CN112121179A

CN112121179A - Composition and application thereof in treating spinal muscular atrophy

Info

Publication number: CN112121179A
Application number: CN202010967367.6A
Authority: CN
Inventors: 刘明录; 张传鹏; 金海锋; 强邦明; 王立新; 冯建海; 王亮; 韩庆梅; 许淼; 卢永灿
Original assignee: Shandong Xinrui Biotechnology Co ltd
Current assignee: Shandong Xinrui Biotechnology Co ltd
Priority date: 2020-09-15
Filing date: 2020-09-15
Publication date: 2020-12-25

Abstract

A composition and application thereof in treating spinal muscular atrophy. The invention provides a composition comprising an SMN adeno-associated viral vector, astrocytes. The combined treatment of the SMN adeno-associated virus vector and the astrocyte can carry out precise treatment and nutritional recovery on nerves, and is more beneficial to the recovery of SMA patients. The invention adopts the astrocytes induced and differentiated by the oral mucosa stem cells of the inventor and AAV vectors carrying SMN gene for injection together to correct SMN gene deletion or mutation and motor neuron dysfunction of patients. The astrocytes induced by the oral mucosa stem cells have high purity and short time, and can provide a large amount of cells. AAV vector carries SMN gene and can treat SMA disease precisely, and the induced astrocyte has the function of secreting nerve factor and promoting neuron recovery.

Description

Composition and application thereof in treating spinal muscular atrophy

Technical Field

The invention relates to a composition and application thereof in treating spinal muscular atrophy, belonging to the technical field of biology and new medicines.

Background

Spinal muscular atrophy is an autosomal recessive motor neuron disease that is accompanied by loss of spinal synapses and motor neurons in addition to neuromuscular junction defects and skeletal muscle atrophy. Deletion or mutation of the motor neuron telomere survival (SMN 1) gene is the major cause of lower full-length levels of SMN protein.

Spinal Muscular Atrophy (SMA) is the most common cause of genetic death in infants and young children. It is estimated that the incidence of live birth is 1/10000, with carriers rates in european and asian progeny ranging from about 1/40 to about 50. SMA is the result of loss of the gene for surviving motoneuron 1 (SMN 1) and other mutations that result in loss of SMN protein. Human has a SMN1 homolog SMN2 which encodes primarily a truncated, rapidly degrading protein product due to nucleotide substitutions that result in exon 7 being excluded.

Astrocytes, the most widely distributed cell type in the mammalian brain, have been considered as supporting cells of the nervous system and function as nutrients and protectors, ion buffers, and transmitter metabolism.

The current SMA drug Spinraza takes SMN2 gene expression as a target point and aims to modify SMN2 splicing and increase the generation of functional full-length SMN protein. SMN is a broadly expressed intracellular protein known to be closely related to the assembly of snrnps and the formation of other cellular RNPs including coding and non-coding RNAs. Also, the method of patent 201810652815.6 to increase SMN protein expression is to disrupt the splice silencer ISS-N1 or ISS +100 on intron 7 of SMN2 using CRISPR/Cas9 technology to achieve increased production of functional full-length SMN protein. Another product, Zolgensma, uses SMN1 gene as target, and uses AAV vector to transfer functional full-length SMN1 gene, to increase SMN protein level of cell. Also, patent CN108795946A provides a series of recombinant adeno-associated viruses carrying an expression cassette of artificially designed SMN1 gene. In vivo experiments show that the recombinant adeno-associated virus vector can be efficiently introduced into the central nervous system, continuously and stably express the SMN1 protein, prolong the life of a Spinal Muscular Atrophy (SMA) model animal, increase the weight of the SMA model animal and restore the growth and development of the SMA model animal.

For Spinraza and Zolgensma, and related patents, because they can only correct SMN protein levels in cells by antisense oligonucleotides or increasing expression of the SMN1 gene, they must be used as early as possible, otherwise, if the motor neuron cells die because of lack of nutrients that ensure cell growth, they are useless.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a composition and an application thereof in treating spinal muscular atrophy, which can correct SMN gene deletion or mutation and motor neuron dysfunction of a patient; better activation and nourishment of the myonerve cells.

In order to solve the technical problems, the invention adopts the following technical scheme:

a composition comprising an SMN adeno-associated viral vector, an astrocyte.

The following is a further improvement of the above technical solution:

the SMN adeno-associated virus vector expresses SMN; the amino acid sequence of the SMN is shown as SEQ ID NO. 1.

The nucleotide sequence of SMN expressed in the SMN adeno-associated virus vector is shown in SEQ ID NO. 2.

The ratio of the SMN adeno-associated virus vector to the astrocytes is 80-120 vg: 1 piece.

The astrocytes are obtained by inducing and differentiating stem cells of human oral mucosa.

The preparation method of the SMN adeno-associated virus vector comprises the steps of plasmid vector preparation, cell transfection and virus collection; and preparing the plasmid vector, namely constructing a nucleotide sequence for expressing the SMN on the adeno-associated virus AAV-IRES-hrGFP to obtain the pAAV plasmid vector.

Transfecting the cell, and infecting HEK293 cells by adopting auxiliary plasmids, packaging plasmids and pAAV plasmids; the mass-volume ratio of the helper plasmid, the packaging plasmid and the pAAV plasmid is 1.5-2.5: 0.8-1.2: 0.8-1.2.

The helper plasmid was pHelper, and the packaging plasmid was pAAV-RC 8.

The SMN adeno-associated virus vector and the astrocytes can be mixed and then used or can be separately and sequentially used for treating spinal muscular atrophy.

The SMN adeno-associated virus vector and the astrocytes can be mixed and injected together, or can be separately and independently injected.

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

(1) the combined treatment of the SMN adeno-associated virus vector and the astrocyte can carry out precise treatment and nutritional recovery on nerves, and is more beneficial to the recovery of SMA patients.

(2) The invention adopts the astrocytes induced and differentiated by the oral mucosa stem cells of the inventor and AAV vectors carrying SMN gene for injection together to correct SMN gene deletion or mutation and motor neuron dysfunction of patients. The autologous oral mucosa stem cells are induced and differentiated into astrocytes, and the astrocytes induced by the autologous oral mucosa stem cells are used for clinical application without ethical problems and the immunological rejection reaction is reduced. The astrocytes induced by the oral mucosa stem cells have high purity and short time, and can provide a large amount of cells. AAV vector carries SMN gene and can treat SMA disease precisely, and the induced astrocyte has the function of secreting nerve factor and promoting neuron recovery.

(3) The composition of the invention is co-cultured with the motor neuron cells (SMN-) under the spinal cord in an in vitro test, the SMN adeno-associated virus vector and the astrocytes play a synergistic role, the growth of the motor neuron axons of the motor neuron cells (SMN-) under the spinal cord is promoted together, the expression of the genes of the markers TUBB3 and ISL1 of the motor neuron cells under the spinal cord is promoted, and the expression of NEFL and ISL1 of the cytokines of the motor neuron cells under the spinal cord is promoted.

Drawings

FIG. 1: microscopic images of astrocytes used in the present invention;

FIG. 2: histograms of axonal length of motor neuron cells of each experimental group;

FIG. 3: detecting the expression quantity of a gene TUBB3 related to a motor neuron cell under the spinal cord by an RT-PCR method;

FIG. 4: detecting the expression quantity of a motor neuron cell related gene ISL1 under the spinal cord by an RT-PCR method;

FIG. 5: detecting the expression quantity of a motor neuron cell related cytokine NEFL under the spinal cord by an ELISA method;

FIG. 6: ELISA method detects the expression level of motoneuron cell-related cytokine ISL1 under spinal cord.

Detailed Description

EXAMPLE 1 preparation of SMN adeno-associated viral vectors

(1) preparation of pAAV-IRES-hrGFP-SMN plasmid vector

SMN amino acid sequence is shown in SEQ ID NO.1, gene sequence is shown in SEQ ID NO.2, and the SMN amino acid sequence is constructed on AAV-IRES-hrGFP and entrusted to Beijing Bomaide biotechnology and scientific Co.

(2) Cell transfection

HEK293 cells were transfected in 15cm dishes until the cells were 70% confluent. 26ug helper plasmid (pHelper), 13. mu.g packaging plasmid (pAAV-RC 8), 13. mu.g pAAV plasmid (pAAV-IRES-hrGFP-SMN), and infection with 96ul liposomes (Henan) for 24 h.

(3) Virus collection

A. 5 days after transfection, the supernatant was collected and then treated with a toluase nuclease for 2 hours (37 ℃).

B. The supernatant was collected by centrifugation (3850 g, 5 min).

C. The filtrate was collected by filtration through a 0.22um filter.

D. The filtrate was concentrated 75-fold after filtration through a 0.22um filter using an Amicon Ultra-15 centrifuge filter, centrifugation (5000 g, 45 min).

E. Iodixanol density gradient purified AAV and 40% viral layer was removed.

1) OptiPrep Using double distilled Water^TM(AXIS-SHIELD PoC AS) to prepare 15%, 25,40 and 54% iodixanol solution;

2) sequentially injecting 10mL of 54,40,25 and 15 percent of the virus into 10-15mL of virus collection liquid;

3) centrifuging at 30500rpm for 9h, 18 ℃;

4) carefully taking out the virus in the 40% iodixanol solution layer, and carrying out heavy suspension on the virus by PBS through a centrifugal filter to obtain an SMN adeno-associated virus vector;

5) viral titer determination was performed using qPCR absolute quantification method.

EXAMPLE 2 culture of astrocytes

In the invention, the method of CN111254114A is adopted, and the stem cells of the oral mucosa of a human are induced and differentiated into astrocytes. The human oral mucosa slice is taken out by professional personnel, and the human oral mucosa stem cells are obtained by culture and induced to differentiate into astrocytes.

Example 3 cellular function assays

(a) Detection of neuronal axon length

The astrocytes, the SMN adeno-associated virus vectors, the astrocytes and the astrocytes (SMN-) are co-cultured with the motor neuron cells under the spinal cord (Cat No. BFN60808759, Qingqi Biotechnology development Co., Ltd.).

The method comprises the following specific steps:

(1) culturing by the method of patent CN111254114A to obtain astrocytes;

(2) according to the invention, an SMN gene in an astrocyte and a subtenon motor neuron is knocked out by a crispr/cas9 knock-out method, so that an astrocyte (SMN-) and a subtenon motor neuron (SMN-) are obtained;

(3) the astrocytes + SMN adeno-associated virus vectors, astrocytes, and astrocytes (SMN-) were co-cultured for 7 days with the subgspinal motor neuron cells (Cat. BFN60808759, Qingqi Biotechnology development Co., Ltd.) or the subgspinal motor neuron cells (SMN-) respectively, and the neurite length was measured.

(4) The length of the axons of the neuronal cells was measured microscopically (the number of measurements was not less than 20).

Normal group of motor neurons

Experimental group 1: adeno-associated virus + astrocytes with integrated SMN gene

1 x 10 of⁵Individual subtenon motor neuron cells were seeded in 24-well plates and then cultured with 1mL of medium (DMEM high glucose +10% FBS) to 30% of the area of the plated dish. Then adding 1 x 10 into 24-hole plate⁸vg (vector genome) adeno-associated viral vector with integrated SMN gene, followed by 1 x 10 addition⁶And (4) astrocytes.

Experimental group 2: adeno-associated virus vector integrating SMN gene

1 x 10 of⁵Inoculating the individual subtenon motor neuron cells in a 24-well plate, adding 1mL of culture medium (DMEM high-sugar +10% FBS), and culturing until the cells cover 30% of the area of the culture dish; then adding 1 x 10 into 24-hole plate⁸vg (vector genome) adeno-associated viral vector incorporating the SMN gene.

Experimental group 3: astrocytes

1 x 10 of⁵Inoculating the individual subtenon motor neuron cells in a 24-well plate, adding 1mL of culture medium (DMEM high-sugar +10% FBS), and culturing until the cells cover 30% of the area of the culture dish; then adding 1 x 10 into 24-hole plate⁶And (4) astrocytes.

Experimental group 4: astrocytes (SMN-)

1 x 10 of⁵Inoculating the individual subtenon motor neuron cells in a 24-well plate, adding 1mL of culture medium (DMEM high-sugar +10% FBS), and culturing until the cells cover 30% of the area of the culture dish; then adding 1 x 10 into 24-hole plate⁶And (c) a plurality of astrocytes (SMN-).

The above groups were co-cultured for 7 days, and the fluid was changed every two days to measure the length of the neuron axons.

Control group: 1 x 10 of⁵Inoculating the individual subtenon motor neuron cells in a 24-well plate, adding 1mL of culture medium (DMEM high-sugar +10% FBS), and culturing until the cells cover 30% of the area of the culture dish; the culture medium was changed every two days, and the neuron axon length was measured at 635 μm after further culture for 7 days

Motor neuron SMN knockout group:

experimental group 1: adeno-associated virus vector and astrocyte integrated with SMN gene

1 x 10 of⁵The individual subtenon motor neuron cells (SMN-) were seeded in 24-well plates, and then 1mL of medium (DMEM high-glucose +10% FBS) was added and cultured until the cells were spread over 30% of the culture dish area; then adding 1 x 10 into 24-hole plate⁸vg (vector genome) adeno-associated viral vector with integrated SMN gene, followed by 1 x 10 addition⁶And (4) astrocytes.

Experimental group 2: adeno-associated virus vector integrating SMN gene

1 x 10 of⁵The individual subtenon motor neuron cells (SMN-) were seeded in 24-well plates, and then 1mL of medium (DMEM high-glucose +10% FBS) was added and cultured until the cells were spread over 30% of the culture dish area; then adding 1 x 10 into 24-hole plate⁸vg (vector genome) adeno-associated viral vector incorporating the SMN gene.

Experimental group 3: astrocytes

1 x 10 of⁵The individual subtenon motor neuron cells (SMN-) were seeded in 24-well plates, followed by addition of 1mL of medium (DMEM high-glucose +10% FBS) and cultured until the cells were plated out over the area of the culture dish30% of; then adding 1 x 10 into 24-hole plate⁶And (4) astrocytes.

Experimental group 4: astrocytes (SMN-)

1 x 10 of⁵The individual subtenon motor neuron cells (SMN-) were seeded in 24-well plates, and then 1mL of medium (DMEM high-glucose +10% FBS) was added and cultured until the cells were spread over 30% of the culture dish area; then adding 1 x 10 into 24-hole plate⁶Astrocytes (SMN-).

The cells were co-cultured, the medium was changed every two days, and the co-culture was carried out for 7 days.

Control group: 1 x 10 of⁵The individual subtenon motor neuron cells (SMN-) were seeded in 24-well plates, and then 1mL of medium (DMEM high-glucose +10% FBS) was added and cultured until the cells were spread over 30% of the culture dish area; the culture medium was changed every two days, and the culture was continued for 7 days to measure the length of neuronal axons, which was 430 μm.

Through detection, the axon length of the motor neuron cells cultured by the normal under-spinal-cord motor neuron cells, the astrocytes (SMN +) and the SMN adeno-associated virus vectors is obviously longer than that of other experimental groups, and is more obviously longer than that of a control group.

Through the co-culture of the SMN knockout group of the motor neuron, the length of the motor neuron axon of the experimental group 1 is obviously longer than that of other experimental groups, the length of the motor neuron axon of the experimental group 1 is 638 micrometers, the length of the motor neuron axon of the experimental group 2 is 596 micrometers, the length of the motor neuron axon of the experimental group 3 is 554 micrometers, the length of the motor neuron axon of the experimental group 4 is 498 micrometers, and the length of the motor neuron axon of the control group is 430 micrometers; the motor neuron axon length of the experiment 2 group is 1.39 times that of the control group; the length of the motor neuron axon of the experiment 3 group is 1.29 times that of the control group, while the length of the motor neuron axon of the experiment 1 group is 1.48 times that of the control group, and the length of the motor neuron axon of the experiment 1 group is close to that of a normal motor neuron cell cultured alone under the spinal cord.

Therefore, the SMN adeno-associated virus vector and the astrocyte can play a synergistic role and jointly promote the growth of motor neuron axons of the motor neuron cells (SMN-) under the spinal cord.

(b) qPCR detects TUBB3, ISL 1.

QPCR detection step: RNA from the motor neuron cells under the spinal cord was first extracted using the RNeasy Plant Mini Kit (available from Qiagen, Inc., cat # 74904) according to the Kit instructions. The cDNA was formed by reverse transcription of both RNAs using PrimeScript RT reagent Kit with gDNA Eraser Kit (purchased from TaKaRa, cat # RR 047A) as described in the Kit instructions. The two cDNAs were used as templates, and the fluorescent quantitative PCR reagents were purchased from Xeno, and the genes to be detected included TUBB3 and ISL1, and the β -atcin gene was used as an internal reference gene. The quantitative PCR primer sequences are shown in Table 1. Each PCR reaction was set to 3 replicates. The RT-PCR was programmed as follows: fluorescence was collected at 94 ℃ for 3min, (94 ℃ for 15s, 58 ℃ for 30s, 72 ℃ for 30 s) x 35cycle during annealing.

TABLE 1 genes and primer sequences for fluorescent quantitation

Through detection, the expression levels of TUBB3 and ISL1 of motor neuron cells cultured together with astrocytes (SMN +) and SMN adeno-associated virus vectors in a normal motor neuron group are obviously higher than those of other experimental groups. Through the co-culture comparison of the detected motor neuron SMN knockout group, the expression levels of TUBB3 and ISL1 in the experimental group 1 are obviously higher than those of other experimental groups in the motor neuron SMN knockout group.

(3) Detecting the cell factor by ELISA method;

a detection step:

after the CO-culture to the sixth day, the medium was changed to 1ML DMEM, and then cultured for 24 hours (37 ℃; 5% CO)₂) And collecting the supernatant. Please refer to the description of the detection reagent in the detection step

Human ISL LIM homeobox protein 1(ISL1) ELISA kit (96T from Shanghai win-win biotechnology Limited)

Human NEFL Elisa test kit (96T from Beijing Donggong Bo Sci Biotechnology Limited)

In the normal group and the knockout group, the marker cytokine ISL1, NEFL of the motor neuron cell under the spinal cord after being co-cultured with the astrocyte and the SMN adeno-associated virus are remarkably increased in comparison in the group through ELISA detection, and have remarkable difference, which proves that the successful astrocyte can effectively promote the growth of the motor neuron cell under the spinal cord.

Sequence listing

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<120> a composition and its use in the treatment of spinal muscular atrophy

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Claims

1. A composition characterized by: the composition comprises an SMN adeno-associated virus vector and astrocytes.

2. A composition according to claim 1, wherein: the SMN adeno-associated virus vector expresses SMN; the amino acid sequence of the SMN is shown as SEQ ID NO. 1.

3. A composition according to claim 1, wherein: the nucleotide sequence of SMN expressed in the SMN adeno-associated virus vector is shown in SEQ ID NO. 2.

4. A composition according to claim 1, wherein: the ratio of the SMN adeno-associated virus vector to the astrocytes is 80-120 vg: 1 piece.

5. A composition according to claim 1, wherein: the astrocytes are obtained by inducing and differentiating stem cells of human oral mucosa.

6. A composition according to claim 1, wherein: the preparation method of the SMN adeno-associated virus vector comprises the steps of plasmid vector preparation, cell transfection and virus collection; and preparing the plasmid vector, namely constructing a nucleotide sequence for expressing the SMN on the adeno-associated virus AAV-IRES-hrGFP to obtain the pAAV plasmid vector.

7. A composition according to claim 6, wherein: transfecting the cell, and infecting HEK293 cells by adopting auxiliary plasmids, packaging plasmids and pAAV plasmids; the mass-volume ratio of the helper plasmid, the packaging plasmid and the pAAV plasmid is 1.5-2.5: 0.8-1.2: 0.8-1.2.

8. A composition according to claim 7, wherein: the helper plasmid was pHelper, and the packaging plasmid was pAAV-RC 8.

9. Use of a composition according to claim 1 for the treatment of spinal muscular atrophy, wherein: the SMN adeno-associated virus vector and the astrocytes can be mixed and then used, or can be used separately and sequentially.

10. Use of a composition according to claim 9 for the treatment of spinal muscular atrophy, wherein: the SMN adeno-associated virus vector and the astrocytes can be mixed and injected together, or can be separately and independently injected.