床应用研究中的诸多不足，本发明提供了一种新型的小干扰RNA(siRNA)的化学修饰方法，本发明所提供的化学修饰方法是异核苷修饰、末端肽缀合及阳离子脂质载体包裹这三种修饰方法的两种或全部修饰方法的联合使用。经该化学修饰方法所获得的产品具有理化性质稳定、生物学行为良好可控、生物活性高效等优点，能够广泛用于抗病毒、抗肿瘤药物研究中。
本发明是在将目前siRNA应用过程中常见的两种策略(结构修饰与载体转运)有机结合的基础上，通过有意识地组装形成可控的超分子复合体系，从而实现对siRNA生物学行为(入胞途径和胞内代谢)的调控，进而使其高效地发挥沉默活性。因此，本发明的研究意在通过调控siRNA/载体复合物的组装结构来指导其生物学行为，最终实现siRNA发挥药效的高效性。这样也有助于减少siRNA及其相关载体的使用剂量，进一步避免或降低其所带来的毒性或免疫原性等副作用。
本发明的一种小干扰RNA(siRNA)的综合化学修饰方法，包括在小干扰RNA的正义链及/或反义链的一个或多个位点掺入D-构型或L-构型的异核苷，在小干扰RNA的正义链及/或反义链的3’-末端进行双肽缀合修饰，以及利用双子类型的阳离子脂质载体进行包载，通过上述的两种或全部修饰方法能够有效提高小干扰RNA的血清稳定性，降低脱靶效应，并实现对其跨膜途径的调控，使小干扰RNA能够按照所期望的途径或所占比例进入细胞(小窝蛋白介导的内 吞和巨胞饮途径)，从而降低小干扰RNA在胞内的破坏和降解程度，进一步提高siRNA的沉默活性，最终实现对siRNA递送的高效性和安全性。
异核苷是一类核苷碱基位置由1’位移至糖基的2’位的核苷类似物，碱基由糖环的1’位移至糖环的2’位，增加了糖苷键的稳定性。本实验室已利用不同原料分别合成了D-(化学式I所示)和L-(化学式II所示)两种构型的异核苷，其通式分别为如下所示：
Figure PCTCN2015000402-appb-000001
其中，n＝1、2、3；B为胸腺嘧啶基(T)、尿嘧啶基(U)、胞嘧啶基(C)、鸟嘌呤基(G)或腺嘌呤基(A)，化学式I所示的异核苷为D-构型的异核苷，化学式II所示的异核苷为L-构型的异核苷。
在本发明以前的工作中，已经证明不同构型的D-/L-异核苷能够对寡核苷酸的局部构象产生不同的影响，从而对寡核苷酸的理化性质和沉默活性产生影响。此外，由于D-/L-异核苷在结构上与天然核苷相似度较高，所以可以最大程度地保留或接近寡核苷酸原有的性质，可以作为一对分子探针应用到核酸与蛋白相互作用等机制的探讨领域。
在本发明中，优选的，在小干扰RNA的正义链及/或反义链的3’末端进行的肽缀合修饰，所缀合的肽片段可以是肽模拟物，所述的肽缀合片段与RNA部分连接的通式为：
Figure PCTCN2015000402-appb-000002
其中X为多肽序列，A为取代或未取代的苯环结构或碳原子，n为0、1、2、3、4或5。
细胞透膜性肽能够介导多种分子跨过细胞膜，采用透膜性肽介导的DNA和PNA细胞内转运已经得到广泛应用并取得较好的效果。本实验室早期从Kaposi  FGF信号肽(15肽)出发，通过计算机模拟得到具有疏水特性的6肽序列H-Leu-Ala-Leu-Leu-Ala-Lys-OH(KALLAL)，分别将其用于PNA和反义核酸共价缀合取得了比较理想的结果，发现该6肽序列不仅能够提高其稳定性和沉默活性，更能够很好地提高修饰寡核苷酸的跨膜能力。在本发明的具体实施例中，化学式III中X所代表的多肽序列为KALLAL或其相似性肽序列。
双子类阳离子脂质载体通常由阳离子头部、脂肪族尾部及连接臂组成。头部结构多为多肽、糖脂等阳离子基团，尾部通常为脂肪族饱和或不饱和长链以及胆固醇等疏水性分子，连接臂则以在体内能降解的二硫键或酰胺键为主。该类载体通过具有正电荷的阳离子水溶性头部与基因药物(DNA/PNA/siRNA)磷酸骨架的负电荷间发生静电吸附作用，将siRNA有效压缩并聚集在载体脂质微粒表面形成纳米复合物。当这种复合物被细胞摄取后，由于胞膜内外环境的变化，复合物结构由层状变为六面体状，致使载体复合物发生解组装，从而从溶酶体中逃逸到细胞质中并释放出DNA/RNA，进而产生沉默作用。
在本发明中，优选的，所述的阳离子脂质载体可以是商用的阳离子脂质载体，如RNAiMax，Oligofectamine等，或是通式为化学式IV的双子类阳离子脂质载体：
Figure PCTCN2015000402-appb-000003
其中，X为硫原子(S)或碳原子(C)，Y为含有正电荷的含氮基团或靶向基团，R为饱和或不饱和脂肪链或疏水性分子。
在本发明的一个具体实施例中，化学式IV中R所代表的不饱和脂肪链为油醇结构。
在本发明中，优选的，在小干扰RNA的正义链及/或反义链的一个或多个位 点掺入D-构型或L-构型的异核苷是通过固相合成实现的，即掺入位使用异核苷亚磷酰胺单体代替天然核苷的亚磷酰胺单体在相应位置进行偶联。
在进行siRNA修饰前，将化学式I和/或化学式II所示的异核苷化合物分别制备成化学式V和/或化学式VI所示的异核苷亚磷酰胺单体，每偶联一个核苷为一个循环，每个循环包括四个反应：脱DMT、偶联、封闭、氧化。
Figure PCTCN2015000402-appb-000004
其中，n＝1、2、3；B为胸腺嘧啶基(T)、尿嘧啶基(U)、氨基保护的鸟嘌呤基(G)、腺嘌呤基(A)或胞嘧啶基(C)。
在本发明中，优选的，合成DNA寡核苷酸链的条件是采用增加异核苷亚磷酰化单体的进样次数至3次；每次进样后的偶联时间为300秒/次；合成异核苷修饰的RNA寡核苷酸链的条件是每个循环进样后的偶联时间增加至900秒/次，偶联3次。
在本发明中，优选的，所述的综合化学修饰方法还包括与其他化学修饰策略的共同使用，包括2’-O-甲氧基(2’-OMe)、2’-氟代(2’-F)、锁定核苷酸(LNA)，磷硫骨架修饰及其他末端缀合方式等。
在本发明的一个具体实施例中，待修饰的小干扰RNA序列为靶向ERK通路中MEK1蛋白的mRNA的siMek1序列和靶向ERK通路中变异型B-Raf激酶蛋白的mRNA的siMB3序列。以上所述siMek1和siMB3修饰前的序列如下：
siMek1：正义链：5’-GCAACUCAUGGUUCAUGCUdtdt-3’；
反义链：5’-AGCAUGAACCAUGAGUUGCdtdt-3’
siMB3：正义链：5’-GCUACAGAGAAAUCUCGAUdtdt-3’
反义链：5’-AUCGAGAUUUCUCUGUAGCdtdt-3’
在本发明的一个具体实施例中，在以上所述siMB3序列的正反义链的3’末端均缀合化学式III的肽片段(PA/PS-siMB3)以及仅正义链3’末端缀合化学式III的肽片段(PS-siMB3)时，相较仅反义链3’末端缀合化学式III的肽片段(PA-siMB3)和未修饰的siMB3序列，PA/PS-siMB3以及PS-siMB3序列具有更 高的血清稳定性。
在本发明的一个具体实施例中，在以上所述siMek1序列和siMB3序列的正义链5’端开始第一个核苷酸掺入化学式I或化学式II所示的异核苷，而在正反义链的3’末端缀合化学式III的肽片段，并由阳离子载体RNAiMax，或阳离子脂质载体Oligofectamine，或化学式IV形成的阳离子脂质体包裹时，相较以上阳离子脂质体包裹未修饰siMek1序列和siMB3序列，具有更高的沉默活性。
在本发明的一个具体实施例中，在以上所述siMB3序列的正反义链的3’末端缀合化学式III的肽片段，并以化学式IV形成的阳离子脂质体材料为递送载体，通过对制剂工艺条件等相关参数的摸索和考察，最终确定了两种不同的siRNA/载体复合物的制备方法，研究表明得到的siRNA/载体复合物均具有较低的细胞毒性和较高的稳定性。
在本发明的一个具体实施例中，在以上所述siMek1序列和siMB3序列的正反义链的3’末端缀合化学式III的肽片段，并由化学式IV形成的阳离子脂质体包裹时，能够以特定的球形囊泡形态均匀、稳定的分布，两者相较由化学式IV形成的阳离子脂质体包裹未修饰siMek1序列和siMB3序列，能够产生更强的分子间相互作用，致使形成的复合物具有较低的表面电位、粒径较大且内部更为致密的纳米组装结构。
在本发明的一个具体实施例中，在以上所述siMB3序列的正反义链的3’末端缀合化学式III的肽片段，并由化学式IV形成的阳离子脂质体包裹形成的纳米复合物，能够使小干扰RNA按照所期望的途径或所占比例进入细胞(小窝蛋白介导的内吞和巨胞饮途径)，从而在一定程度上避开细胞内溶酶体的降解过程，实现更为高效的发挥沉默活性。
在本发明的一个具体实施例中，在以上所述siMB3序列的正反义链的3’末端缀合化学式III的肽片段，并由化学式IV形成的阳离子脂质体包裹形成的纳米复合物，相较以上阳离子脂质体包裹未修饰siMB3序列时，具有更高的沉默活性。
本发明通过对3’，3”-双肽-siRNA缀合物与阳离子脂质载体(通式为化学式IV)形成制剂时处方工艺条件的摸索优化，实现对其组装体的可控性，包括粒子表观形貌和内部结构、粒径和电位等参数的优化。
所述综合化学修饰方法，最优选的，包括以下步骤：
(1)称取CLD固体粉末，加无水乙醇溶解，得到CLD的乙醇溶液，4℃保 存；
(2)小干扰RNA的正反义链的3’-末端进行肽缀合修饰，使用DEPC水水化；
(3)按照VsiRNA/VCLD＝5/1，N/P＝3/1或5/1，加入步骤(1)和(2)得到的CLD的乙醇溶液以及DEPC水水化后的小干扰RNA，在超声温度为70℃下，超声40min，涡旋1min，得到纳米复合物颗粒。
形成的纳米复合物颗粒可以通过小窝蛋白介导的内吞和巨胞饮途径入胞方式等调控其进入细胞的途径或比例。
相较于现有技术，本发明的优点在于：
1.本发明提供的结合异核苷修饰、末端肽缀合及阳离子脂质载体包裹的化学修饰策略，能够发挥所用三种化学修饰方式各自的优势，取长补短，所获得的siRNA具有更高的血清稳定性和生物活性，且表现出良好可控的跨膜转运能力和靶mRNA的沉默效应，为siRNA技术的临床应用奠定了良好的基础。
2.3’，3”-双肽-siRNA缀合物与阳离子脂质载体存在双重结合作用，在较少的载体用量情况下，即可达到与商用载体相近的生物活性，从而实现更为高效的递送siRNA并降低载体自身所引起的生物毒性。
3.通过全面的考察D-，L-异核苷在siRNA的不同位点修饰后的血清稳定性和生物活性结果，可以获得siRNA不同位点受构象变化的影响差异。
4.通过对siRNA与阳离子脂质载体形成纳米复合物制备工艺条件和参数的摸索和优化，可以获得均一稳定的组装体系，并实现对其跨膜途径的调控，进而影响其在胞内的代谢行为，最终高效地发挥沉默活性，进一步推进siRNA的临床应用。
附图说明
图1为异核苷结合3’，3”-双肽-siMB3缀合物的血清稳定性结果(50％FBS)。
图2为异核苷结合3’，3”-双肽-siMek1缀合物的沉默活性结果(30nM，24h)，转染试剂为阳离子脂质体RNAiMax，上图为Western Blotting结果，下图为Real-time PCR结果。
图3为异核苷结合3’，3”-双肽-siMB3缀合物的Real-time PCR结果(30nM，24h)，转染试剂为阳离子脂质体RNAiMax。
图4为异核苷结合3’，3”-双肽-siMB3缀合物的Real-time PCR结果(30nM)， 转染试剂为阳离子脂质体Oligofectamine。
图5为异核苷结合3’，3”-双肽-siMB3缀合物的Western blotting结果(30nM)，转染试剂为阳离子脂质体Oligofectamine。
图6为琼脂糖凝胶阻滞电泳检测阳离子脂质载体/3’，3”-双肽-siRNA缀合物结合稳定性结果，其中siMek1**表示3’，3”-双肽siMek1缀合物，依次将N/P＝1、2、4、6、8、10、12的siMek1与阳离子脂质载体混匀，室温下孵育30min后，110v电压下电泳30min。
图7为两相混合法制备工艺(MT法)各参数对天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD制剂形成的影响。
图8为MT制备工艺的最优条件组合形成的制剂。
图9为天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的四种制剂的体外性质评价(血清稳定性实验、红细胞溶血实验和稀释稳定性实验)。
图10为天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的四种制剂的动态光扫描图(DLS)。
图11为阳离子脂质载体与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成制剂(AT法制剂)的透射电镜(TEM)图。其中，A为阳离子脂质载体CLD形态图；B为阳离子脂质载体CLD与天然siRNA形成的离子图；C为阳离子脂质载体CLD与3’，3”-双肽-siRNA缀合物结合形态图(N/P＝20，msiRNA＝200ng)。Bar：200nm；Bar in square：100nm。
图12为阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成制剂(AT法)的原子力显微镜(AFM)图。其中图A为单独阳离子脂质载体CLD形态图；图B为阳离子脂质载体CLD与天然siRNA结合形态图；图C为阳离子脂质载体CLD与3’，3”-双肽-siRNA缀合物结合形态图(N/P＝20，msiRNA＝200ng)。
图13为阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成四种制剂的细胞摄取情况。
图14为跨膜途径及各通道小分子细胞通路抑制剂的选择。
图15为阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成的四种制剂被抑制剂处理后的细胞摄取情况。
图16为阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成的四种制剂的入胞途径比例图。
图17为阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成四种制剂不同时间和不同浓度下的沉默活性RT-PCR结果。
图18为阳离子脂质载体CLD转染天然及异核苷联合3’，3”-双肽-siRNA缀合物的沉默活性结果。其中Blank为天然siRNA，Na为阳离子脂质载体CLD转染天然siRNA，PP为阳离子脂质载体CLD转染3’，3”-双肽-siRNA缀合物，D1PP为阳离子脂质载体CLD转染D-构型异核苷修饰的3’，3”-双肽-siRNA缀合物，L1PP为阳离子脂质载体CLD转染L-构型异核苷修饰的3’，3”-双肽-siRNA缀合物。
图19为阳离子脂质载体CLD分别与天然siRNA和异核苷联合修饰3’，3”-双肽-siRNA缀合物形成纳米复合物的制剂稳定性结果。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随具体实施例的描述更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明精神和范围下可以对本发明的技术方案和细节形式进行修改或替换，但这些修改和替换均落入本发明保护的范围内。
实施例1.异核苷结合末端肽缀合修饰的siRNA的及其血清稳定性评价
1、异核苷结合末端肽缀合修饰的siRNA的合成
在本实施例中，待修饰的小干扰RNA为靶向ERK通路中变异型BRaf激酶蛋白的mRNA的siMB3序列，修饰前的siMB3序列如下：
siMB3：正义链：5’-GCUACAGAGAAAUCUCGAUdtdt-3’
反义链：5’-AUCGAGAUUUCUCUGUAGCdtdt-3’
修饰策略选择以下任意一种：1)在以上所述siMB3序列正义链的5’端开始第一个核苷酸掺入化学式I或化学式II所示的异核苷。
Figure PCTCN2015000402-appb-000005
其中，n＝1；B为鸟嘌呤基(G)。
2)在以上所述siMB3序列的正反义链的3’末端均缀合化学式III的肽片段(PA/PS-siMB3)或仅正义链或正反义链的3’末端缀合化学式III的肽片段(PS-siMB3/PA-siMB3)。
Figure PCTCN2015000402-appb-000006
其中X为6肽序列H-Leu-Ala-Leu-Leu-Ala-Lys-OH(KALLAL)，A为碳原子，n为1。
3)在以上所述siMB3序列的正反义链的3’末端均缀合化学式III的肽片段(PA/PS-siMB3)或仅正义链或反义链3’末端缀合化学式III的肽片段(PS-siMB3/PA-siMB3)的同时，在以上所述siMB3序列正义链的5’端开始第一个核苷酸掺入化学式I或化学式II所示的异核苷。
经过上述修饰策略后得到的修饰后的siRNA如下表1所示：
表1
Figure PCTCN2015000402-appb-000007
Figure PCTCN2015000402-appb-000008
2、血清稳定性实验
分别考察经不同修饰后的siMB3序列在10％(体积分数)的胎牛血清中的稳定性。分别取天然siMB3、不同修饰后的siMB3序列4μL(20μM)+20μL FBS+16μL PBS至200μL微量离心管中，分别移取10μL至3个微量离心管中，置于37℃水浴孵育。随后在相同时间点取出样品立即放入-80℃冰箱或液氮中。在冰上骤冷后，用20％变性聚丙烯酰胺凝胶电泳进行分析，核酸染料进行染色15min，最后用化学发光凝胶成像系统对电泳结果进行成像分析。
3、结果
实验结果见图1。从该结果中可以看出：单独进行正义链5’末端异核苷修饰和肽缀合修饰时，D-异核苷对血清稳定性的影响较小，L-异核苷则会一定程度降低血清稳定性；而进行单肽缀合时，正义链末端缀合和反义链末端缀合出现了不同的结果，正义链单肽缀合与双肽缀合的结果相一致，均能够明显地提高血清稳定性，但在反义链进行单肽缀合则对血清稳定性影响较小。当在正义链5’端进行异核苷修饰时，无论D-/L-异核苷修饰，均使在反义链单肽缀合的血清稳定性提高，而使正义链单肽缀合的血清稳定性降低。说明核酶的端基选择性与热力学有关，而异核苷能改变平衡。肽缀合修饰弥补了异核苷修饰对血清稳定性的影响，能够得到使稳定性大幅提高的修饰策略。
实施例2.异核苷结合末端肽缀合修饰的siRNA/阳离子脂质体RNAiMax的生物活性评价
实验操作：Hela细胞10万/孔铺6孔板，过夜培养。将5μL RNAiMAX与终浓度为30nM的修饰后的siRNA混合，常温孵育15min，加入到细胞培养板中，孵育24h。利用TRizol提取总RNA，将总RNA反转录成为cDNA，进而利用Gotaq Green Mix进行Real-time实验，考察siRNA在mRNA水平基因沉默作用。提取总蛋白，利用BSA对总蛋白定量，利用聚丙烯酰胺凝胶(分离胶为10％，浓缩胶为5％)分离蛋白，转膜后，进行一抗和二抗免疫反应，最终通过 bio-rad公司的化学发光凝胶检测系统检测蛋白敲除效果。
实验结果：根据PCR结果和Real-time PCR结果(图2)，当对靶向ERK通路中MEK1蛋白的mRNA的siMek1序列进行修饰时：在正义链的5’末端进行异核苷修饰时，无论在无肽缀合或是在不同的肽缀合修饰情况下，都发现L-异核苷修饰后相较于D-异核苷修饰对siRNA沉默活性的提高更为明显。而对比不同肽缀合情况时，发现对活性提高的顺序依次为正义链单肽缀＜反义链单肽缀＜正反义链双肽缀，而且无论在结合正义链5’末端D-异核苷修饰还是L-异核苷修饰时，结果都是这样的活性变化规律。在仅仅是肽缀合而没有异核苷修饰时，便得到了同样的活性变化规律，说明异核苷修饰和肽缀合修饰在活性的影响上相互加成的。因此，通过PCR结果和Real-time PCR结果发现了活性提高最为明显的修饰策略：正义链5’末端L-异核苷修饰的3’，3”-双肽-siRNA缀合物。
当进行靶向Braf-mutant mRNA的siMB3的修饰及活性评价时，根据Real-time PCR结果(图3)，在所有siMB3先导结构中，仍然发现正义链5’末端L-异核苷修饰结合3’，3”-双肽-siRNA缀合物的沉默活性最好。但与修饰的siMek1序列结果不同的是，在siMB3修饰结果中发现3’，3”-双肽-siRNA缀合物的沉默活性明显提高，甚至高于结合了正义链5’末端L-异核苷修饰siRNA的沉默活性结果。同时，D-/L-异核苷修饰对沉默活性的影响也没有在siMek1中明显。通过之前对比siMek1和siMB3的血清稳定性，可以发现siMek1相较于siMB3在血清中要明显的更为稳定。由于siMB3的血清稳定性更低，因此双肽缀合在siMB3中对血清稳定性的提高也更为明显。siRNA的血清稳定性提高，意味着在体内存在的完整siRNA更多，从而发挥沉默活性的siRNA也变更多。因此，由于血清稳定性的提高对siMB3更为明显，因此双肽缀合对于siMB3沉默活性的提高也更为明显。而D-/L-异核苷的影响较小，说明正义链5’末端异核苷修饰对沉默活性的提高存在序列特异性。这可能与不同序列的正反义双链在进入RISC复合体时，所存在的竞争性程度不同有关。初步推测，当正反义链5’末端热力学差异较大时(siMB3)，在形成RISC复合体时，反义链将占据明显的优势，因此利用正义链5’末端isoNA修饰限制正义链进入RISC复合体的效果则不会很明显；而当正反义链5’末端热力学差异较小时，在形成RISC复合体时，正反义链进入RISC复合体的能力接近，因此当正义链5’末端异核苷修饰阻碍了正义链进入RISC复合体的能力时，反义链进入RISC复合体的能力将增加更多，从而沉默活性提高的更为明显。
实施例3.异核苷结合修饰的3’，3”-双肽-siRNA缀合物/阳离子脂质体Oligofectamine的生物活性评价
实验操作：将A375细胞以10万/孔铺6孔板，过夜培养。将4μL Oligofectamine与终浓度为30nM的修饰后的siRNA混合，常温孵育20min，加入到细胞培养板中，孵育24h-72h。利用TRizol提取总RNA，将总RNA反转录成为cDNA，进而利用Gotaq Green Mix进行Real-time实验，考察siRNA在mRNA水平基因沉默作用。提取总蛋白，利用BSA对总蛋白定量，利用聚丙烯酰胺凝胶(分离胶为10％，浓缩胶为5％)分离蛋白，转膜后，进行一抗和二抗免疫反应，最终通过bio-rad公司的化学发光凝胶检测系统检测蛋白敲除效果。
实验结果：根据Oligofectamine转染30nM siRNA先导结构的Real-time PCR结果(图4)，孵育24h时，先导结构PA/PS-siMB3-S01D和PA/PS-siMB3-S01L的沉默活性均略好于天然siMB3和3’，3”-双肽-siRNA缀合物(PA/PS-siMB3)，而其中PA/PS-siMB3的沉默活性相对最弱。当孵育时间延长至48h时，天然siMB3的沉默活性明显降低，先导结构PA/PS-siMB3-S01D和PA/PS-siMB3-S01L的沉默活性虽然也略有降低，但其沉默活性仍好于天然siMB3，但3’，3”-双肽-siRNA缀合物的沉默活性却相较孵育24h时有明显提高。当孵育时间延长至72h时，mRNA的水平均已明显恢复，这应该是由于随着孵育时间的延长，在开始被siRNA降解的mRNA又经过不断地转录得到了恢复。但通过不同修饰序列间的对比，仍能发现双肽缀合及双肽缀合结合正义链5’末端L-异核苷修饰的沉默活性最好，这说明双肽缀合在一定程度上能够延长siRNA发挥沉默活性的时间。初步分析，沉默时间的延长应该与双肽缀合明显提高了siRNA的胞内稳定性有关。而且，结合双肽缀合siRNA的血清中降解的规律，可以发现缀合肽在血清中大多是先于siRNA双链自身内部的降解，也即是肽缀合能够阻碍核酶对siRNA末端的进攻，当缀合肽未被切割下来前，都能够延缓核酶对siRNA的进攻。从另一方面，也可能能够延缓Dicer蛋白及TRBP蛋白的识别，从而起到延缓siRNA发挥沉默活性的作用。因此，当延长孵育时间至72h时，3’，3”-双肽-siRNA缀合物序列相较于天然siRNA均具有着更好地沉默活性。根据Western blotting结果(图5)，当孵育48h时，根据Braf下游蛋白p-ERK的表达情况，同样发现先导结构PA/PS-siMB3-S01L的沉默活性明显好于天然siMB3和3’，3”-双肽-siRNA缀合物的。而当孵育时间延长至72h时，3’，3”-双肽-siRNA缀合物 和先导结构PA/PS-siMB3-S01L的沉默活性仍均好于天然siMB3。
实施例4.利用琼脂糖凝胶电泳考察3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD结合的结合能力
3’，3”-双肽-siRNA缀合物的制备：
修饰前的siMek1序列如下：
siMek1：正义链：5’-GCAACUCAUGGUUCAUGCUdtdt-3’；
反义链：5’-AGCAUGAACCAUGAGUUGCdtdt-3’
在以上所述siMek1序列的正反义链的3’末端均缀合化学式III的肽片段(PA/PS-siMek1)：
Figure PCTCN2015000402-appb-000009
其中X为6肽序列H-Leu-Ala-Leu-Leu-Ala-Lys-OH(KALLAL)，A为碳原子，n为1。
CLD分子具体结构如下所示：以赖氨酸作为阳离子头部，中间连接臂由胱氨酸残基组成，尾部由油醇(9，10-顺式双键的十八碳的饱和脂肪链烃)组成亲脂性尾链。
Figure PCTCN2015000402-appb-000010
实验操作(AT法)：称取CLD 4.2mg于已硅烷化处理的锥形瓶中，溶于一定量有机溶剂中(氯仿/甲醇＝1∶1，v/v)，震荡溶解后，以适当流速的氮气或是氩气吹干溶剂，使溶解物成贴壁薄膜状，残留的溶剂减压慢慢蒸去，取出，放 于真空干燥器中干燥一晚。于第二天取出，加入1mL DEPC水，加塞封口。于50℃的水浴中超声30min，过0.25μm无菌滤膜，即制得阳离子脂质体。取已配制好的阳离子脂质体复合物(4.2mg/mL，相当于N/P＝20时CLD的浓度)及已有浓度为20μM的siRNA水溶液，Opti作为溶剂，依次配制相当于N/P＝1、2、4、6、8、10、12转染试剂，加入3’，3”-双肽-siRNA缀合物水溶液后，室温孵育30min，即完成CLD/3’，3”-双肽-siRNA缀合物复合物的制备。通过琼脂糖凝胶电泳阻滞试验对3’，3”-双肽-siRNA缀合物与CLD的结合能力进行考察，实验条件如下：1％的琼脂糖凝胶；1×TAE电解溶液；电压110V；电泳时间30min；染料是Goldview；阳离子载体为CLD。
实验结果：根据图6可以看出，随着N/P的不断增加，可见阳离子脂质载体(式IV，R＝油醇)能够有效的与3’，3”-双肽-siRNA缀合物相结合，并最终能够完全结合。当N/P＝1时，阳离子脂质载体就已经开始能够有效的与3’，3”-双肽-siRNA缀合物相结合。随着N/P的升高，游离3’，3”-双肽-siRNA缀合物条带亮度进一步逐渐变弱。当N/P＝12时，游离双3’，3”-双肽-siRNA缀合物条带亮度相对于对照组siRNA亮度已经变的非常暗了，此时阳离子脂质载体化合物已能完全结合3’，3”-双肽-siRNA缀合物(见图6)。与3’，3”-双肽-siRNA缀合物结合能力考察结果再次说明双重作用的存在能够提高siRNA与阳离子脂质载体阳离子脂质载体的结合能力。
实施例5.两相混合法(MT法)制备天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD纳米复合物的工艺参数摸索及确定
参数条件的设定：将制备一定浓度的CLD乙醇溶液缓慢滴加至具有一定浓度的siRNA水溶液中，涡旋一定时间后，超声制得纳米颗粒。在两相混合法的操作过程中，纳米颗粒的形成和稳定受到5种因素的相互制约：A.体积比VsiRNA/VCLD；B.材料浓度比(N/P)(N是指CLD中含有的可质子化N个数，P是指siRNA中含有的磷酸量)；C.超声温度；D.超声时间；E.涡旋时间。因此，为摸索两相混合法的最优条件组合，每个因素设定四个参数：
A.A1：5/1、A2：10/1、A3：15/1、A4：20/1
B.B1：3/1、B2：5/1、B3：7/1、B4：10/1
C.C1：40℃、C2：50℃、C3：60℃、C4：70℃
D.D1：10min、D2：20min、D3：30min、D4：40min
E.E1：20s、E2：40s、E3：1min、E4：2min
设计L16(45)正交实验，共16组平行实验，通过考察每种因素对多分散性系数(Poly Dispersity Index，PDI)的影响，确定最优的制剂处方工艺。
实验操作：准确称取一定量CLD固体粉末，加无水乙醇溶解，终浓度为1.142mg/mL(100μM)，4℃保存。siRNA使用DEPC水水化，终浓度10μM(备用)。依次按照不同体积比(A)和不同N/P比(B)将CLD乙醇溶液滴入siRNA水溶液，涡旋混匀一定时间(E)。之后，按照不同超声温度(C)和不同超声时间(D)制备，完成后测定粒径和电位。
实验结果及分析(图7)：以PDI作为基准，对5种因素进行了分析可以得出两种最优的条件组合A和B，即：体积比VsiRNA/VCLD＝5/1，N/P＝3/1(A)和5/1(B)，超声温度为70℃，超声时间40min，涡旋时间1min。在这种制剂工艺条件下，3’，3”-双肽-siRNA缀合物与CLD可以形成稳定的纳米颗粒(图8)，且B组能够得到最优的纳米复合物颗粒。
实施例6.天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的四种制剂的体外性质评价(血清稳定性实验、红细胞溶血实验和稀释稳定性实验)
实验操作：天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的四种制剂按照实施例4和实施例5进行制备。
血清稳定性实验：在96孔板中分别加入100μL的不同制剂和5％葡糖糖溶液，与100μL胎牛血清混匀，37℃孵育，分别于0min、5min、10min、30min、1h、3h、5h、10h、24h、33h、48h利用Bio-Rad酶标仪测定630nm处的吸光度(OD值)，每个样品设置3个复孔。
红细胞溶血实验：从SD大鼠眼静脉丛取血，4℃1500g离心10min，弃血清，用0.9％的生理盐水洗涤分离得到的血红细胞后，用pH 7.38的PBS磷酸盐缓冲液混悬制成2％(v/v)血红细胞混悬液。96孔板中加入血细胞悬液100μL，再加入100μL的PBS(阴性对照)、1％Triton X-100(阳性对照)或者系列浓度的样品，37℃孵育1h。完整的血红细胞通过离心去除，用Bio-Rad酶标仪测定上清液540nm处吸光度值。相对溶血率用下面的公式计算：
([Abs]sample-[Abs]buffer)/([Abs]Triton X-100-[Abs]buffer)×100％.
稀释稳定性实验：按照siRNA(200μL，2μM)/CLD(40μL，50μM)制备的四种制剂，之后分别将制剂稀释5倍、10倍、20倍、40倍，观察其表征变化(测 定电位时再稀释5倍)。
实验结果(图9)：可以看出四种制剂组和FBS组的吸光度值并无明显差别，这可以说明四种制剂在血清中不会和血清白蛋白等发生凝集反应，是十分安全的；MT法制得的天然siRNA/CLD复合物制剂的溶血率最高，这与其表面电位最高有关。此外，MT法制剂的溶血率相对高于AT法，这是和组装方式有关的，即：MT法中siRNA主要在内层，而AT法中的siRNA更多在外层，进而电位就会有所差别，溶血率也会有所差别。总之，四种制剂的溶血率都相对偏低，比较安全；就两种制备工艺方法来讲，AT法制剂在稀释的过程中电位和粒径变动较大，这说明MT法制得制剂的稀释稳定性明显大于AT法，这也从侧面反映出在MT法中无论是天然siRNA还是3’，3”-双肽-siRNA缀合物与CLD的相互作用强于AT法。在同一MT方法制得的制剂中，3’，3”-双肽-siRNA缀合物形成的制剂经稀释后的表面电位低于天然siRNA形成的电位。
实施例7.天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的四种制剂的动态光扫描图(DLS)
实验操作：天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的四种制剂按照实施例4和实施例5进行制备。各取一定量的水溶液放入EP管中，在动态光散射仪(Dynamic Light Scattering Instrument，型号：Zetasizer Nano ZSP)测定相应的水合粒径、表面电位和多分散系数等相关参数。
实验结果(如图10及表2所示)：经过制备工艺条件的摸索和考察，目前通过两种制备工艺均能够制得相对均一、稳定的类球形纳米颗粒复合物。四种制剂的多分散系数(Polydispersity index，PDI)均小于0.3，这说明形成的制剂具有良好的动态分布。四种制剂的表面电位均能够控制在+20～30mV范围区间，而这个范围的表面电位有利于细胞对制剂的有效摄取，同时最大程度地降低了因正电荷所带来的细胞毒性问题。其中在同一制剂工艺方法中，3’，3”-双肽-siRNA缀合物/CLD形成制剂的表面电位均低于天然siRNA/CLD形成的制剂，这在保证细胞有效摄取的基础上，3’，3”-双肽-siRNA缀合物具有更低的细胞毒副作用，能够更为安全的发挥其沉默活性。就水合粒径来讲，通过该处方工艺可以使粒径控制在100nm～150nm左右，这种粒径的粒子在体内具有较好的被动靶向作用，也就是增强渗透滞留效应(Enhanced Permeability and Retention Effect，EPR)。在旋膜水化法中(AT法)，3’，3”-双肽-siRNA缀合物与阳离子脂质载体 CLD可以形成粒径较大且表面电位较低的纳米复合物，这种特征与天然siRNA的组装体系有着明显的差别，因此也会由此产生后期生物学行为的特异性。
表2
Figure PCTCN2015000402-appb-000011
实施例8.阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物相结合的超分子结构表征(AT法制剂)
实验操作：采用透射电子显微镜(TEM)和原子力显微镜(AFM)共同考察CLD脂质体及其与天然siRNA(siMek1)和3’，3”-双肽-siRNA缀合物(PA/PS-siMek1，实施例4方法制备)形成复合物的粒子内部结构和表观形貌。其操作如下：先按照实施例4的操作分别制备单独脂质体为例、天然siRNA与CLD纳米复合物、3’，3”-双肽-siRNA缀合物与CLD形成的纳米复合物。分别取三种复合物水溶液4μL，滴注于方孔铜网(型号为GilderGrids 400目)，室温下自然挥干。采用透射电子显微镜(型号：Philips Tacnai G2 20 S-TWIN Microscope operating at 200kV)对其进行内部结构扫描，得三者的TEM图像。分别取三种复合物水溶液1mL，滴注与1cm2的石英覆盖片上，室温挥干溶剂后，采用AFM(型号：Multimode IIIa AFM Veeco Metrology，USA)设备对其进行表观形貌的分析和成像。
实验结果：图11为单独阳离子脂质载体微粒、阳离子脂质载体CLD与天然siRNA复合物、阳离子脂质载体CLD与3’，3”-双肽-siRNA缀合物复合物三组样品形态的透射电镜扫描结果。在单独阳离子脂质载体微粒组中，阳离子脂质载体呈现球状，为典型的球状脂质囊泡。当阳离子脂质载体CLD分别与天然siRNA及3’，3”-双肽-siRNA缀合物结合后，脂质微粒的结构形态出现了明显的变化：就图11B而言，在球中心部位颜色较深，而边缘部分相对较浅。说明在形成复合物后，天然siRNA能够有效的被阳离子脂质载体CLD所包载，使siRNA处于复合物的内部，被阳离子脂质载体CLD所保护。从图11C中可以看到，阳离子脂质载体CLD包载3’，3”-双肽-siRNA缀合物形成复合物的表观形貌与上述两 种有着明显的差别：形成颜色更深的类球状结构粒子，且没有明显的边缘化现象(如图11B)。当带正电荷的阳离子脂质载体微粒与带负电荷siRNA相互作用时，由于电荷间的相互作用，siRNA将会被压缩结合于脂质微粒表面。这种压缩作用势必会在脂质微粒的表层形成siRNA压缩层，从而在脂质微粒的表面形成颜色更深的色带。根据TEM的工作成像原理可以推出这时的粒子压缩得更为紧密。因此，通过TEM的结果可以看到：相对于天然siRNA，3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的复合物压缩得更为紧密，这种现象的产生是由于3’-末端共价缀合的肽序列所引起的。
阳离子脂质微粒与天然siRNA(siMek1)及3’，3”-双肽-siRNA缀合物(siMek1)相互作用所形成复合物的原子力显微镜(AFM)结果显示(图12)，阳离子脂质载体与天然siRNA及3’，3”-双肽-siRNA缀合物所形成复合物，呈现均匀的圆形且分布均匀。这样的结果与TEM扫描结果是一致的。当两者实验条件一致时(N/P＝20，msiRNA＝200ng)，阳离子脂质载体与3’，3”-双肽-siRNA缀合物的结合形态不同于阳离子脂质载体与天然siRNA的结合形态。图A可以看出，阳离子脂质载体CLD的水平距离(即直径)约120nm，垂直距离约6nm。垂直距离和水平距离差距很大的原因是由于制样时采用挥干溶剂法，即将配制一定浓度的材料溶液滴加到云母片上，使溶剂挥发后进行检测，故被测粒子在干燥过程中会受重力影响向下塌陷，从而使垂直距离变低。图B显示阳离子脂质载体CLD与天然siRNA形成复合物的直径约100nm，垂直距离约5nm。由于阳离子脂质载体CLD与天然siRNA发生静电吸附作用，使阳离子脂质载体CLD进一步被压缩，故形成复合物的粒径较阳离子脂质载体CLD略小。图3中显示阳离子脂质载体CLD与3’，3”-双肽-siRNA缀合物形成的复合物的粒径在130nm左右，垂直距离约6.6nm。其粒径和垂直距离均大于天然siRNA与阳离子脂质载体CLD形成的复合物，针对这种现象并结合TEM数据可以得出以下结论：3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD间的相互作用力大于天然siRNA与阳离子脂质载体CLD间单纯的静电作用力，使其形成的复合物压缩更紧密的同时形成夹层更多，使单一复合物的粒径增大。天然siMB3与3’，3”-双肽-siRNA缀合物和阳离子脂质载体CLD形成的复合物存在着差异性，这种差异性体现在双肽缀siRNA能够更为相对紧密的与阳离子脂质载体CLD相结合，这种现象的产生是由于3’-末端共价缀合的肽序列所引起的。综上所述，由于末端肽的引入，使3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成超分子复 合物的作用模式与天然siRNA和阳离子脂质载体CLD的作用模式具有显著差异，这种差异所带来的化学和生物学的意义将结合后续阳离子脂质载体/天然siRNA与阳离子脂质载体/3’，3”-双肽-siRNA缀合物的生物活性评价结果做进一步的分析。
实施例9.阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成四种制剂的细胞摄取情况
实验操作：天然及3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的四种制剂按照实施例4和实施例5进行制备。将黑色素瘤A375细胞以30万/孔接种于6孔板中，培养24h使细胞贴壁，37℃孵育15min。用OPTI-MEM稀释10倍，双肽siRNA及天然siRNA的终浓度为100nM。把培养基吸弃，用1mL PBS洗2遍，每孔加入2mL将上述包载Cy3标记的3’，3”-双肽-siRNA缀合物及天然siRNA的制剂，培养4h。之后按照流式细胞术法在流式细胞仪中检测制剂的摄取情况。
实验结果(如图13)：可以看到，黑色素瘤A375细胞对3’，3”-双肽-siRNA缀合物/CLD复合物和天然siRNA/CLD复合物的摄取情况时有显著性差别的。无论是在给药后的4h还是6h，在同一处方工艺条件下制得的制剂中，3’，3”-双肽-siRNA缀合物的细胞摄取情况显著性地高于天然siRNA的细胞摄取情况，而在相同siRNA条件下，两种制备工艺方法对细胞摄取的情况并没有显著的差异性。这些结果在一定程度上暗示了3’，3”-双肽-siRNA缀合物的细胞跨膜方式不同于天然siRNA。
实施例10.阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成的四种制剂被抑制剂处理后的细胞摄取情况
1.细胞通路及相应抑制剂的选择
细胞摄取外源性物质的途径主要分为以下几种：细胞吞噬、特异性受体介导入胞、巨胞饮、网格蛋白介导内吞和小窝蛋白介导内吞等等。通过这些途径进入细胞的外源性物质绝大部分都经过溶酶体的酸化过程，最终被降解或排出胞外。近期研究表明：在这些细胞通路中，当外源性物质以小窝蛋白介导的内吞形式进入细胞后，可直接被释放到细胞质中，在一定程度上能够避开溶酶体的降解过程；此外，巨胞饮途径虽然经过溶酶体过程，但其逃逸速度相对较快。 因此，药物以小窝蛋白介导的内吞或者巨胞饮途径进入细胞时有利于实现高效地发挥其生物活性。以内吞方式为重点考察对象，设计并考察四种入胞的途径，即：巨胞饮途径、网格蛋白介导内吞、小窝蛋白介导内吞和能量依赖性内吞。并整理相关研究资料总结出每种入胞途径相对应的小分子抑制剂(图14所示)。
2.各小分子细胞通路抑制剂对细胞摄取双肽siRNA及天然siRNA的考察
实验操作：人黑褐色素瘤细胞A375分别以每孔30万接种于12孔板中，培养24h后洗净原培养基，用1mL预冷PBS洗2遍，分别以2mL/孔加入不同浓度的抑制剂(如图14)，37℃、5％CO2孵育30min。之后，以200μL/孔加入不同CLD/Cy3-siRNA复合物(制剂操作同实施例4和实施例5)，Cy3-siRNA终浓度为100nM。37℃、5％CO2孵育6h后，用300μL胰酶室温消化3min，加入1mL培养基终止消化，吸至2mL离心管离心，弃培养基，加入1mL预冷的PBS洗涤两遍并使用400μL PBS重悬细胞，使用流式细胞仪测量细胞中Cy3荧光强度，采集10000个细胞，激发波长为550nm，发射波长为590nm，采集数据使用FCS Express V3软件分析。
实验结果(如图15所示)：就巨胞饮途径来讲，相对于旋膜水化法(AT法)制得的两种制剂，两相混合法(MT法)制得的制剂占有比例的较多，其中MT法制得的3’，3”-双肽-siRNA缀合物占有的比例最大。就网格蛋白介导的内吞途径来讲，旋膜水化法(AT法)制得的天然siRNA以金刚烷胺通道占有的比例最大；此外，采用氯丙嗪对细胞通道抑制后，其细胞摄取反而增大，这说明单独抑制该通路后，会激活其他通路从而增加细胞对siRNA的摄取。就小窝蛋白介导的内吞途径来讲，在同一制剂处方工艺条件下(无论是AT法还是MT法)，3’，3”-双肽-siRNA缀合物占有的比例均大于天然siRNA。ATP依赖的途径中，四种制剂都对该途径有较大的依赖，彼此间并不存在显著性差异。
经过对四种制剂入胞机制的探讨可以总结出如下几点(如图16所示)：1)四种制剂的细胞摄取并不是单一途径；2)在同一制剂处方条件下(无论是AT法还是MT法)，3’，3”-双肽-siRNA缀合物依赖小窝蛋白介导内吞的比例要显著高于天然siRNA，而依赖网格蛋白介导的内吞比例显著低于天然siRNA；3)在旋膜水化法(AT法)制得的制剂中，3’，3”-双肽-siRNA缀合物和天然siRNA依赖巨胞饮的内吞所占比例显著低于两相混合法(MT法)中对应比例；4)由2)和3)的结论可以推测两相混合法(MT法)制得的制剂会更多绕过溶酶体(即小窝蛋白介导和巨胞饮会绕过溶酶体或快速实现溶酶体逃逸)，而且在两相混合法 (MT法)中3’，3”-双肽-siRNA缀合物和天然siRNA的差异性显著高于旋膜水化法(AT法)制得的制剂，这种结果会导致更多的基因沉默效应差异。
因此，通过处方工艺条件的摸索和优化，能够可控的得到均一稳定的3’，3”-双肽-siRNA缀合物及天然siRNA与阳离子脂质载体CLD形成的超分子组装体，进而调控3’，3”-双肽-siRNA缀合物的入胞途径，使其更为高效的发挥RNAi的效应。同时，根据对跨膜机制的解析，也可以对3’，3”-双肽-siRNA缀合物及天然siRNA的沉默活性进行推测：1)无论是MT法还是AT法，3’，3”-双肽-siRNA缀合物的沉默活性都高于天然siRNA(因为3’，3”-双肽-siRNA缀合物在小窝蛋白和巨胞饮占有的比例相对天然siRNA较多)；2)四种制剂中，MT法制得的3’，3”-双肽-siRNA缀合物/CLD复合物的沉默活性是最好的(因为小窝蛋白和巨胞饮途径占据了99％)；3)AT法制得的天然siRNA/CLD复合物的沉默活性是最差的(因为网格蛋白占有的比例最大，这意味着有较多的siRNA被溶酶体降解的机率)。
实施例11.阳离子脂质载体CLD与天然siRNA及3’，3”-双肽-siRNA缀合物结合形成四种制剂不同时间和不同浓度下的沉默活性RT-PCR结果
实验操作：3’，3”-双肽-siRNA缀合物及天然siRNA与阳离子脂质载体CLD形成的四种制剂按照实施例4和实施例5进行制备。人黑色素瘤A375细胞10万/孔铺6孔板，过夜培养。将5μL CLD分别与终浓度为30nM、60nM、和100nM的修饰后的siRNA混合，常温孵育15min，加入到细胞培养板中，孵育24h。在不同时间点(24h、48h、72h)条件下，利用TRizol提取总RNA，将总RNA反转录成为cDNA，进而利用Gotaq Green Mix进行Real-time实验，考察siRNA在mRNA水平基因沉默作用。提取总蛋白，利用BSA对总蛋白定量，利用聚丙烯酰胺凝胶(分离胶为10％，浓缩胶为5％)分离蛋白，转膜后，进行一抗和二抗免疫反应，最终通过bio-rad公司的化学发光凝胶检测系统检测蛋白敲除效果。
实验结果(如图17所示)：在60nM和100nM给药浓度时四种制剂具有较好的沉默效果，且呈现出显著性差异。在60nM和100nM浓度时(与细胞摄取浓度一致)，MT法制得的3’，3”-双肽-siRNA缀合物制剂的沉默效果优于其他三组制剂，且呈显著性差异。两种制剂来讲，MT法的两组制剂对靶mRNA的沉默活性优于AT法两组对应的制剂。此外，四种制剂的沉默效应呈浓度依赖性的增加。
沉默效应的实验结果与我们根据跨膜通路所预测的结果相一致。这说明我们可以通过对3’，3”-双肽-siRNA缀合物与阳离子脂质载体CLD形成的纳米复合物的跨膜通路进行调控，实现对后期沉默活性的预知和判断，在一定程度上改变siRNA的胞内代谢行为，实现基因沉默的高效性。
实施例12.利用Real-time实验考察异核苷联合修饰的3’，3”-双肽-siRNA缀合物/阳离子脂质体CLD的生物活性
异核苷联合修饰的3’，3”-双肽-siRNA缀合物的制备：
修饰前的siMek1序列如下：
siMek1：正义链：5’-GCAACUCAUGGUUCAUGCUdtdt-3’；
反义链：5’-AGCAUGAACCAUGAGUUGCdtdt-3’
在以上所述siMek1序列的正反义链的3’末端均缀合化学式III的肽片段(PA/PS-siMek1)；
Figure PCTCN2015000402-appb-000012
其中X为6肽序列H-Leu-Ala-Leu-Leu-Ala-Lys-OH(KALLAL)，A为碳原子，n为1。
同时在siMek1序列正义链的5’端开始第一个核苷酸掺入化学式I所示的异核苷(D1PP)或是在在siMek1序列正义链的5’端开始第一个核苷酸掺入化学式II所示的异核苷(L1PP).
实验操作：将Hela细胞以20万/孔铺6孔板，孵育12-14h后进行转染在siRNA浓度相同(30nM)的情况下，商用转染试剂RNAiMAX的用量为5μL，阳离子脂质载体的用量为2.6μL(浓度为6.3mg/mL)。将转染试剂与siRNA混合后室温孵育25min，加入到培养板中，培养24h。利用TRizol提取总RNA，将总RNA反转录成为cDNA，进而利用Gotaq Green Mix进行Real-time实验，考察siRNA在mRNA水平基因沉默作用。
实验结果：Real-time PCR考察阳离子脂质载体CLD包载异核苷联合修饰的3’，3”-双肽-siRNA缀合物对靶mRNA的沉默结果表明(图18)，与对照组的沉默 效果相比，采用阳离子脂质载体CLD转染天然siRNA、3’，3”-双肽-siRNA缀合物及异核苷联合修饰的3’，3”-双肽-siRNA缀合物都能有效的抑制相关靶mRNA。其中在阳离子脂质载体CLD作为转染试剂条件下，3’，3”-双肽-siRNA缀合物与天然siRNA的沉默效果相当，这说明在确保沉默活性不受影响的条件下双肽缀siRNA能够更为有效的提高血清稳定性，降低核酶的降解情况。此外，经异核苷修饰的3’，3”-双肽-siRNA缀合物较3’，3”-双肽-siRNA缀合物具有更好地沉默活性，说明在提高稳定性的基础上异核苷联合双肽缀的修饰策略能够显著提高其对靶标的沉默能力。在图中可以看到以D-构型异核苷联合合修饰的3’，3”-双肽-siRNA缀合物在采用阳离子脂质载体CLD转染时具有最好的沉默活性。这说明了异核苷结合肽缀合修饰、以及阳离子脂质载体包载的小干扰RNA具有良好的应用前景和继续开发的价值。
实施例13.利用凝胶电泳实验考察异核苷联合缀修饰的3’，3”-双肽-siRNA缀合物/阳离子脂质体CLD及天然siRNA/阳离子CLD的制剂稳定性
实验操作：异核苷联合修饰的3’，3”-双肽-siRNA缀合物/CLD制剂的制备参见实施例4。采用聚阴离子置换实验考察异核苷联合修饰的3’，3”-双肽-siRNA缀合物/阳离子脂质体CLD的稳定性。本实验的聚阴离子选择的是肝素。肝素是一种磺酸化的粘多糖，在人体无论是在细胞内环境还是在细胞外环境，都存在着多种酸性蛋白。这些蛋白带有负电荷，可以与siRNA发生相互竞争从而破坏siRNA/载体系统的稳定性。如果载体复合物中的siRNA被肝素置换的速度和程度较差，这说明该siRNA/载体复合物是稳定的，这增加了该复合物在体内的稳定性，利于更好地发挥沉默活性。
按照实施例4的操作制备CLD脂质体。以一定比例制备异核苷联合修饰的3’，3”-双肽-siRNA缀合物/CLD复合物和天然siRNA/CLD纳米复合物，形成稳定制剂。将不同的制剂分别与0.1、0.2、0.3、0.4、0.5、0.75、1.0、1.5、3.0IU肝素/μg siRNA混合，37℃孵育30min后，加入5×上样缓冲液，在含有0.5μg/mLEB的1％琼脂糖凝胶上80V电压电泳3min，再100V电泳15min，凝胶成像系统观察EB/siRNA荧光。游离siRNA用作对照组。
通过研究结果表明(图19)：天然siRNA与阳离子脂质体CLD形成的复合物稳定性是最差的，因为天然siRNA在肝素0.2IU/μg的条件下就开始出现游离现象，说明天然siRNA最早被聚阴离子肝素置换出来；就3’，3”-双肽-siRNA缀 合物来讲，在肝素0.3IU/μg的条件下开始出现轻微的游离现象，这说明由于在siRNA末端双肽的引入，导致其与阳离子脂质体CLD的相互作用增强，从而整个系统的稳定性比天然siRNA相比有明显的提升；就异核苷联合修饰的3’，3”-双肽-siRNA缀合物来讲，无论是D-构型还是L构型异核苷的掺入，都使得其与CLD的相互作用进一步增强，但增强幅度不大(与3’，3”-双肽-siRNA缀合物的稳定性相类似)。这是因为在核酸药物与CLD载体间相互作用过程中主要依靠静电吸附和一定的疏水力作用，而异核苷的掺入对电性并没有较大的改变，因为异核苷掺入siRNA的主要目的是体现后续生物学的优势。总之，异核苷联合修饰的3’，3”-双肽-siRNA缀合物/阳离子脂质体CLD具有较好的稳定性。
本文显示并详细描述的信息足以实现本发明的上述目的，因此本发明的优选实施方案代表本发明的主题，该主题为本发明所广泛涵盖。本发明的范围完全涵盖其它对本领域技术人员来说显而易见的实施方案，因此，本发明的范围不被除所附权利要求之外的任何内容所限制，其中除了明确说明外，所用元素的单数形式并不是指“一个和唯一”，而是指“一个或更多”。对本领域一般技术人员来说，所有公知的上述优选的实施方案和附加实施方案部分的结构、组成和功能上的等价物因此引入本文作参考，而且试图被本发明的权利要求所涵盖。
此外，不需要某种设备或方法来表达本发明所解决的每个问题，因为它们都已包括在本发明的权利要求之内。另外，无论本发明公开事实中的所有部分、成分，或者方法步骤是否在权利要求中被明确叙述，它们都没有贡献给公众。但是，对本领域普通技术人员来说，很明显在不背离如所附权利要求中所阐明的本发明的实质和范围的前提下，可以在形式、试剂和合成细节上做出各种改变和修饰。

Claims (11)

  1. 一种小干扰RNA(siRNA)的综合化学修饰方法，其特征在于，其中包括在小干扰RNA的正义链及/或反义链的一个或多个位点掺入D-构型或L-构型的异核苷，在小干扰RNA的正义链及/或正反义链的3’-末端进行肽缀合修饰，以及利用阳离子脂质载体实现所修饰的小干扰RNA跨膜转运中的两种或全部修饰方法阳离子脂质载体。
  2. 如权利要求1所述的综合化学修饰方法，其特征在于，所述D-构型或L-构型异核苷，分别具有如下化学式所示的结构：
    Figure PCTCN2015000402-appb-100001
    其中，n＝1、2、3；B为胸腺嘧啶基(T)、尿嘧啶基(U)、胞嘧啶基(C)、鸟嘌呤基(G)、腺嘌呤基(A)；化学式I所示的异核苷为D-构型的异核苷，化学式II所示的异核苷为L-构型的异核苷。
  3. 如权利要求1所述的综合化学修饰方法，其特征在于，所述的在小干扰RNA的正反义链的3’末端进行的肽缀合修饰，其中肽缀合片段与RNA部分的连接的通式为：
    Figure PCTCN2015000402-appb-100002
    其中X为多肽序列，A为取代或未取代的苯环或碳原子，n为0、1、2、3、4或5。
  4. 如权利要求3所述的综合化学修饰方法，其特征在于，所述的X为H-Leu-Ala-Leu-Leu-Ala-Lys-OH。
  5. 如权利要求1所述的综合化学修饰方法，其特征在于，所述的阳离子脂质载体为商用的阳离子脂质载体，包括RNAiMax，Oligofectamine，或是通式为化学式IV的阳离子脂质载体：
    Figure PCTCN2015000402-appb-100003
    其中，X为硫原子(S)或碳原子(C)，Y为其他含有氨基结构或靶向基团，R为饱和或不饱和脂肪链或疏水性分子。
  6. 如权利要求5所述的综合化学修饰方法，其特征在于，R所代表的不饱和脂肪链为油醇结构。
  7. 如权利要求1所述的综合化学修饰方法，其特征在于，异核苷掺入小干扰RNA的合成是通过固相合成实现的，掺入位使用异核苷亚磷酰胺单体代替天然核苷的亚磷酰胺单体在相应位置进行偶联。
  8. 如权利要求7所述的综合化学修饰方法，其特征在于，在进行siRNA修饰前，将化学式I和/或化学式II所示的异核苷化合物分别制备成化学式V和/或化学式VI所示的异核苷亚磷酰胺单体，在DNA合成仪上利用亚磷酰胺法掺入；
    Figure PCTCN2015000402-appb-100004
    其中，n＝1、2、3；B为胸腺嘧啶基(T)、尿嘧啶基(U)和氨基保护的鸟嘌 呤基(G)、腺嘌呤基(A)、胞嘧啶基(C)。
  9. 如权利要求1中所述的综合化学修饰方法，其特征在于，所述的综合化学修饰策略还包括与其他化学修饰策略的共同使用，包括2’-O-甲氧基(2’-OMe)、2’-氟代(2’-F)、锁定核苷酸(LNA)，磷硫骨架修饰及其他末端缀合方式等。
  10. 如权利要求1中所述综合化学修饰方法，其特征在于，包括以下步骤：
    (1)称取CLD固体粉末，加无水乙醇溶解，得到CLD的乙醇溶液，4℃保存；
    (2)小干扰RNA的正反义链的3’-末端进行肽缀合修饰，使用DEPC水化；
    (3)按照VsiRNA/VCLD＝5/1，N/P＝3/1或5/1，加入步骤(1)和(2)得到的CLD的乙醇溶液以及DEPC水水化后的小干扰RNA，在超声温度为70℃下，超声40min，涡旋1min，得到纳米复合物颗粒。
  11. 如权利要求1中所述综合化学修饰方法，其特征在于，形成的纳米复合物颗粒可以通过小窝蛋白介导的内吞和巨胞饮途径入胞方式等调控其进入细胞的途径或比例。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899092A (zh) * 2009-06-01 2010-12-01 北京大学 一种新型肽-链接基-缀合物及其固相合成方法
CN102660543A (zh) * 2012-01-21 2012-09-12 北京大学 一种利用异核苷对siRNA序列进行化学修饰的方法、及其产品和应用
CN102925487A (zh) * 2012-10-11 2013-02-13 北京大学 一种阳离子脂质载体及其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899092A (zh) * 2009-06-01 2010-12-01 北京大学 一种新型肽-链接基-缀合物及其固相合成方法
CN102660543A (zh) * 2012-01-21 2012-09-12 北京大学 一种利用异核苷对siRNA序列进行化学修饰的方法、及其产品和应用
CN102925487A (zh) * 2012-10-11 2013-02-13 北京大学 一种阳离子脂质载体及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YE HUANG ET AL.: "Modification of Oligonucleotides by Isonucleosides Incorporation and Peptides Conjugation", JOURNAL OF CHINESE PHARMACEUTICAL SCIENCES, 31 December 2012 (2012-12-31), pages 499 - 508, XP055240622, ISSN: 1003-1057 *

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