CN117821508A - Engineered circRNA for encoding NGF protein, pharmaceutical composition, preparation method and application thereof - Google Patents

Abstract

The invention discloses an engineering circRNA for encoding NGF protein, a pharmaceutical composition, a preparation method and application thereof. The engineered circRNA encoding Nerve Growth Factor (NGF) has the advantage of enhancing NGF stability and expression efficiency. By packaging engineered circrnas in nanolipid particles (LNPs), efficient delivery and release of drugs can be achieved. The pharmaceutical composition can be applied by topical administration, gastrointestinal administration, parenteral administration, etc., to provide neuroprotection and treatment of nerve injury. The administration has obvious neuroprotective effect, does not need continuous administration, and avoids the risk of virus infection of animal-derived proteins. One advantage of circRNA-based therapy compared to viral gene delivery is that mRNA is not transported to the nucleus, thereby mitigating the risk of sequence insertion into the genome and thus mutagenesis of cancer.

Description

Engineered circRNA for encoding NGF protein, pharmaceutical composition, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to an engineering circRNA for encoding NGF protein, a pharmaceutical composition, a preparation method and application thereof.

Background

Nerve growth factor (nerve growth factor, NGF), an NGF protein, is widely used clinically for treating optic nerve injury, keratitis, and the like. However, the existing animal-derived NGF protein has the risk of prion infection, the recombinant protein fermentation process and the purification process are expensive, and daily intramuscular injection is required, which brings great pain to patients.

circRNA is a class of closed RNA that does not have a 5 'or 3' end. It has long been recognized as a rare and aberrant splicing byproduct of the 5 'splice junction and the upstream 3' splice junction. Recently, due to the rapid development of high throughput RNA sequencing and bioinformatic analysis, a large number of biological functions of natural circRNA have been revealed, from proteins and gene sponges, cell activity modulators to protein translation templates. In addition, as the understanding of the physiological functions of natural circRNAs continues to increase, there has been increasing interest in developing circRNA synthesis techniques and exploring the use of synthetic circRNAs in the treatment of disease in recent years. So far, synthetic circRNAs have been used not only for therapy, e.g. as alternative therapeutic proteins and polypeptides and vaccines, but also as biosensors. Meanwhile, in order to optimize the therapeutic effect while reducing side effects, various methods have been attempted to synthesize circRNA. Previously, circrnas were primarily considered non-coding RNAs, but recent studies have shown that they are capable of encoding and translating proteins and polypeptides through natural and synthetic circrnas. The circRNA has unique advantages in the development of vaccines and nucleic acid pharmaceuticals. First, it is less prone to degradation and is more stable than mRNA vaccines and nucleic acid drugs. Second, the amount required for rolling circle translation is lower than that of mRNA and therefore less toxic. The covalent closed loop structure surrounded by the circRNAs can protect it from exonuclease degradation and can address the vulnerability of mRNA vaccines to degradation. These properties make circRNA have great potential as a polypeptide/protein replacement therapy and vaccine. However, no relevant report on the engineering of the circRNA of NGF proteins has been found.

Disclosure of Invention

The present invention aims to overcome the above-mentioned drawbacks and deficiencies of the prior art and to provide a nucleic acid promoting the formation of engineered circRNA encoding NGF proteins.

A second object of the present invention is to provide a recombinant expression vector containing the nucleic acid

A third object of the present invention is to provide a recombinant bacterium comprising the recombinant expression vector.

The fourth object of the present invention is to provide a method for preparing an engineered circRNA encoding NGF protein.

It is a fifth object of the present invention to provide an engineered circRNA encoding NGF protein prepared by the above preparation method.

The sixth object of the present invention is a lipid nanoparticle coated with the above-described engineered circRNA encoding NGF proteins.

The seventh object of the present invention is a method for preparing the lipid nanoparticle.

An eighth object of the present invention is the use of the engineered circRNA encoding NGF proteins or the lipid nanoparticle.

The above object of the present invention is achieved by the following technical solutions:

a nucleic acid that promotes the formation of engineered circRNA encoding NGF protein, having the structure: 5 '. Fwdarw.3' is in sequence an in vitro transcription promoter-3 'type I intron and 3' homology arm-exon sequence E1-Spacer (Spacer) 1-Internal Ribosome Entry Site (IRES) -NGF protein coding sequence-Spacer (Spacer) 2-exon sequence E2-5 'type I intron and 5' homology arm; the NGF protein coding sequence is a CDS sequence for coding human or murine NGF, and the nucleotide sequence of the NGF protein coding sequence is shown as SEQ ID NO.2 or SEQ ID NO.4 respectively.

The nucleic acid can be based on the self-splicing of the I-type intron, can promote the cyclization of the circRNA under the catalysis of GTP (guanosine), and can solve the difficult problem of the cyclization of a long coding RNA sequence and obtain better cyclization efficiency in order to further improve the cyclization efficiency of the self-splicing precursor RNA.

Further, the in vitro transcription promoter is a T7, T3 or SP6 promoter.

Preferably, the in vitro transcription promoter is a T7 promoter.

Further, the 3 'type I intron and 3' homology arm-exon sequence E1, the exon sequences E2-5 'type I intron and 5' homology arm are derived from Anabaena.

Further, the exon sequence E1 is AAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA and the exon sequence E2 is AGACGCTACGGACTT.

Further, the spacer sequence (spacer) 1 is CGCCGGAAACGCAATAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAA AAACCAAAAAAACAAAACACA; spacer sequence 2 is AAAAAACAAAAAACAAAACGGCTATTATGCGTTACCGGCG.

Further, the internal ribosome entry site is the internal ribosome entry site IRES of Coxsackie virus B3.

Furthermore, the coding sequence of the NGF protein is a sequence of human NGF CDS sequence after codon optimization, and the nucleotide sequences of the coding sequence are respectively shown in SEQ ID NO. 6.

Further, the 3' -end of the NGF protein coding sequence is also connected with a protein tag sequence.

Preferably, the protein tag sequence is 3×flag.

Further, an enzyme cutting site is inserted between the NGF protein coding sequence and two adjacent components. Can be used for gene replacement, and saves cost.

Further, the sequence of the nucleic acid is shown as SEQ ID NO.1 or SEQ ID NO.11 or SEQ ID NO. 12.

The invention also provides a recombinant expression vector containing any one of the nucleic acids.

The invention also provides recombinant bacteria containing the recombinant expression vector.

The invention also provides a preparation method of the engineering circRNA for encoding NGF protein, which comprises the following steps:

s1, synthesizing any one of the nucleic acid sequences;

s2, connecting the nucleic acid sequence in the step S1 into a basic plasmid template, converting host bacteria, extracting plasmids, and obtaining a transcription template;

s3, linearizing the transcription template obtained in the step S2, carrying out in-vitro transcription, digesting and removing DNA, precipitating to obtain a circRNA precursor, adding GTP for incubation, realizing cyclization, and purifying to obtain cyclized RNA, thus obtaining the engineering circRNA for encoding NGF protein.

Further, the sequence of the circRNA precursor is shown as SEQ ID NO:8 to 10.

Further, the purification in step S3 is to digest and purify the circularized circRNA with an RNA purification kit and RNaseR enzyme. The purified circRNA was dissolved in acidic sodium citrate buffer and purified by HPLC, the second peak product of HPLC was collected and purified by RNA purification kit.

The invention also provides the engineering circRNA for encoding the NGF protein prepared by the preparation method.

The invention also provides Lipid Nanoparticles (LNPs) coated with the engineered circRNA encoding NGF proteins.

The invention also provides a preparation method of the lipid nanoparticle, which is to mix the engineering circRNA for encoding NGF protein with a cationic or polycation compound and package the mixture with lipid.

Preferably, the lipid includes, but is not limited to, a lipid capable of promoting self-assembly to form virus-sized particles (-100 nm), a lipid that allows for the release of circRNA from endosomes into cells, a lipid that supports the phospholipid bilayer structure, or a lipid that acts as a stabilizer. More preferably, the lipid may further comprise a pegylated lipid in order to increase the half-life of the LNP.

The circRNA or the lipid nanoparticle containing the circRNA has the advantages that: 1. in-vitro synthesis is carried out, cell culture is not needed, and the risk of animal source pollution is avoided; 2. the research and development and production are faster, the standardized production is easy, the mass production and the quality control are easy, and the same production flow is applicable to a plurality of different products; 3. can be expressed continuously over a period of time; 4. supporting multiple protein forms including intracellular proteins, transmembrane proteins, VLPs, etc., and circumventing purification problems caused by low yields of VLPs; 5. no risk of infection and genome integration.

The research of the invention shows that the engineering circRNA encoding NGF protein or the Lipid Nanoparticle (LNPs) has the effects of neuroprotection and treatment of nerve injury. Therefore, the invention also provides application of the engineered circRNA encoding NGF protein or the Lipid Nanoparticle (LNPs) in preparing medicines for preventing and/or treating nerve injury and/or neurodegenerative diseases.

"prevention" as used herein refers to all actions of avoiding symptoms or delaying stress of a particular symptom by administering the product of the present invention before or after the onset of disease.

"treatment" as used herein refers to a therapeutic intervention that ameliorates signs, symptoms, etc. of a disease or pathological condition after the disease has begun to develop.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides an engineered circRNA encoding NGF protein and lipid nanoparticles wrapping the engineered circRNA, and the engineered circRNA encoding Nerve Growth Factor (NGF) has the advantage of enhancing NGF stability and expression efficiency. By packaging engineered circrnas in nanolipid particles (LNPs), efficient delivery and release of drugs can be achieved. The pharmaceutical composition can be applied by topical administration, gastrointestinal administration, parenteral administration, etc., to provide neuroprotection and treatment of nerve injury. The administration has obvious neuroprotective effect, does not need continuous administration, and avoids the risk of virus infection of animal-derived proteins. One advantage of circRNA-based therapy compared to viral gene delivery is that mRNA is not transported to the nucleus, thereby mitigating the risk of sequence insertion into the genome and thus mutagenesis of cancer.

Drawings

FIG. 1 is a schematic diagram of the structure of linear RNA and circular RNA encoding NGF protein.

FIG. 2 shows the result of sequence sequencing of recombinant protein encoding wild-type murine NGF.

FIG. 3 shows the sequencing results of the sequences encoding wild-type human NGF proteins.

FIG. 4 shows the sequencing result of the sequence hNGF-MAhek2 encoding the optimized human NGF protein.

FIG. 5 shows the detection of the loop RNA preparation flow.

FIG. 6 shows HPLC purification of the cyclized product.

FIG. 7 shows the protein expression of hNGF sequences before and after optimization.

FIG. 8 shows the WB assay results of cell lysates of cells transfected with circ-mNGF.

FIG. 9 shows the neuroprotective effects of LNP-circNGF.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation and detection of engineered circRNA encoding NGF proteins

1. FIG. 1 is a schematic diagram of the structure of linear RNA and circular RNA of the NGF protein, specifically, the gene sequence of the chemically synthesized circ-CVB3-mNGF-3xFlag, and the sequence structure is as follows: t7 pro+ (3 'intron+homology) +E1+spacer1+CVB3+mNGF-3xFlag+spacer2+E2+ (5' intron+homology), the specific sequence is shown in SEQ ID NO. 1; wherein 1-19nt is T7 master, 20-183nt is (3 'intron+homology), 184-234nt is E1, 235-304nt is spacer1, 305-1045nt is CVB3, 1046-2062nt is mNGF-3xFlag,2063-2102nt is spacer2, 2103-2117nt is E2, 2118-2268nt is (5' intron+homology); after looping, E1+spacer+CVB3+mNGF-3xFlag+spacer2+E2.

In addition, the wild type murine NGF (mNGF) sequence is replaced by wild type human NGF (hNGF) or optimized human NGF (hNGF-MAhek 3) respectively to obtain the Circ-CVB3-hNGF-3xflag (the specific sequence is shown as SEQ ID NO. 11) and the Circ-CVB3-hNGF-MAhek3-3xflag (the specific sequence is shown as SEQ ID NO. 12).

2. Short nucleotide chains (primers) were synthesized by the solid phase phosphoramidite triester method, and the primers are shown below:

primer1(F)：gctagcTAATACGACTCACT, therein "gctagc"is NheI cleavage site;

Primer2(R)：AAGCTTCTAGATATGCTGTT, therein "AAGCTT"HindIII cleavage site.

3. And (3) carrying out PCR amplification by using the gene sequence synthesized in the step (1) as a template and using the primer synthesized in the step (2).

4. The amplified product of step 3 was ligated into pUC57 vector and sequenced by transformation.

5. The results are shown in FIGS. 2-4 after sequencing verification. Specifically, fig. 2 shows the sequencing result of the wild-type murine NGF recombinant protein encoding the signal peptide, the nucleotide sequence of which is shown in SEQ ID NO:2, the amino acid sequence is shown as SEQ ID NO: 3. FIG. 3 shows the result of sequence sequencing of the protein encoding wild-type human NGF, the nucleotide sequence of which is shown in SEQ ID NO:4, the amino acid sequence of which is shown as SEQ ID NO: shown at 5. FIG. 4 shows the sequencing result of the sequence hNGF-MAhek2 encoding the optimized human NGF protein, the nucleotide sequence of which is shown in SEQ ID NO:6, the amino acid sequence of which is shown as SEQ ID NO:7, the display sequence is consistent with expectations.

6. Fermenting the strain by shaking bottles, and purifying by using an endotoxin-free plasmid large extraction kit to obtain a transcription template. The transcription template is linearized with the restriction endonuclease BbsI. Transcription is carried out by using a T7 in vitro transcription kit to obtain SEQ ID NO:8 to 10. The transcription templates were digested with DNaseI, respectively, and the circRNA precursor was purified by precipitation. The circularized circRNA was circularized with GTP, and digested and purified with RNA purification kit and RNaseR enzyme, respectively (FIG. 5). The purified circRNA was dissolved in acidic sodium citrate buffer and purified by HPLC (FIG. 6), the second peak product of HPLC (F2) was collected and purified using RNA purification kit.

RNA from each stage was assayed with formaldehyde denaturing gel, as shown in FIG. 6, where F2 was circularized, and the correct size of RNA was seen with substantially no degradation.

Example 2 alignment of protein expression efficiency of hNGF sequence before and after optimization

The circ-hNGF and circ-hNGF-MAhek2 circular RNAs were transfected into hek293 cells, respectively, and the supernatants were used for ELISA. As shown in FIG. 7, it was revealed that the protein expression level of circ-hNGF-MAhek2 was higher than that of circ-hNGF.

EXAMPLE 3 preparation of mRNA drug

1. Raw material preparation

1) The cationic lipid SM-102, distearoyl phosphatidylcholine DSPC, cholesterol and PEGylated lipid PEG-DMG are dissolved and mixed in ethanol according to the molar ratio of 50:10:38.5:1.5.

2) The preparation of example 1 was provided which encodes mNGF-containing circRNA (circ-mNGF).

2. Expression detection

HEK293 cells were plated in 6-well plates, transfected with 0.5. Mu.g of circularized purified NGF-encoding circRNA (circ-mNGF) and negative controls were supplemented with lipofectamine messagerMax transfection reagent alone. After transfection for 6h, 12h, 24h, 48h and 72h, cell lysates were taken for WB detection, and the WB detection results are shown in FIG. 8. The expressed protein is correct in size and can be expressed continuously for 48 hours.

3. Lipid nanoparticle preparation of circRNA

Lipid mixture in step 1: the circRNA is respectively mixed and packaged in a nano particle preparation instrument Benchtop of Precision Nanosystems according to the flow rate ratio of 1:3. The packaged circRNA-LNP was dialyzed and concentrated to DPBS by ultrafiltration, and samples for animal experiments were obtained after aseptic filtration. The average particle size of the packaged sample was 78.83nm, PDI value was 0.028, and intercept (intercept) was 0.953.

EXAMPLE 4 neuroprotection test

1. Method of

C57 male mice of around 6 weeks old were intravitreally injected with 200ng of the circRNA-LNP prepared in example 3 on day 0, ONC operated on day 4, murine-killing retinal patches on day 18, stained with RBPMS antibody, confocal microscopy photographed GCL, and rbpms+ cells were counted.

2. Experimental results

The results of RGC counts are shown in FIG. 9, which shows that the RGC number in a single field of normal mice is about 350, the RGC number in a single field of view after ONC injury is about 60, the RGC number of injected NGF protein is about 90, and the RGC number in a single field of view after ONC injury of mice injected with the circRNA-LNP drug is about 140, and the number of surviving RGCs reaches 2 times that of the non-dosed group, indicating that the engineered circRNA of NGF protein has better effect of preventing/treating nerve injury and/or neurodegenerative disease than NGF protein.

Claims (10)

1. A nucleic acid that promotes the formation of engineered circRNA encoding NGF protein, characterized in that it has the structure: the 5 '. Fwdarw.3' is in turn an in vitro transcription promoter-3 'end I-type intron and 3' end homology arm-exon sequence E1-interval sequence 1-internal ribosome entry site-NGF protein coding sequence-interval sequence 2-exon sequence E2-5 'end I-type intron and 5' end homology arm; the NGF protein coding sequence is a CDS sequence for coding human or murine NGF, and the nucleotide sequence of the NGF protein coding sequence is shown as SEQ ID NO.2 or SEQ ID NO.4 respectively;

preferably, the in vitro transcription promoter is a T7, T3 or SP6 promoter;

preferably, the in vitro transcription promoter is a T7 promoter;

preferably, the 3 'type I intron and 3' homology arm-exon sequence E1, the exon sequences E2-5 'type I intron and 5' homology arm are derived from Anabaena;

preferably, the internal ribosome entry site is the internal ribosome entry site of coxsackievirus B3;

preferably, the 3' -end of the NGF protein coding sequence is also connected with a protein tag sequence;

preferably, the protein tag sequence is 3 xflag;

preferably, an enzyme cleavage site is inserted between the NGF protein coding sequence and two adjacent modules;

preferably, the exon sequence E1 is AAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA and the exon sequence E2 is AGACGCTACGGACTT;

preferably, the spacer sequence 1 is CGCCGGAAACGCAATAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAA AAACCAAAAAAACAAAACACA and the spacer sequence 2 is AAAAAACAAAAAACAAAACGGCTATTATGCGTTACCGGCG.

2. The nucleic acid of claim 1, wherein the NGF protein coding sequence is a codon optimized sequence of a CDS sequence of human NGF, and the nucleotide sequence is shown in SEQ ID No. 6.

3. The nucleic acid according to claim 1 or 2, characterized in that the sequence of the nucleic acid is shown in SEQ ID No.1 or SEQ ID No.11 or SEQ ID No. 12.

4. A recombinant expression vector comprising the nucleic acid of any one of claims 1 to 3.

5. A recombinant bacterium comprising the recombinant expression vector according to claim 4.

6. A method for preparing an engineered circRNA encoding NGF protein, comprising the steps of:

s1, synthesizing the nucleic acid sequence of any one of claims 1 to 3;

s2, connecting the nucleic acid sequence in the step S1 into a basic plasmid template, converting host bacteria, extracting plasmids, and obtaining a transcription template;

s3, linearizing the transcription template obtained in the step S2, carrying out in vitro transcription, digesting and removing DNA, precipitating to obtain a circRNA precursor, adding GTP for incubation, realizing cyclization, and purifying to obtain cyclized RNA, thus obtaining the engineering circRNA for encoding NGF protein.

7. The engineered circRNA encoding NGF protein prepared by the method of claim 6.

8. A lipid nanoparticle comprising an engineered circRNA encoding NGF protein of claim 7.

9. The method for preparing the lipid nanoparticle according to claim 8, wherein the engineered circRNA encoding NGF protein according to claim 7 is mixed with a cationic or polycationic compound and packaged with a lipid.

10. Use of the engineered circRNA encoding NGF protein of claim 7 or the lipid nanoparticle of claim 8 for the manufacture of a medicament for the prevention and/or treatment of nerve damage and/or neurodegenerative disease.