KR20150015615A - Nucleic acid delivery composition comprising an antibody and a nucleic acid-binding peptide - Google Patents

Abstract

Provided are: a composition for delivering nucleic acids, comprising nucleic acid binding peptides including cationic amino acids and hydrophobic amino acids and a stabilizing carrier including antibodies; a nucleic acid complex comprising nucleic acids and the composition for delivering nucleic acids; and a method for delivering nucleic acids using the composition for delivering nucleic acids. The present invention relates to a novel nucleic acid delivery system which efficiently delivers nucleic acids into cells by being combined with specific surface parameters (antigens) of specific cells, and inducing internalization into cells by the combination, and to efficient disease treatment techniques using the same.

Description

[0001] The present invention relates to a nucleic acid delivery composition comprising an antibody and a nucleic acid-binding peptide,

A composition for nucleic acid delivery comprising a nucleic acid-binding peptide and an antibody comprising a cationic amino acid and a hydrophobic amino acid, a nucleic acid conjugate nanoparticle comprising the nucleic acid delivery composition and the nucleic acid, and a nucleic acid delivery method using the nucleic acid delivery composition will be.

Gene transfer is a technique of introducing a nucleic acid material such as DNA or RNA into a cell using a carrier and then changing the characteristics of the cell by functions such as expression of the introduced gene or RNA interference. When a circular DNA (plasmid DNA) is introduced, it is a technology that can promote the expression of a new protein by transcription and expression of the introduced DNA.

RNA interference is a technique for reducing or suppressing gene expression by introducing exogenous double stranded RNA (dsRNA) into a cell to specifically destroy or block the expression of a specific messenger RNA (mRNA) (A. Fire et al., Nature, 391, 806-11, 1998). Specific RNA types such as small interfering ribonucleic acid (siRNA) and microRNA (miRNAs) are known to exhibit such RNA interference effects. In particular, it is known that siRNA has various applications such as drug screening and disease treatment because it shows a specific interference effect on a specific target mRNA. However, in clinical applications, efficient and cell-specific delivery of the RNA interfering substances remains a challenge.

Lipid-based, cationic, low molecular weight-based, cationic polymer-based techniques and combinations thereof have been extensively tried for intracellular delivery of small interfering ribonucleic acid (siRNA), but most have been applied to nonspecific delivery methods. As a method for improving this, target delivery using a lipid-based, antibody fragment-based, and polymer-based delivery vehicle in which a cell-specific substance has been introduced has been attempted (WO 2006/023491, US 2010/0209440, , And the introduction of targeting moieties has shown improved siRNA delivery efficiency.

Therefore, for the application of small interfering ribonucleic acid (siRNA) as a successful clinical therapeutic agent, there is a need to develop a novel delivery system that exhibits excellent target transfer efficiency while forming a more stable small interfering ribonucleic acid complex. In addition, the development of novel functional delivery vehicles (delivery vehicles with biological efficacy) that can maximize the effect of small interfering ribonucleic acid delivery is expected to bring a significant turning point in the development of therapeutic agents for diseases using siRNA.

One example provides a composition for nucleic acid delivery comprising an antibody and a nucleic acid-binding peptide comprising a cationic amino acid and a hydrophobic amino acid.

Another example provides a nucleic acid complex comprising the nucleic acid delivery composition and the nucleic acid.

Another example provides a nucleic acid delivery method using the nucleic acid delivery composition.

The present invention relates to a novel nucleic acid delivery system capable of binding to a specific surface factor (antigen) of a specific cell and inducing internalization of the binding into the cell, thereby effectively transferring the nucleic acid into a cell, Treatment technology.

In the nucleic acid delivery system, an antibody serving as a targeting carrier and a nucleic acid-binding peptide serving as a stabilizing carrier may be mixed at a specific ratio to form a nanoparticle-type complex upon binding with a nucleic acid.

Antibodies that are included as targeting vehicles in the delivery system exhibit not only very good specific cell targeting properties due to their high antigen specificity, but they may also exhibit the biological activities inherent in antibodies (e.g., inhibiting cancer growth and killing) A synergistic effect with a nucleic acid (e.g., siRNA) can be obtained. In addition, the nucleic acid-binding peptide contained as a stabilizing carrier can form a highly stable complex by binding to a nucleic acid and introducing a functional group such as PEG, and can form nanoparticles stable in vivo (e.g., blood) Can be formed.

A composition for nucleic acid delivery comprising an antibody and a nucleic acid-binding peptide according to an embodiment of the present invention is a novel new concept nucleic acid delivery system combining two functional sites of targeting (antibody) and stabilization (pegylated nucleic acid-binding peptide) In addition to excellent efficiency of targeting and transferring nucleic acid, it also protects nucleic acid very stably even in environments where there are many nucleic acid destabilizing factors such as nucleic acid degrading enzyme, and the stable and uniform particles in the blood without aggregation of nanoparticles . Therefore, the nucleic acid delivery system (composition for nucleic acid delivery) according to one aspect is not decomposed and aggregated in the blood even after systemic delivery such as intravenous injection, and forms a highly stable form of circulation, After binding to the tissue alone, the nucleic acid can be efficiently transferred into the cell through internalization of the cell.

In one example, a composition for nucleic acid delivery comprising a stabilizing carrier and a targeting carrier is provided.

In this specification, the stabilizing carrier may be a nucleic acid-binding peptide, and the nucleic acid-binding peptide may be a nucleic acid-binding peptide in which a functional group such as PEG is not introduced or introduced, and a (second) nucleic acid- May be referred to as " first nucleic acid binding peptide " The stabilization carrier can be represented illustratively as shown in the figure below:

<Stabilization vehicle>

: 핵산 결합 펩타이드;

: 작용기(e.g., PEG))(

: Nucleic acid binding peptides;

: Functional group (eg, PEG))

The targeting carrier may be a conjugate in which a nucleic acid-binding peptide is bound to an antibody or an antibody, and the nucleic acid-binding peptide may be a nucleic acid-binding peptide in which a functional group such as PEG is not introduced or introduced. The nucleic acid-binding peptide bound to the antibody in the targeting carrier is hereinafter referred to as a 'second nucleic acid-binding peptide' in order to distinguish it from the (first) nucleic acid-binding peptide as the stabilizing carrier described above. The targeting vehicle may be represented illustratively as shown below:

<Targeting Carrier>

: 핵산 결합 펩타이드,(

: Nucleic acid binding peptide,

: 작용기(e.g., PEG),

: Functional group (eg, PEG),

: 항체 또는 항원 결합 단편(e.g., scFvFc)).

: Antibody or antigen binding fragment (eg, scFvFc)).

The nucleic acid-binding peptide serving as the stabilizing carrier includes a cationic amino acid capable of ionic bonding with a nucleic acid and a hydrophobic amino acid for enhancing the binding strength between the cationic amino acid and the nucleic acid and enhancing stability. In addition, the stabilizing carrier may include a functional group such as PEG (polyethylene glycol) to improve stability in an aqueous solution or an in vivo environment so that the complexes can be maintained in an evenly dispersed form without aggregation . PEG binding to a peptide or protein prevents the peptide or protein from being degraded by enzymes in the body or lost in organs such as liver to be maintained in the body for a long period of time (increased half-life) Lt; / RTI &gt; In addition, the present invention also provides a method of binding a peptide or protein to a functional group such as PEG, thereby allowing the complex to form stable nanoparticles so that the nanoparticle-like complexes do not aggregate to form aggregates, So as to be uniformly dispersed and / or transported in an aqueous or environment environment. As shown in Example 3, in the case where PEG is not bound, the nanoparticle-like complex is aggregated in a salt solution environment similar to the body to form a large-sized aggregate and can not maintain a stable nanoparticle form. On the other hand, (100 nM or less) when the functional group such as PEG is bound as shown in FIG. 3B, and the size difference of the salt solution is not greatly increased even when the concentration of the salt solution is increased. That is, the present invention can increase the stability in the body (the prevention of aggregation and the uniform dispersion of the aqueous solution) by combining the peptide or the protein with a functional group such as PEG It has one feature. In addition, by intramolecular bonding of functional groups such as PEG, intracellular absorption of the complex can be increased.

More specifically, the nucleic acid-binding peptide is a peptide capable of forming an ion complex with a nucleic acid material such as DNA or RNA, and is a peptide selected from the group consisting of one cationic amino acid selected from the group consisting of arginine, lysine, ornithine, histidine, , Tyrosine, phenylalanine, leucine, and the like.

The total number of amino acids contained in the nucleic acid-binding peptide molecule may be about 2 to 100, specifically about 10 to 60, more specifically about 15 to 40.

The cationic amino acid contained in the first nucleic acid-binding peptide molecule is capable of forming an ionic complex with a nucleic acid by binding with an anion of the nucleic acid by an electrostatic attraction. The number of cationic amino acids in the nucleic acid-binding peptide molecule may be from 1 to 35, in particular from 10 to 30, more specifically from 15 to 25, in order to make it more suitable for ionic complex formation with the nucleic acid. Each of the cationic amino acids may be the same as or different from each other, and may be independently selected from the group consisting of arginine, lysine, and histidine.

The hydrophobic amino acid in the nucleic acid-binding peptide molecule enhances the stability of the ionic complex with the nucleic acid in the aqueous solution, and enhances the intracellular delivery efficiency of the nucleic acid during nucleic acid delivery. To adequately represent this stabilizing efficacy, the hydrophobic amino acid may comprise a benzene ring. In order to appropriately indicate such stabilizing effect, the content of the hydrophobic amino acid in the nucleic acid-binding peptide molecule is 1 to 50% by weight, specifically 10 to 48% by weight, more specifically 15 To 45% by weight. The hydrophobic amino acid is a hydrophobic amino acid including a benzene ring and may be at least one selected from the group consisting of tryptophan, tyrosine, phenylalanine, etc., and each hydrophobic amino acid contained in the nucleic acid binding peptide may be the same or different.

In an embodiment, the nucleic acid-binding peptide molecule may be represented by the following formula 1:

[Chemical Formula 1]

_{[(X) p1 (Z)} q1] 1 - [(X) p2 (Z) q2] 2 ... _{[(X) p (n-} 1) (Z) q (n-1)] n-1 - [(X) pn (Z) qn] n,

In the formula

X is a cationic amino acid,

Z is a hydrophobic amino acid comprising an aryl group, such as a benzene ring,

n is an integer of 1 to 20, specifically 2 to 10,

'p1, p2 ... pn 'is the number of cationic amino acids contained in the unit, and is an integer of 1 to 10, specifically 1 to 5 (where n represents the position of the corresponding unit)

'q1, q2 ... qn ', q is the number of hydrophobic amino acids including the benzene ring included in the unit, and is an integer of 1 to 10, specifically 1 to 5 (where n represents the position of the corresponding unit)

The p1, p2 ... pn and q1, q2 ... qn may be the same or different from each other for each unit,

The total amino acid length is 2 to 100, in particular 10 to 60, more particularly 15 to 40,

When two or more Xs are present, they may be the same or different amino acids,

When two or more Z are present, they may be the same or different amino acids.

As described above, in the nucleic acid-binding peptide, not only the cationic amino acid residue and the anion of the nucleic acid form an ionic complex by the electrostatic attraction, but also the hydrophobic amino acid residue in the formed ionic complex improves the stability of the complex in the aqueous solution Show efficacy.

In addition, the nucleic acid-binding peptide may further include at least one functional group selected from the group consisting of PEG (polyethyleneglycol) at one end, both ends, or a bondable position. As described above, by introducing a functional group such as PEG, nucleic acid delivery efficiency and stability can be further improved. The PEG that can be used at this time may be, for example, a mass average molecular weight ranging from 100 to 10,000, specifically from 500 to 5,000. In one embodiment, the PEG may be one wherein the reducing group, such as an aldehyde group, is functionalized by the incorporation of one or more, e. G., One or two reducing groups. The reducing group may be liberated after PEG is bound to the nucleic acid-binding peptide or antibody.

When a functional group-introduced nucleic acid-binding peptide is used, the molar ratio of functional group (for example, PEG) / nucleic acid-binding peptide is 1 to 5, specifically 2 to 5, more specifically To 3 to 5 times.

An antibody that serves as a targeting moiety may be any antibody that specifically binds to an antigen expressed on the surface of a target cell in nucleic acid delivery, or a fragment derived from the antibody. Unless otherwise specified herein, an antibody is interpreted to mean an antigen-binding fragment derived from said antibody. The antibody may be specifically bound to an antigen and internalized into a cell to increase the efficiency of cell therapy by transferring the target nucleic acid into the cell. The antibody may be a mouse antibody, a chimeric antibody, a humanized antibody, Lt; / RTI &gt; The antigen-binding fragment derived from the antibody may be selected from the group consisting of scFv, (scFv) ₂ , scFvFc, Fab, Fab 'and F (ab') ₂ derived from the antibody.

As used herein, "specifically binding" or "specifically recognizing" means the same as commonly known to those skilled in the art, meaning that the antigen and antibody specifically interact to effect an immunological response.

As used herein, the term "antibody" or "antibody as a targeting agent", which is a component of the nucleic acid delivery composition and the second nucleic acid binding protein-antibody conjugate, includes a complete IgA form, a full IgD form, (scFv) ₂ , scFvFc, Fab, Fab 'and F (ab') ₂ , which are antigen binding fragments derived from the above antibodies, and / or antibodies of the full IgG form, full IgG form, And the like.

Antibodies that are included in the composition for nucleic acid delivery to act as targeting carriers can be used without any particular limitation if they are antibodies that specifically bind to any one of all the antigens expressed in the target nucleic acid transfer target cell. For example, the antibody may be an antibody of the full form (full IgA form, full IgD form, full IgE form, full IgG form, full IgM form, e.g. full IgG form), and antigen binding fragment fragments scFvFc, (scFv) ₂ , Fab, Fab 'and F (ab') ₂ , and the like. The antibody may be a protein that specifically expresses all the proteins in vivo, particularly cancer cells or cancer tissues, such as various growth factors or receptor tyrosine kinase. For example, the antibody may be an anti-c-Met antibody, an epidermal growth factor receptor (anti-EGFR) antibody, a human epidermal growth factor receptor 2 (HER2) antibody, a human epidermal growth factor receptor (HER3) ScFv Fc, (scFv) ₂ , Fab, Fab ', and F (F)) derived from the above antibody are selected from the group consisting of one or more antibodies selected from the group consisting of an antibody, a growth factor, an angiopoietin 2 antibody, (ab ') ₂ , and the like, but is not limited thereto.

The anti-c-Met antibody may be a c-Met derived from a mammal such as a primate such as a human, a monkey, a rodent such as a mouse or a rat, or the like, for example, GenBank Accession Nos. It is an antibody that specifically recognizes c-Met having the amino acid sequence provided in NP_000236, NP_001162100, NP_032617.2, NP_113705.1, and the like.

The anti-EGFR antibody may be an EGFR derived from a mammal such as a primate such as a human, a monkey, a rodent such as a mouse or a rat, for example, GenBank Accession Nos. (MRNA) provided by the nucleotide sequence (mRNA) provided in the nucleotide sequence (mRNA) provided in the nucleotide sequence (SEQ ID NO: The antibody may be at least one selected from the group consisting of cetuximab, panitumumab, and the like.

The anti-HER2 antibody may be a HER2 derived from a mammal such as a primate such as a human, a monkey, a rodent such as a mouse or a rat, for example, GenBank Accession Nos. An antibody that specifically recognizes HER2 having an amino acid sequence encoded by a nucleotide sequence (mRNA) provided in NM 004448.2 (human), NM_001003817 (mouse), NM_017003 (rat), etc., includes Transtuzumab (Herceptin) Pertuzumab, Trastuzumab emtansine (T-DM1), and the like.

The anti-HER3 antibody may be HER3 derived from mammals such as primates such as humans and monkeys, rodents such as mice and rats, and the like, for example, GenBank Accession Nos. May be an antibody that specifically recognizes HER3 having the amino acid sequence encoded by the nucleotide sequence (mRNA) provided in NM_001982.2, NM_001982.3, NM_001005915.1, NM_010153.1, NM_017218.2, or NM_001103105.1.

The anti-VEGF antibody may be a VEGF derived from a mammal such as a primate such as a human, a monkey, a rodent such as a mouse or a rat, and the like, for example, GenBank Accession Number NM_001025366.2. NM_001025367.2. NM_001025368.2. NM_001025369.2. NM_001025370.2. NM_001033756.2. NM_001171622.1. NM_001171623.1. NM_001171624.1. NM_001171625.1. NM_001171626.1. NM_001171627.1. NM_001171628.1. NM_001171629.1. NM_001171630.1. NM_001204384.1. NM_001204385.1. Or an antibody that specifically recognizes VEGF encoded by the nucleotide sequence (mRNA) provided in NM_003376.5 or the like, such as avastin.

In an embodiment, the antibody as the targeting vehicle may be in the form of a second nucleic acid binding protein-antibody conjugate linked to a second nucleic acid binding peptide separately from the (first) nucleic acid binding peptide used as the stabilizing carrier described above have. The second nucleic acid binding protein-antibody conjugate can be prepared such that the second nucleic acid binding protein binds to the N-terminus, C-terminus or both termini of the antibody (in the case of the dimeric form of IgG type antibody, , Or both N-terminus, C-terminus, or both ends of each of the two monomers). For example, the second nucleic acid binding protein-antibody conjugate may be such that the second nucleic acid binding protein binds to the N terminus of the antibody (in the case of dimeric IgG type antibody, the N terminus of one monomer or the N terminus of each of the two monomers) It can be. The second nucleic acid binding protein-antibody conjugate may be one to four, or one to two, or one second nucleic acid binding protein per antibody. As described above, the antibody may be an antibody of full type IgG, full IgD form, full IgE form, full IgG form, full IgM form, such as full IgG form, and / or an antibody fragment scFv, scFvFc, (scFv) ₂ , Fab, Fab 'and F (ab') ₂ .

The second nucleic acid binding protein may be one in which at least one functional group selected from the group consisting of polyethyleneglycol (PEG) is introduced at one end or both ends thereof to further enhance nucleic acid transfer efficiency and stability. When a functional group such as PEG is introduced into one end or both ends of the second nucleic acid binding protein, the second nucleic acid binding protein can bind to the antibody through the functional group (see FIG. 2). The second nucleic acid binding protein-antibody conjugate structure may exhibit a more advantageous effect on the target transfer of the nucleic acid through the antibody by binding the second nucleic acid binding protein to the nucleic acid and binding the nucleic acid to the antibody, It can contribute to the stability enhancement effect described above.

The type of peptide usable as the second nucleic acid-binding peptide linked to the antibody may be the same as or different from that of the first nucleic acid-binding peptide serving as the stabilizing transporter, as described above for the nucleic acid-binding peptide serving as a stabilizing transporter.

The composition for nucleic acid delivery may contain the antibody and the nucleic acid-binding peptide as described above in a predetermined ratio. For example, the ratio of the content of the antibody or the second nucleic acid binding protein-antibody conjugate as a targeting vehicle to the first nucleic acid binding protein as a stabilizing carrier is 1: 9 to 3: 7 (Targeting Vehicle Mall: Stabilizing Delivery Mall). When a second nucleic acid binding protein-antibody conjugate is used as the targeting vehicle, the second nucleic acid binding protein-antibody conjugate may comprise from 1 to 4, or from 1 to 2, second nucleic acid binding proteins per antibody, or And the second nucleic acid binding protein may be bound to the N-terminus, C-terminus, or both ends of the antibody. Expressed as the ratio of the amount of the antibody to the nucleic acid binding peptide (including the second nucleic acid binding protein contained in the conjugate when the second nucleic acid binding protein-antibody conjugate is used as the targeting vehicle) is about 1/10 To 3/10 (antibody mol / nucleic acid binding peptide mole).

As described above, the composition for nucleic acid delivery is excellent in targeting efficiency in nucleic acid delivery by including an antibody, and also includes a nucleic acid-binding peptide including a cationic amino acid and a hydrophobic amino acid, It can be usefully used as a new nucleic acid transducer capable of overcoming the limitations of existing nucleic acid transporters.

Another example provides a nucleic acid complex comprising the above nucleic acid delivery composition and a nucleic acid. The antibody contained in the nucleic acid complex as a component of the nucleic acid delivery composition may be a complete antibody (full IgA form, full IgE form, full IgE form, full IgG form, full IgG form, for example, full IgG form) an antigen-binding fragment selected from the group consisting of scFv, scFvFc, (scFv) ₂ , Fab, Fab 'and F (ab') ₂ or the like, or an antigen-binding fragment derived from the antibody or antibody conjugated with a second nucleic acid- A second nucleic acid-binding peptide-antibody conjugate. In the complex, a nucleic acid is positioned at the center, and a nucleic acid-binding peptide electrostatically bound to the nucleic acid (including a first nucleic acid-binding peptide as a stabilizing carrier and a second nucleic acid-binding peptide bonded to the antibody) surrounds the nucleic acid, Or a stable nanoparticle morphology.

The site where the cationic amino acid of the nucleic acid-binding peptide is distributed can be bound to the nucleic acid by the electrostatic attraction, and one or more nucleic acid-binding peptides can bind to the nucleic acid to form a nanoparticle- have. At this time, the antibody may bind to the nucleic acid-binding peptide or to the nucleic acid through a second nucleic acid-binding peptide (when the antibody is in the form of a second nucleic acid-binding peptide-antibody conjugate) linked to the antibody (see FIG.

The nucleic acid may be at least one selected from the group consisting of single-stranded or double-stranded DNA, RNA, small interfering RNA (siRNA), antisense-oligonucleotide, shRNA, microRNAs (miRNAs)

The nucleic acid complex may be in the form of a uniform and stable nanoparticle. For example, the nanoparticles may have an average particle diameter of 100 nm or less, for example, 10 to 100 nm or 50 nm to 100 nm.

The amount of the nucleic acid that can be delivered by the nucleic acid delivery composition or the amount of the nucleic acid in the nucleic acid complex is 0.1 to 1 mole of nucleic acid relative to 1 mole of the composition for nucleic acid delivery (targeting agent + stabilization transfer agent) For example, 0.1 to 0.5 mole. As described above, the content ratio between the targeting carrier (the antibody or the second nucleic acid binding protein-antibody conjugate) and the stabilizing carrier (nucleic acid binding protein) in the nucleic acid delivery composition is 1: 9 to 3: Carrier mall).

Another example provides a nucleic acid delivery method comprising the step of administering the nucleic acid complex to a patient.

The patient may be a mammal such as a primate such as a human, a monkey, a rodent such as a rat, a rodent, etc., or a cell, a tissue or a culture thereof derived from the animal (separated) Or a cell or tissue derived therefrom. The nucleic acid delivery method may further comprise identifying a patient in need of delivery of the nucleic acid prior to the administering step.

The nucleic acid complex can be administered orally or parenterally to a patient in need of such nucleic acid administration, or to the cell or tissue.

The nucleic acid complex may further comprise a pharmaceutically acceptable carrier. The carrier is typically used in the formulation of a drug containing nucleic acid, and may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, starch, But are not limited to, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, , Mineral oil, and the like, but is not limited thereto. The pharmaceutical composition may further comprise at least one member selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent,

Administration of the nucleic acid complex can be via an oral or parenteral route. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above.

The nucleic acid complex may be in the form of a solution, suspension, syrup or emulsion in an oil or an aqueous medium, or may be formulated in the form of an extract, powder, granule, tablet or capsule, May additionally include topics.

The present invention provides a novel nucleic acid delivery system capable of binding a nucleic acid (for example, siRNA) to form a stable complex and capable of specific cell-specific delivery, thereby enabling delivery of a novel nucleic acid target that can be clinically applied Do.

The outline effects of the present invention are as follows.

- Nucleic Acid Target Transmission: Transferring and functioning only to specific cells through the targeting transporter

- Stabilization of nucleic acid complexes: Stable complexes with nucleic acids can be formed through stabilizing transporters and can be maintained as stable nanoparticles in the blood.

- Synergy of biological activity: Because the targeting carrier has a biological effect inherent to the antibody, rather than simple targeting, synergism with the therapeutic effect of the nucleic acid and the antibody specific effect can occur.

1 is a schematic diagram of a nucleic acid delivery vehicle according to one embodiment.
FIG. 2 is a schematic view illustrating a process for preparing an antibody conjugate into which nucleic acid-binding peptides are introduced according to one embodiment.
FIG. 3 is a graph showing the size exclusion chromatography result after purification of an antibody conjugate into which a nucleic acid-binding peptide according to an embodiment is introduced.
FIG. 4 is a graph comparing changes in nano particle behavior of NaCl concentration of a nucleic acid-binding peptide and a small interfering ribonucleic acid (siRNA) complex before and after the introduction of PEGylation.
5 shows the results of evaluating the cell-specific transfer efficiency of a 1: 9 (molar ratio) mixed carrier of an anti-HER2 scFvFc antibody conjugate and a R3WW-PEG conjugated with a nucleic acid-binding peptide (R3W) according to one embodiment and a fluorescent siRNA complex (R3W = RRRWRRRWRRRWRRRWRRRWRRRW (SEQ ID NO: 6), R3WW = RRRWWRRRWWRRRWWRRRWWRRRWWRRRWW (SEQ ID NO: 4), PEG = polyethylene glycol (Mw = 2000).
FIG. 6 is a schematic view schematically showing a process for producing a nucleic acid-binding peptide (R3WW) _6- PEG.
FIG. 7 is a graph showing the results of separation and purification of (R3WW) _6- PEG by HPLC.
Figure 8 is a graph showing product levels tested with varying moles of PEG and (R3WW) ₆ peptides.
Figure 9 is a graph showing the results of ion exchange column performance for anti-Her2 scFvFc.
Figure 10 shows the isolation and purification of the (R3WW) _6- PEG-scFvFc conjugate [(R3WW) _6- conj-scFvFc].
Fig. 11 shows the affinity measurement results of the scFvFc and (R3WW) _6- PEG-scFvFc conjugates to the HER2 antigen.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

[ Example  1] nucleic acid binding Peptide [(R3WW) ₆ -PEG]  Produce

A peptide having the following amino acid sequence was prepared:

RRRWWRRRWWRRRWWRRRWWRRRWWRRRWW (SEQ ID NO: 4)

[Hereinafter referred to as '(R3WW) ₆ ' or 'R3WW'].

PEG (MW 2000) was added to the synthesized peptide [(R3WW) ₆ ] through a reductive amination reaction using an aldehyde-functionalized PEG (ALW-PEG-ALD) functionalized with an aldehyde group ) Were conjugated. Specifically, a mixture with ALD-PEG-ALD (MW 2000 ) and the synthesized (R3WW) ₆ peptide and the molar ratio of 1 to 5 (PEG / (R3WW) 6 peptide) were dissolved in 10mol / ml concentration in DMSO (Dimethyl sulfoxide) After reacting at room temperature for 3 hours, it was purified by high performance liquid chromatography (HPLC) to obtain a pegylated nucleic acid-binding peptide [(R3WW) ₆ -PEG-ALD] (see FIG. 6).

The obtained HPLC results are shown in FIG. 7 (mobile phase: H ₂ O + 0.1% (v / v) trifluoroacetic acid / ACN (acetonitrile) + 0.1% (v / v) TFA; column: C18 , Shisheido)).

The results obtained by performing HPLC (C18 column, UV 280 nm detection, H2O / acetonitrile linear gradient) test in order to confirm an appropriate molar ratio between (R3WW) ₆ peptide and PEG are shown in FIG. The Y-axis of FIG. 8 represents the integration value of each corresponding peak on the HPLC. As shown in FIG. 8, it was confirmed that the product amount was optimized when the molar ratio of PEG / (R3WW) ₆ peptide was around 4.

The (R3WW) _6- PEG thus prepared was used as a stabilizing carrier in the following experiment or conjugated to an antibody and used as a conjugation type targeting vehicle.

[ Example  2] nucleic acid binding Peptides  Introduced antibody conjugate ( antibody conjugate ; Targeting  Carrier) production and separation-analysis

2.1. term- HER2 scFvFc  Produce

An antibody against HER2, a breast cancer-specific factor, was used as a representative example of an antibody used as a targeting moiety. Specifically, an Fc fusion protein (scFvFc; SEQ ID NO: 1) into which a single chain antibody of Transtuzumab (Herceptin), which is an antibody binding to HER2, was introduced using HindIII / XhoI restriction enzyme was used as an expression vector pCEP4 vector; Invitrogen), and then the protein was produced through an animal cell culture process.

SEQ ID NO: 1:

EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR

IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG

GDGFYAMDYW GQGTLVTVSS GGGGSGGGGS GGGGSDIQMT QSPSSLSASV

GDRVTITCRA SQDVNTAVAW YQQKPGKAPK LLIYSASFLY SGVPSRFSGS

RSGTDFTLTI SSLQPEDFAT YYCQQHYTTP PTFGQGTKVE IKREPKSCDK

THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE

VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK

VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF

YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV

FSCSVMHEAL HNHYTQKSLS LSPGK

FreeStyle^TM 293 Expression Medium; Invitrogen).The animal cell culture process was performed as follows. ^TM FreeStyle 293-F cells (suspension human embryonal kidney cells, Cat no.R790-07;.; Invitogen hereinafter 'HEK293F') to 1.0X10 37 ℃ and 8% CO ₂ in a 1L conical flask to a concentration of ⁶ cells / ml And cultured under culture conditions for 12 days. The DNA (1.250 mg) cloned into the pCEP4 vector was transfected into the HEK293F cells using Freestyle MAX reagent (Promega). The transfected cells were cultured for 2 weeks, and the culture medium in which the protein was expressed once every 3-4 days was collected and replaced with fresh medium (medium: Gibco

FreeStyle ^TM 293 Expression Medium; Invitrogen).

The protein (scFvFc) was finally isolated through affinity chromatography (protein A column) and ion exchange chromatography. Specifically, the protein was purified from the cell culture medium by FPLC (Fast protein liquid chromatography). The FPLC conditions were tabulated as shown in Table 1 below.

Protein A
column
(HiTrap Protein A HP; GE Healthcare) Loading buffer: 1XPBS + 15% (v / v) glycerol
Wash buffer: 30 mM acetate / 400 mM NaCl (pH 5.2) + 15% (v / v) glycerol
Elution buffer: 100 mM glycine-HCl (pH 2.7) + 500 mM NaCl
+ 15% (v / v) glycerol 1 M sodium acetate (pH 5.2) 1 ml (fraction volume, 5 ml): preventing aggregate formation after elution Desalting column
Desalting
Column) 20 mM Tris (pH 8.5) + 15% glycerol IEX (Ionic Exchange)
column
(source 15Q column; GE Healthcare) A buffer: 20 mM Tris (pH 8.5) + 15% glycerol
B buffer: 20 mM Tris (pH 8.5) + 15% glycerol + 1 M NaCl
→ B 5-15% gradient
Length: 80ml
flow rate: 4 ml / min
fraction size: 10ml
injection volume: 5ml
column: source 15Q After concentration, the final buffer 20 mM sodium PBS (pH 6.0) + 15% glycerol
Centricon enrichment

After primary purification with Protein A column, the buffer was replaced with NaCl-free buffer using a desalting column, and finally a peak fraction was collected through an ion exchange column (IEX) column. Ion exchange column was prepared by using GE Healthcare's source 15Q resin. After loading the protein into column with A buffer, B buffer was flowed with 5 ~ 15% gradient to separate impurities and anti-Her2 scFvFc. The separated final protein (anti-Her2 scFvFc) was concentrated to a concentration of 1 mg / ml in 20 mM sodium PBS (pH 6.0) + 15% (v / v) glycerol buffer and used for the experiment and stored at 4 ° C. The expressed protein showed a yield of 8 mg per liter of average culture medium.

The result of ion exchange column performance for the anti-HER2 scFvFc is shown in FIG. As shown in FIG. 9, when the gradient of 5 to 15% of the B buffer was 7.5 minutes, it was confirmed that the anti-HER2 scFvFc is the most optimal condition for complete separation from impurities.

2.2. Nucleic acid binding Peptides and  term- Her2 scFvFc ( R3WW ) ₆ - scFvFc ] Produce

The pegylated nucleic acid-binding peptide [(R3WW) ₆ -PEG-ALD] prepared in Example 1 was conjugated to the N-terminus of the separated and purified anti-Her2 scFvFc, and one (R3WW ) _6- PEG-ALD conjugate was purified through affinity chromatography and ion exchange chromatography (see FIG. 2). Specifically, the molar amount of 10 moles (mole) of the separated purified anti-Her2 scFvFc and (R3WW) _6- PEG-ALD (25-100 micromoles (uM)) prepared in Example 1 were mixed, For 2 days to prepare a chemically coupled conjugate [(R3WW) _6- PEG-scFvFc]. At this time, (R3WW) 6-PEG-ALD reacts with the amine group at the N-terminal of scFvFc to bind. After the reaction, unreacted peptides were removed by Protein A column separation and purified by ion exchange chromatography (source 15S column; B gradient 0-100%, A buffer: 20 mM Tris (pH 8.5) + 15% glycerol / B buffer : 20 mM Tris (pH 8.5) + 15% glycerol + 4M NaCl) to remove unreacted antibody and byproducts. (See Figs. 2 and 10).

The results of the analysis using the ion exchange chromatography (source 15S column) are shown in FIG. The results of the ion exchange chromatography analysis of the conjugate prepared above and the results of the ion exchange chromatography analysis of the prepared conjugate were also shown in FIG. 3, except that the amino acid sequence of the nucleic acid-binding peptide was changed as follows :

9Rd: RRRRRRRRR-GPG-RRRRRRRRR (SEQ ID NO: 5: nucleic acid-binding peptide of the scFvFc-9Rd conjugate shown in FIG. 3)

(R3W) ₆ : RRRWRRRWRRRWRRWRRRWRRRW (SEQ ID NO: 6: nucleic acid binding peptide of the scFvFc-R3W conjugate shown in Fig. 3).

(With reference to Figure 3, the PEG is omitted for the sake of brevity)

As shown in Fig. 3, the scFvFc [9Rd-scFvFc junction, (R3W) _6- scFvFc junction, or (R3WW) ₆ -scFvFc junction to which the nucleic acid binding peptide having the cationic amino acid R (Arg) Product peak of the (R3WW) _6- scFvFc conjugate was more prominent than that of the (R3WW) _6- scFvFc conjugate, since product cation was eluted at a higher salt concentration, Appeared at the back. 9Rd-scFvFc conjugate, (R3W) From the above results, ₆ -scFvFc conjugate, and (R3WW) ₆ -scFvFc conjugates, in particular (R3W) ₆ is -scFvFc conjugates and (R3WW) ₆ -scFvFc conjugate successfully obtained may be confirmed that .

2.3. ( R3WW ) ₆ - PEG - scFvFc  Junction HER2  Affinity measurement

ScFvFc (Example 2.1), (R3WW) _6- PEG-scFvFc conjugate (Example 2.2) prepared above on a CM5 chip (10000 RU immobilization; GE healthcare) immobilized with human Fc binding antibody (human Fc binding antibody) (~ 150 RU capture), and the association / dissociation of HER2 (HER2 concentration: 0 ~ 100 nM; Sino Biological Inc.) was observed and the KD value was measured.

The results obtained are shown in Fig. 11 (KD values: scFvFc = 0.582 nM, (R3WW) 6-conj-scFvFc = 0.561 nM). As shown in FIG. 11, the affinity of the anti-Her2 scFvFc for Her2 was measured, and it was confirmed that there was almost no change in the affinity for the specific antigen (Her2) before and after the conjugation.

[ Example  3] Pegylation ( PEGylation ) After introduction, nucleic acid binding Peptides and Small interference  Ribonucleic acid ( siRNA ) Evaluation of particle stability improvement of composite

The enhancement of particle stability in the NaCl solution of pegylated nucleic acid binding peptide and small interfering ribonucleic acid (siRNA) complexes serving as a stabilizing transporter was confirmed by nanoparticle behavior.

The (R3WW) ₆ peptide prepared in Example 1 and the siRNA were mixed at a molar ratio of 1: 5 and incubated at room temperature for 30 minutes to form a complex. Thereafter, they were incubated in NaCl solutions at different concentrations (0, 75, 150, 300 nM) for 30 minutes. The size of the resulting complex nanoparticles was measured by DLS (dynamic light scattering) equipment.

The sequence of the siRNA used is as follows:

Sense sequence: 5'-CCU ACG CCA CCA AUU UCG U (dTdT) -3 '(SEQ ID NO: 2-dTdT)

Antisense sequence: 5'-ACG AAA UUG GUG GCG UAG G (dTdT) -3 '(SEQ ID NO: 3-dTdT)

The results obtained are shown in Fig. As shown in FIG. 4, aggregation phenomenon in which the nanoparticle size increases as the concentration of NaCl increases is observed in the complex of the nucleic acid-binding peptide and the siRNA before the pegylation, and the NaCl concentration is lowered to 75 mM It can be seen that the pegylated peptide-siRNA complex stably maintains homogeneous nanoparticles of 100 nm or less even in the 300 mM NaCl solution, while the size is already aggregated and changed into large particles of several μm.

[ Example  4] Cell-specific transfer efficiency evaluation

Analysis of FACS (Fluorescence-activated cell sorting) analysis of siRNA intracellular delivery efficiency of a nucleic acid carrier mixed with (R3WW) _6- PEG-scFvFc conjugate (targeting carrier) and (R3WW) _6- PEG Respectively.

Specifically, the evaluation before, SK-BR-3 (HER2 positive cells; ATCC, accession number HTB-30 ), and MCF7 (HER2 negative cells; ATCC, accession number: HTB- 22) each 2 X 10 ⁵ cells / well Lt; RTI ID = 0.0 &gt; well-plates. &Lt; / RTI &gt; (Targeting Molar: Stabilizing Delivery Mole) of 1: 9 on the molar basis (R3WW) ₆ -PEG-scFvFc conjugate (targeting vehicle; Example 2.2) and (R3WW) _6- PEG And FITC-siRNA were prepared and used for the test.

The FITC-siRNA was prepared by labeling FITC (Fluorescein isothiocyanate) at the 5 'end of the siRNAs of SEQ ID NOS: 2 and 3 used in Example 3. (R3WW) ₆ -PEG-scFvFc conjugate (Example 2.2) was mixed with the prepared FITC-siRNA at a molar ratio of 1: 1 (conjugate moles: FITC siRNA moles), followed by complexation at room temperature for 30 minutes, (R3WW) ₆ -PEG peptide was added to the PEG-scFvFc conjugate at a molar ratio of 1: 9 (target transfer moles: stabilizing transfer moles) and further complexed at room temperature for 30 minutes to prepare a complex.

The obtained complexes were treated with the cells prepared previously (SK-BR-3 and MCF7, respectively) (complex treatment concentration: 20 nM or 100 nM on the basis of siRNA concentration). The cells were incubated at 37 ° C for 4 hours, harvested with trypsin, and the cells were washed with PBS / 0.3% (v / v) BSA (pH 3.8) to remove the complex remaining on the cell surface without internalization into the cells . FACS analysis was performed on the final obtained cell solution on PBS (using a BD FACS Canto II instrument).

The results obtained are shown in Fig. As shown in FIG. 5, it was confirmed that a large amount of siRNA was specifically introduced into only HER2 overexpressing cells (SK-BR-3) through a nucleic acid delivery system (complex).

<110> SAMSUNG ELECTRONICS CO., LTD. <120> Nucleic acid delivery composition comprising an antibody and a          nucleic acid-binding peptide <130> DPP20124526 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> Anti-HER2 scFvFc <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr              20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Ser Ser     130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 145 150 155 160 Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly                 165 170 175 Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly             180 185 190 Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu         195 200 205 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln     210 215 220 Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 225 230 235 240 Ile Lys Arg Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro                 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro             260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr         275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn     290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val                 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser             340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys         355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu     370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu                 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe             420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly         435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr     450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense trand of siRNA <400> 2 ccuacgccac caauuucgu 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Antisense strand of siRNA <400> 3 acgaaauugg uggcguagg 19 <210> 4 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> nucleic acid binding peptide (R3WW) 6 <400> 4 Arg Arg Arg Trp Trp Arg Arg Arg Trp Trp Arg Arg Arg Trp Trp Arg   1 5 10 15 Arg Arg Trp Trp Arg Arg Arg Trp Trp Arg Arg Arg Trp Trp              20 25 30 <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> nucleic acid binding peptide 9Rd <400> 5 Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Pro Gly Arg Arg Arg Arg   1 5 10 15 Arg Arg Arg Arg Arg              20 <210> 6 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> nucleic acid binding peptide (R3W) 6 <400> 6 Arg Arg Arg Trp Arg Arg Arg Trp Arg Arg Arg Trp Arg Arg Arg Trp   1 5 10 15 Arg Arg Arg Trp Arg Arg Arg Trp              20

Claims (11)

A composition for nucleic acid delivery comprising a first nucleic acid-binding peptide and an antibody comprising a cationic amino acid and a hydrophobic amino acid. The method of claim 1, wherein the antibody consisting of scFv, scFvFc, (scFv) _2, Fab, Fab 'and F (ab') ₂ derived from the antibody or the antibody specifically binding to the antigen expression on the cell surface Binding fragment selected from the group consisting of: 3. The composition of claim 2, wherein said antibody is in the form of a nucleic acid-linked peptide-antibody conjugate linked to a second nucleic acid-binding peptide comprising a cationic amino acid and a hydrophobic amino acid. 4. The method according to any one of claims 1 to 3, wherein the first nucleic acid-binding peptide or the second nucleic acid-binding peptide is independently selected from the group consisting of peptides represented by the following formula (1) Composition:
[Chemical Formula 1]
_{[(X) p1 (Z)} q1] 1 - [(X) p2 (Z) q2] 2 ... _{[(X) p (n-} 1) (Z) q (n-1)] n-1 - [(X) pn (Z) qn] n,
In the formula
X is a cationic amino acid,
Z is a hydrophobic amino acid comprising a benzene ring
n is an integer of 1 to 20,
'p1, p2 ... pn ', p is the number of cationic amino acids contained in the unit, and is an integer of 1 to 10,
'q1, q2 ... qn ', q is the number of hydrophobic amino acids including the benzene ring contained in the unit, and is an integer of 1 to 10,
The p1, p2 ... pn and q1, q2 ... qn are the same or different values for each unit,
The total amino acid length is 2 to 100,
When two or more X are present, they are the same or different amino acids,
Z is an amino acid which is the same or different from each other when two or more Z are present. 5. The nucleic acid delivery composition according to claim 4, wherein the cationic amino acid is selected from the group consisting of arginine, lysine and histidine, and the hydrophobic amino acid comprising the benzene ring is selected from the group consisting of tryptophan, tyrosine and phenylalanine . 4. The nucleic acid delivery composition according to any one of claims 1 to 3, wherein the first nucleic acid-binding peptide or the second nucleic acid-binding peptide further comprises PEG (polyethyleneglycol). 4. The method according to any one of claims 1 to 3, wherein the mixing ratio of the antibody or the second nucleic acid binding protein-antibody conjugate to the first nucleic acid binding peptide is 1: 9 to 3: 7 (the ratio of the antibody or the second nucleic acid Binding protein-antibody conjugate mol: first nucleic acid-binding peptide moles). A nucleic acid delivery composition comprising the nucleic acid delivery composition according to any one of claims 1 to 3,
Wherein the nucleic acid is electrostatically bound to a cationic amino acid of a first nucleic acid binding peptide or a second nucleic acid binding &lt; RTI ID = 0.0 &gt;
Nucleic acid complex. 9. The nucleic acid complex of claim 8, wherein the first nucleic acid-binding peptide or the second nucleic acid-binding peptide further comprises PEG. The method of claim 8, wherein the mixing ratio of the antibody or the second nucleic acid binding protein-antibody conjugate and the first nucleic acid binding peptide in the nucleic acid delivery composition is 1: 9 to 3: 7 (the antibody or the second nucleic acid binding protein- Antibody conjugate mol: first nucleic acid binding peptide moles). 9. The nucleic acid complex according to claim 8, wherein the nucleic acid conjugate comprises 0.1 to 1 mole of nucleic acid relative to 1 mole of the nucleic acid delivery composition.

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