CN116590342A - AAV vector and application thereof - Google Patents
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The invention provides an AAV vector and application thereof. The AAV vectors include a nucleic acid segment encoding a tat-degrading peptide. The AAV vector comprising the nucleic acid segment for encoding the secretory tat-degrading peptide designed by the invention can be continuously expressed in the periphery, and the expressed degrading peptide can realize long-acting intervention of central diseases across a blood brain barrier and a cell membrane or play a role in AAV infected cells and non-infected cells when the expressed degrading peptide is continuously expressed in the center across the cell membrane. That is, the AAV vectors of the present invention achieve broad and long-lasting delivery of degraded peptide drugs.
Description
Technical Field
The invention relates to an AAV vector and application thereof, belonging to the field of adeno-associated virus vector medicines.
Background
The traditional polypeptide medicine, especially the polypeptide medicine composed of pure natural amino acids, is easy to be rapidly degraded in vivo, so that the in vivo effective medicine concentration maintenance time is short, the medicine treatment effect is difficult to be reflected, and the traditional polypeptide medicine cannot be orally taken and needs repeated intramuscular injection. In addition, traditional polypeptide drugs often cannot efficiently cross the blood brain barrier, and it is difficult to achieve therapeutic effects in the central system. Traditionally, degrading peptides, which are used clinically in acute treatment (e.g., stroke), exist as follows: the effective drug concentration is difficult to maintain in vivo, the treatment effect of chronic diseases is low, and the compliance of long-term injection patients is poor.
The polypeptide drug composed of pure natural amino acids has the problems of easy and rapid degradation in vivo, short in vivo effective drug concentration maintenance time, difficult stable drug effect, high individual heterogeneity and the like, and can be clinically used for selecting adeno-associated virus (AAV) treatment technology. The core of the technology is AAV vector medicine for simply carrying target genes, and the aim is to express the target genes in infected cells to supplement the missing/reduced proteins of the infected cells. The AAV vector drug is administered once, and the drug effect can be as long as 5-10 years. But this technique has significant drawbacks: (1) infection is inefficient and intervention is limited to a small fraction of infected cells. (2) Traditional adeno-associated viruses (AAV) or their encoded proteins cannot cross the blood brain barrier and cell membrane, nor can they achieve extensive intervention across infected cells, brain regions, and even peripheral centers.
Disclosure of Invention
To solve the problems in the prior art, we propose an AAV vector and its use.
The present invention first provides an AAV vector comprising a nucleic acid fragment encoding a tat-degrading peptide.
The degradation peptide disclosed by the invention refers to a peptide capable of targeting endogenous proteins and degrading the endogenous proteins.
The tat-degrading peptide is a secretory tat-degrading peptide, the AAV vector comprising the nucleic acid fragment for encoding the secretory tat-degrading peptide designed by the invention can be continuously expressed in the periphery, and the expressed degrading peptide can cross the blood brain barrier and cell membrane to realize long-acting intervention of central diseases or can continuously express the polypeptide in the central to cross the cell membrane to play a role in AAV infected cells and non-infected cells. That is, the AAV vectors of the present invention achieve broad and long-lasting delivery of degraded peptide drugs.
The tat refers to a transcription activator (human immunodeficiency virus-1transcription activator) of human immunodeficiency virus type 1, also can be abbreviated as HIV-1tat, is a cell penetrating peptide, and enters cells in a nontoxic and efficient way.
According to a specific embodiment of the present invention, preferably, the degradation peptide is: peptides carrying a deg tag or CTM tag that mediate degradation of endogenous proteins;
preferably, the amino acid sequence of the deg tag is shown in SEQ ID NO. 1;
SEQ ID NO.1：RRRG；
preferably, the amino acid sequence of the CTM tag is shown as SEQ ID NO. 2;
SEQ ID NO.2：KFERQ。
according to a specific embodiment of the present invention, preferably, the amino acid sequence of tat is shown in SEQ ID NO. 3;
SEQ ID NO.3：YGRKKRRQRRR；
preferably, the nucleic acid sequence for encoding tat is shown in SEQ ID NO. 4;
SEQ ID NO.2：TATGGCAGGAAGAAGCGGAGACAGCGACGAAGA。
according to a specific embodiment of the invention, preferably, the degrading peptide is βsyn-deg;
wherein, the amino acid sequence of the beta syn-deg is shown as SEQ ID NO. 5;
SEQ ID NO.5：YGRKKRRQRRRRTKSGVYLVGRRRG；
preferably, the nucleic acid sequence encoding βsyn-deg is shown in SEQ ID NO. 6;
SEQ ID NO.6：
CGTACTAAATCTGGTGTTTATTTGGTTGGTCGACGACGAGGC。
the deg is also called degron, and mediates the combination of alpha-synuclein (alpha-synuclein) and proteasome in a tat-beta syn-degron structure to induce alpha-synuclein degradation. The tat-beta syn-degron can effectively and specifically reduce alpha-synuclein.
Physiological status synuclein includes three types of alpha-synuclein (alpha-syn), beta-synuclein (beta-syn) and gamma-synuclein (gamma-syn). The alpha-synuclein in physiological state is disordered monomer, and the synuclein in pathological state is pathological polymerization state of monomer, namely toxic alpha-synuclein. The secretory tat-degradation peptide provided by the invention can identify and bind alpha-synuclein through the coded beta syn sequence, and mediate targeted degradation.
According to a specific embodiment of the present invention, preferably, the degradation peptide is βsyn-CTM;
wherein, the amino acid sequence of the beta syn-CTM is shown as SEQ ID NO. 7;
SEQ ID NO.7：YGRKKRRQRRRRTKSGVYLVGKFERQ。
according to a specific embodiment of the invention, preferably, the AAV vector further comprises a promoter sequence;
preferably, the promoter is a CMV promoter or a cell type specific promoter;
preferably, the cell type specific promoter is: EF1a or hSyn.
According to a specific embodiment of the invention, preferably, the AAV vector is of serotype: AAV9, AAV2, AAV5 or AAV8. The present invention is not limited specifically to the serotype of AAV used, and in practical application, AAV of corresponding serotype can be used according to the injection site, for example, AAV9 of serotype having the ability to cross the blood brain barrier is selected for peripheral injection, AAV2, AAV5 or AAV8 of serotype not having the ability to cross the blood brain barrier is selected for central local injection.
In a second aspect, the invention provides the use of an AAV vector in the manufacture of a medicament for the intervention of a central disorder. The intervention effect of the drug on the central disease is extensive and long-lasting.
In a third aspect, the invention provides a medicament comprising the AAV vector, and pharmaceutically acceptable excipients.
According to a specific embodiment of the present invention, preferably, the drug is an injection; further preferably, the medicament is a brain injection.
The invention has the beneficial effects that:
1. the AAV vector comprising the nucleic acid fragment for encoding the tat-degrading peptide is superior to the traditional nucleic acid intervention with low infection efficiency and limited intervention effect on a small part of infected cells, and can realize wide delivery and long-acting intervention by continuously secreting the degrading peptide through the infected cells and crossing blood-brain barrier and cell membrane. The AAV vector effectively solves the problems that the traditional peptide-degrading drug is difficult to maintain the effective drug concentration in vivo, the treatment effect of chronic diseases is low, the compliance of patients after long-term injection is poor and the like in practical application. Namely, the AAV vector realizes the wide and long-acting delivery of the traditional degradation peptide drug, and improves the application scene thereof.
2. The AAV vector comprising the nucleic acid segment encoding the tat-degrading peptide designed by the invention can be injected at fixed points during brain surgery, and can be continuously expressed, so that diseases can be intervened across cell membranes and brain regions. That is, the AAV vectors of the present invention can be injected into a single brain region and can function in a plurality of brain regions.
3. The AAV vector comprising the nucleic acid segment for encoding the tat-degrading peptide designed by the invention can express target genes in a host for a long time, and achieves the purpose of long-term central intervention of once administration. The AAV vector solves the problems that the AAV vector only acts on transfected cells, the central system diseases are difficult to interfere, and the effective concentration of the natural polypeptide drug is difficult to maintain in vivo due to the rapid degradation of the natural polypeptide drug in vivo, so that the effectiveness is poor.
4. The AAV vector designed by the invention is an improvement on the traditional AAV vector and the traditional degradation peptide treatment mode, and also has the advantages of the treatment method of the traditional AAV vector, such as good safety, treatment potential, long-term efficacy, clinical feasibility and the like, and the characteristics of the polypeptide drug that can be coded by genes.
Drawings
FIG. 1 is a schematic diagram showing the structure of a vector in which a nucleic acid fragment of tat-. Beta.syn-degron is inserted in this example.
FIG. 2 is a schematic diagram showing expression of AAV vectors constructed in example 1 in animal models.
FIG. 3 shows the location and coordinates of the intracerebral injection of Experimental example 1, and the right graph in FIG. 3 shows the distribution of the expression of green fluorescent protein and tat.
Figure 4 shows the levels of alpha-synuclein in the right substantia nigra and striatum of the parkinson's disease mouse model 3 months after injection.
Figure 5 shows the time and distance of locomotion on the rotating rod of parkinson's disease mice after 3 months of injection.
Figure 6 shows the time of parkinson's disease mice in the open field central zone 3 months after injection.
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention.
Example 1
This example provides a method of constructing an AAV vector comprising a tat- βsyn-degron structure. It comprises the following steps:
(1) Nucleic acid fragments encoding tat-ss syn-degron were synthesized in vitro. The sequence is as follows: SEQ ID NO.8: ATGTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACGTACTAAATCTGGTGT TTATTTGGTTGGTCGACGACGAGGCTGA.
(2) Constructing an expression vector.
Serotype AAV2/9 is used to infect local tissues such as local brain regions; AAV expression vectors (alternatively referred to as pAAV viruses) were constructed by driving tat- βsyn-degron transcription and expression via the CMV promoter.
FIG. 1 is a schematic diagram showing the structure of a vector in which a nucleic acid fragment of tat-. Beta.syn-degron is inserted in this example. The carrier structure is recorded as: pAAV-CMV-tat-beta Syn-Deg-ef1a-egfp-3 Xflag. The virus can be obtained by Shanghai and metabiological purchasing.
Experimental example 1
This experimental example provides the case where the AAV vector constructed in example 1 was expressed across membranes in an animal model.
A schematic representation of the expression of AAV vectors constructed in example 1 in animal models is shown in FIG. 2. In fig. 2, the left side shows that cells transfected and infected by the whole brain through local intracerebral injection or intravenous injection (the injection mode is shown on the right side of fig. 2) can express and secrete the tat-beta syn-deg degradation peptide with pharmacodynamic action for a long time, the degradation peptide can secrete host cells under the mediation of a tat transmembrane sequence, and enter surrounding infected or uninfected cells to play a role in pharmacodynamic action, and the system plays a long-acting role based on the characteristic of AAV long-term expression target genes. We call this technology a cell pharmaceutical factory in the body brain.
As shown in FIG. 3, 1. Mu.l, 2X 10, was injected into the striatum of the mice 12 After 30 days of injection of g/ml of the aforementioned pAAV virus, animals were perfused with fixed staining of tat, high expression of fluorescent protein in striatal bodies (EGFP) was detected and the distribution of tat was significantly broader due to its transmembrane diffusion effect than that of host cells expressing EGFP alone, and the enlarged picture on the right side of fig. 3 also shows the phenomenon that red tat-positive cells were produced and diffused from green molding cells. The feasibility of the in vivo cell pharmaceutical factory technology was demonstrated.
Experimental example 2
This experimental example provides the case where the AAV vector constructed in example 1 is expressed and acts in an animal model.
The literature has demonstrated that tat-beta Syn-deg drugs can target down the level of protein alpha-synuclein. However, the effect of the existing tat-beta Syn-deg medicine is rapidly reduced after a single injection, the medicine needs to be intravenously injected every day, and only a small improvement on dyskinesia is realized, so that the movement and non-movement disorder of the central degenerative disease cannot be improved for a long time and prominently.
This experimental example examined the expression of AAV vectors constructed in example 1 in animal models.
(1) AAV control viruses (hereinafter referred to simply as control viruses) encoding tat-polypeptides of the same length and disorder were injected into the left-hand black matter (substania nigra) of a 9-month-old Parkinson's disease mouse model (A53T mouse), and the right-hand black matter was injected with the aforementioned pAAV-CMV-tat-beta Syn-deg-ef1a-egfp-3xflag, which was continuously produced by in vivo cell pharmaceutical factories.
As shown in fig. 4, the AAV vector of the present invention significantly reduced the level of α -synuclein (a-syn) in the right substantia nigra and downstream striatum of the parkinson's disease mouse model (a 53T mouse) as compared to the control virus on its left hand side after 3 months of injection, GAPDH was an internal control. It was confirmed that the AAV vector of the present invention can exert its drug effect by the drug produced in the brain by the cell factory.
(2) Selecting 3 month-old, 6 month-old and 9 month-old A53T mice, injecting control virus (ctl) into the left substantia nigra of the 3 month-old (3M), 6 month-old (6M) and 9 month-old (9M) A53T mice, injecting the pAAV-CMV-tat-beta Syn-deg-ef1a-egfp-3xflag virus (deg) into the right substantia nigra, and after 3 months, remarkably improving the movement level of the 6 month-old, 9 month-old and 12 month-old animals in a rotating rod (as shown in figures 5 and 5 times) and remarkably reducing the anxiety level in an open field (figures 6 and 10 minutes) compared with the control group of the right substantia nigra injection control virus A53T mice. The method comprises the following steps:
FIG. 5 shows that the control virus was injected into the left black matrix of the A53T mice of 3 months and 6 months of age, the pAAV-CMV-tat-beta Syn-deg-ef1a-egfp-3xflag virus was injected into the right black matrix, and the movement time and movement distance on the rotating rod were remarkably increased after 3 months, as compared with the control mice. In the 9 month-old group, since the intervention time is too late, the dopamine neurons in the substantia nigra region die in a large quantity before the intervention, so that the tat-beta Syn-deg has no obvious effect of improving the dyskinesia of the 9 month-old group A53T mice. Namely, the tat-beta Syn-deg medicine degrades toxic proteins, and is more suitable for improving the middle and early PD dyskinesia.
FIG. 6 shows that the left-side substantia nigra of the A53T mice of the 3 month-old (3M), 6 month (6M), 9 month (9M) age groups were injected with the control virus (ctl) and the right-side substantia nigra was injected with the pAAV-CMV-tat- βSyn-deg-ef1a-egfp-3xflag virus (deg) described above, and that the time ratio in the open field center was significantly increased after 3 months. Namely, the tat-beta Syn-deg medicine can obviously improve the non-dyskinesia with low dependence on dopaminergic neurons in substantia nigra region in PD, and is suitable for the advanced treatment scheme of PD.
The experimental results not only prove the feasibility of the technology provided by the invention, but also prove that the AAV vector drug can obviously improve the dyskinesia and the non-dyskinesia of mice with parkinsonism models. The AAV vector medicine of the invention achieves the effect of long-term treatment by once administration, solves the problem that the common AAV vector medicine can not break through transfected cells, can break through transfected cells, and plays a role in treating a plurality of brain regions of the whole brain. The AAV vector drug also solves the problems that the traditional natural polypeptide drug is difficult to maintain the effective drug concentration for a long time in vivo, so that the effectiveness is poor, and the improvement of dyskinesia and non-dyskinesia is not obvious.
Claims (10)
1. An AAV vector comprising a nucleic acid fragment encoding a tat-degrading peptide.
2. The AAV vector of claim 1, wherein the degrading peptide is: peptides carrying a deg tag or CTM tag that mediate degradation of endogenous proteins;
preferably, the amino acid sequence of the deg tag is shown in SEQ ID NO. 1;
SEQ ID NO.1：RRRG；
preferably, the amino acid sequence of the CTM tag is shown as SEQ ID NO. 2;
SEQ ID NO.2：KFERQ。
3. the AAV vector of claim 1, wherein the tat has the amino acid sequence shown in SEQ ID No. 3;
SEQ ID NO.3：YGRKKRRQRRR；
preferably, the nucleic acid sequence for encoding tat is shown in SEQ ID NO. 4;
SEQ ID NO.2：TATGGCAGGAAGAAGCGGAGACAGCGACGAAGA。
4. the AAV vector of claim 1, wherein the degrading peptide is βsyn-deg;
wherein, the amino acid sequence of the beta syn-deg is shown as SEQ ID NO. 5;
SEQ ID NO.5：YGRKKRRQRRRRTKSGVYLVGRRRG；
preferably, the nucleic acid sequence encoding βsyn-deg is shown in SEQ ID NO. 6;
SEQ ID NO.6：
CGTACTAAATCTGGTGTTTATTTGGTTGGTCGACGACGAGGC。
5. the AAV vector of claim 1, wherein the degradation peptide is βsyn-CTM;
wherein, the amino acid sequence of the beta syn-CTM is shown as SEQ ID NO. 7;
SEQ ID NO.7：YGRKKRRQRRRRTKSGVYLVGKFERQ。
6. the AAV vector of claim 1, wherein the AAV vector further comprises a promoter sequence;
preferably, the promoter is a CMV promoter or a cell type specific promoter;
preferably, the cell type specific promoter is: EF1a or hSyn.
7. The AAV vector of claim 1, wherein the serotype of the AAV vector is: AAV9, AAV2, AAV5 or AAV8.
8. Use of an AAV vector according to any one of claims 1-7 in the manufacture of a medicament for broad and long-acting intervention in a central disease.
9. A medicament comprising the AAV vector of any one of claims 1-7, and a pharmaceutically acceptable adjuvant.
10. The medicament of claim 9, wherein the medicament is an injection;
preferably, the medicament is a brain injection.
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