CN108795985A - A kind of mucopolysaccharidosis slow virus carrier, slow virus and its preparation method and application - Google Patents
A kind of mucopolysaccharidosis slow virus carrier, slow virus and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of slow virus carrier of mucopolysaccharidosis, slow virus and its preparation method and application, the slow virus carrier is that the donor splicing site at 5 ' ends of pTYF slow virus carriers is transformed, wherein, the slow virus carrier further includes MPS II genes.MPS II genes are specifically connected on the basis of the improved slow virus carrier of the application; more efficient gene delivery can be realized while guaranteeing safety; so that expression quantity of the MPS II genes in the brain relevant cell of transgenosis obviously increases, the transmission of normal gene during completion mucopolysaccharidosis gene therapy that can be more efficient.
Description
Technical field
The invention belongs to gene engineering technology field, be related to a kind of mucopolysaccharidosis slow virus carrier, slow virus and its
Slow virus carrier Optimal Expression MPS II genes after preparation method and application more particularly to a kind of application improvement are used to prepare and control
Treat the drug of Gaucher disease.
Background technology
Mucopolysaccharidosis II types are also referred to as special (Hunter) syndrome of the Chinese, are described earliest by Hunter (1917).
Mekusick (1965) is classified as mucopolysaccharidosis type Ⅱ, belongs to sex-linked recessive inheritance, due to idose -2- sulfuric esters
Caused by enzyme (iduornate-2-sulphate sulphatase, IDS) gene mutation.Patient is male, mother of patient
It is carrier but does not fall ill.It is this type of to be divided into two hypotypes again：II-A of MPS are heavy type, and mental incapacity is heavier, dead mostly before 15 years old
It dies；II-B of MPS are light-duty, and normal intelligence lasts a long time, and can survive by 40 years old or more.
Due to lacking iduronate-2-sulfatase in patient body, and make acidic mucopolysaccharide degradation that obstacle, the enzyme gene occur
It is located in Xq27.3~q28, since the shortage of enzyme makes excessive glutinous polysaccharide be deposited in histocyte, and with urine ejection.With
Increasing for polysaccharide deposition is sticked in histocyte, leads to dysfunction.Breathing has snore sound, is often accompanied by chronic respiratory tract infection.Lung is dynamic
Arteries and veins high pressure and coronary artery infarct are also much shown in.Often there is Heart enlargement and with systole phase and diastolic murmur.Heavy person's intelligence
It is incomplete more apparent.
How normal nascent is, occurs Development retardation, bone and face about since 2 years old in slight Le syndrome recklessly, but
Relatively light, generation is later, and progress is also relatively slow.There can be arthrocleisis, claw-like hand, short and small, but without kyphosis.It is saved in 2nd and 5 phalanges
Short and small, metacarpal bone proximal end does not come to a point.Radius, ulna distal joint face can relative tilt and angulation, wrist joint gap mild stenosis.At
People often has secondary osteoarticular change.Skin is thickened in wrinkle or nodositas, especially apparent with upper limb and chest, can be in pair sometimes
The distribution of title property, and have crinosity.It can blind because of retinosis.Often there is progressive deaf.Hepatosplenomegaly, with chronic diarrhea.
Occur excessive dermatan sulfate and Heparan sulfate in urine.
Discharge the ratio between dermatan sulfate and heparitin sulfate are 1 in diagnosis basis urine:1.Fibroblast cell-culture, 35S are viscous
Polysaccharide put aside, the Hunter syndromes factor of purifying, which is added, to be corrected, this can indirect proof be iduronate-2-sulfatase
Activity lacks.As can directly surveying serum and intracellular enzyme activity can then make a definite diagnosis.
Mucopolysaccharidosis is the disease of iduronate-2-sulfatase loss of activity caused by single gene mutation, therefore base
Because the thorough treatment to the disease theoretically may be implemented in therapy.Intracerebral injection intraventricular injection will
Drug is directly injected into the ventricles of the brain.By trace drug injection-tube people's telocoele when experiment.This method is easy to operate, and expense is relatively low, can
For observing various physical signs and variation behind different pharmaceutical note human brain room.
Although there are many gene therapy methods of the gene delivery of domestic and international application viral vectors progress at present, different diseases
Gene transfer efficiency difference is apparent caused by the different preparation methods of poisonous carrier or even same vehicle, and gene transfer efficiency will
Directly affect the therapeutic effect of disease.There is efficiency mistake using the method that hereditary disease is treated in gene therapy mostly now
It is low and modified only for blood stem cell so that expection is not achieved in the clinical effectiveness of disease treatment always, therefore is badly in need of
The method of viral gene transmission efficiency can be improved to the full extent come the effect of improving genetic disease.
Invention content
In view of the deficiencies of the prior art, the present invention provides a kind of mucopolysaccharidosis slow virus carrier, slow virus and its
Preparation method and application, the mucopolysaccharidosis slow virus carrier transfection efficiency higher, stability is stronger, and safety is more preferable.
For this purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of slow virus carrier, the slow virus carrier is in pTYF slow virus carriers
5 ' end donor splicing sites be transformed, specific reforming mode is as follows：
(a) donor splicing site at its 5 ' end is deleted or is transformed, and improved slow virus carrier donor splicing site is not
It is the package carrier and the potential site with reference to the homologous recombination between slow virus；
(b) it still has the function of viral packaging signal；
Wherein, the slow virus carrier further includes MPS II genes.
In the present invention, by the donor splicing site at 5 ' ends being deleted or being transformed so that slow virus carrier pTYF
Donor splicing site is not the package carrier and the potential site with reference to the homologous recombination between slow virus ', i.e., it is not
Have again pathogenic so that inhibition of HIV loses the function of self-replacation, therefore substantially increase whole gene treatment use it is slow
The security performance of viral vectors itself, this is a kind of safe function improvement not available for other all slow virus carriers, transfection
More efficient, stability is stronger, and safety is more preferable, the transmission of normal gene during completion gene therapy that can be more efficient,
Improved slow virus carrier is specifically loaded into MPS II genes, can successfully be realized in neuronal cell after transducer cell
MPS II genes stablize expression so that slow virus carrier can realize the treatment to mucopolysaccharidosis gene.
According to the present invention, the donor splicing site at 5 ' ends of the slow virus carrier is deleted or the nucleotide sequence of transformation
It is exemplified below：
In a particular embodiment, 5 ' donor splicing site GT of wild type sports CA, and particular sequence is as follows：Wild type (SEQ
ID NO.3)：GGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTA；
Saltant type (SEQ ID NO.4)：GGCAAGAGGCGAGGGGCGGCGACTGCAGAGTACGCCAAAAATTTTGACT
AGCGGAGGCTA.
In the particular embodiment, 5 ' donor splicing site GT of wild type sports GG, and particular sequence is as follows：Wild type
(SEQ ID NO.5)：GGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTA；
Saltant type (SEQ ID NO.6)：GGCAAGAGGCGAGGGGCGGCGACTGGGGAGTACGCCAAAAATTTTGACT
AGCGGAGGCTA.
According to the present invention, the nucleotide sequence of the MPS II genes has at least as shown in SEQ ID NO.1 or with it
80% homology, preferably at least 85% homology, the further preferably at least nucleotide sequence of 95% homology.
In some embodiments, the nucleotide sequence of the MPS II genes and nucleotides sequence shown in SEQ ID NO.1
Arrange the nucleotide sequence at least 80% homology.
In some embodiments, the nucleotide sequence of the MPS II genes and nucleotides sequence shown in SEQ ID NO.1
Arrange the nucleotide sequence at least 82% homology.
In some embodiments, the nucleotide sequence of the MPS II genes and nucleotides sequence shown in SEQ ID NO.1
Arrange the nucleotide sequence at least 85% homology.
In some embodiments, the nucleotide sequence of the MPS II genes and nucleotides sequence shown in SEQ ID NO.1
Arrange the nucleotide sequence at least 88% homology.
In some embodiments, the nucleotide sequence of the MPS II genes and nucleotides sequence shown in SEQ ID NO.1
Arrange the nucleotide sequence at least 90% homology.
In some embodiments, the nucleotide sequence of the MPS II genes and nucleotides sequence shown in SEQ ID NO.1
Arrange the nucleotide sequence at least 92% homology.
In some embodiments, the nucleotide sequence of the MPS II genes and nucleotides sequence shown in SEQ ID NO.1
Arrange the nucleotide sequence at least 95% homology.
In the present invention, inventor has found there is at least 80% homology with nucleotide sequence shown in SEQ ID NO.1
Sequence, be improved MPS II genes its still have the function of MPS II genes, it may be possible to iduronate-2-sulfatase
Length shortening or only with the functional domain partial sequence in iduronate-2-sulfatase, by these improved nucleotide
Sequence is loaded into the function that MPS II genes can be realized in the slow virus carrier, to be repaiied to MPS II genes
Multiple, nucleotide sequence (IDS) is as follows shown in the SEQ ID NO.1：
atgccgccaccccggaccggccgaggccttctctggctgggtctggttctgagctccgtctgcgtcgccctcggatc
cgaaacgcaggccaactcgaccacagatgctctgaacgttcttctcatcatcgtggatgacctgcgcccctccctgg
gctgttatggggataagctggtgaggtccccaaatattgaccaactggcatcccacagcctcctcttccagaatgcc
tttgcgcagcaagcagtgtgcgccccgagccgcgtttctttcctcactggcaggagacctgacaccacccgcctgta
cgacttcaactcctactggagggtgcacgctggaaacttctccaccatcccccagtacttcaaggagaatggctatg
tgaccatgtcggtgggaaaagtctttcaccctgggatatcttctaaccataccgatgattctccgtatagctggtct
tttccaccttatcatccttcctctgagaagtatgaaaacactaagacatgtcgagggccagatggagaactccatgc
caacctgctttgccctgtggatgtgctggatgttcccgagggcaccttgcctgacaaacagagcactgagcaagcca
tacagttgttggaaaagatgaaaacgtcagccagtcctttcttcctggccgttgggtatcataagccacacatcccc
ttcagataccccaaggaatttcagaagttgtatcccttggagaacatcaccctggcccccgatcccgaggtccctga
tggcctaccccctgtggcctacaacccctggatggacatcaggcaacgggaagacgtccaagccttaaacatcagtg
tgccgtatggtccaattcctgtggactttcaggaggaccaaagttccacaggtttcagactgaagacttcatctacc
agaaagtataagtag.
Further include promoter sequence before the MPS II genes according to the present invention, the promoter sequence be EF1 α and/
Or CMV, preferably EF1 α.
In the present invention, as long as the promoter that can start MPS II gene expressions is all feasible, inventor has found to use
EF1 α overall length promoters, can realize more efficient gene delivery while guaranteeing safety.
According to the present invention, the nucleotide sequence of the EF1 α has at least 90% same as shown in SEQ ID NO.2 or with it
Source property, the preferably at least nucleotide sequence of 95% homology.
In some embodiments, the nucleotide sequence of the EF1 α has with nucleotide sequence shown in SEQ ID NO.2
At least nucleotide sequence of 90% homology.
In some embodiments, the nucleotide sequence of the EF1 α has with nucleotide sequence shown in SEQ ID NO.2
At least nucleotide sequence of 92% homology.
In some embodiments, the nucleotide sequence of the EF1 α has with nucleotide sequence shown in SEQ ID NO.2
At least nucleotide sequence of 95% homology.
In the present invention, inventor has found there is at least 90% homology with nucleotide sequence shown in SEQ ID NO.2
Sequence, be improved EF1 α its still have the function of promoter, it may be possible to the shortening to EF1 α length, after these are transformed
Nucleotide sequence be loaded into the function that promoter can be realized in the slow virus carrier, to start the table of ARSA genes
It reaches, nucleotide sequence is as follows shown in the SEQ ID NO.2：
gcggccgctagcatgcctaggtcgaccaattctcatgtttgacagcttatcatcgataagctttggagctaagccag
caatggtagagggaagattctgcacgtcccttccaggcggcctccccgtcaccaccccccccaacccgccccgaccg
gagctgagagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaa
cgtagaacccagagatcgctgcgttcccgccccctcacccgcccgctctcgtcatcactgaggtggagaagagcatg
cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggc
aattgaaccggtgcctagagaaagtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcc
cgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaa
cacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattact
tccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggcctt
gcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctgg
tggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgct
ttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcg
gcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgg
gggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaag
gctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatgga
ggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgct
tcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtcttt
aggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggc
acttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggtt
caaagtttttttcttccatttcaggtgtcgtgaaaactctagagcggccgcggaggccgaattccgtcga.
Second aspect, the present invention provide a kind of recombinant slow virus, will include slow virus carrier as described in relation to the first aspect
The recombinant slow virus that pTYF is obtained with packaging helper plasmid pNHP and pHEF-VSV-G cotransfection mammalian cell.
Preferably, the mammalian cell is HEK293T cells and/or TE671 cells.
The third aspect, the present invention provide a kind of method preparing the slow virus as described in second aspect, include the following steps：
(1) slow virus carrier pTYF is transformed；
(2) in full genome synthetic promoter and MPS II gene orders and the slow virus carrier of inserting step (1) point mutation；
(3) slow virus carrier of structure and packaging helper plasmid cotransfection mammalian cell are obtained into recombinant slow virus.
According to the present invention, the site of step (2) described insertion can be the restriction enzyme site that any genetic engineering can synthesize, this
Invention preferably uses between BamHI and SpeI restriction enzyme sites.
According to the present invention, the packaging helper plasmid described in step (3) is pNHP and pHEF-VSV-G.
According to the present invention, the mammalian cell is HEK293T cells and/or TE671 cells.
According to the present invention, incubation time after the cotransfection mammalian cell is 24-72h, for example, can be for 24 hours, 25h,
26h、27h、28h、29h、30h、31h、32h、33h、34h、35h、36h、37h、38h、39h、40h、41h、42h、43h、44h、
45h, 46h, 47h, 48h, 50h, 52h, 55h, 58h, 60h, 62h, 65h, 68h, 70h or 72h.
Fourth aspect, the present invention provide a kind of recombinant cell, and the recombinant cell includes slow disease as described in relation to the first aspect
Poisonous carrier and/or the recombinant slow virus as described in second aspect.
According to the present invention, the recombinant cell is recombination stem cell, and preferably peripheral hematopoietic stem cells and/or mesenchyma are dry thin
Born of the same parents.
In the present invention, the stem cell after slow-virus transfection can stablize great expression MPS II genes, pass through by
Recombinant slow virus imports peripheral hematopoietic stem cells and mescenchymal stem cell, forms dual stem cell line, can further increase MPS
II genes are lived in transmission efficiency and the expression quantity of brain, can realize faster mucopolysaccharidosis remission and more comprehensively
Lasting gene therapy.
5th aspect, the present invention provide a kind of pharmaceutical composition, and described pharmaceutical composition includes as described in relation to the first aspect
Slow virus carrier, the recombinant slow virus as described in second aspect or any one in the recombinant cell as described in fourth aspect or
At least two combination.
6th aspect, the present invention provide slow virus carrier as described in relation to the first aspect, the recombinant lentiviral as described in second aspect
Virus, the recombinant cell as described in fourth aspect or the pharmaceutical composition as described in terms of the 5th are controlled preparing mucopolysaccharidosis
The drug for the treatment of and/or the purposes in reagent.
In a specific embodiment, the stem cell be by collect peripheral blood in patients after detach wherein stem cell,
It after slow virus carrier transfecting stem cells, will be fed back in a manner of intravenous injection again and carry out mucopolysaccharidosis disease into patient's body
Treatment.
It in a specific embodiment, can be by the way that slow virus carrier direct injection to sick cell position be sticked
Polysaccharide stores up the treatment of disease.
In a specific embodiment, mucopolysaccharidosis disease can be carried out by the way that slow virus carrier is transfected blood
Treatment.
According to the present invention, the composition further includes the auxiliary material pharmaceutically received, the auxiliary material be excipient, diluent,
In carrier, flavoring agent, adhesive and filler any one or at least two combination.
Compared with prior art, the present invention has the advantages that：
(1) the application slow virus carrier has carried out special transformation so that inhibition of HIV loses the function of self-replacation, greatly
The big security performance for improving the slow virus carrier itself that whole gene treatment uses, improved slow virus carrier transfection efficiency
Higher, stability is stronger, and safety is more preferable, the transmission of normal gene during completion gene therapy that can be more efficient；
(2) MPS II genes are specifically connected on the basis of the improved slow virus carrier of the application, in EF1 α promoters
Startup under, more efficient gene delivery can be realized while guaranteeing safety so that MPS II genes are in transgenosis
Expression quantity in brain relevant cell obviously increases, normal during completion mucopolysaccharidosis gene therapy that can be more efficient
The transmission of gene；
(3) the MPS II genes that the application slow virus carrier can be directly damaged in repair cell, can effectively improve
MPS II genes are lived in the transmission efficiency of brain with expression quantity, this has great importance to the validity of guarantee gene therapy,
To realize that faster mucopolysaccharidosis remission and more comprehensively lasting gene therapy are laid a good foundation.
Description of the drawings
Fig. 1 is the transformation schematic diagram of slow virus carrier pTYF；
Fig. 2 is the structural schematic diagram of slow virus carrier；
Fig. 3 is the purifying flow diagram of slow virus carrier；
Fig. 4 carries normal MPS II genes for slow virus carrier and is injected directly into controlling for brain treatment mucopolysaccharidosis disease
Treat flow diagram.
Specific implementation mode
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, according to technology or condition described in document in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
The structure of 1 slow virus carrier of embodiment
The present embodiment provides a kind of construction methods of slow virus carrier, specifically comprise the following steps：
(1) the transformation schematic diagram of slow virus carrier pTYF is as shown in Figure 1, specifically sport 5 ' donor splicing site of wild type
Site GT sports CA, and the enhancer in U3 is deleted, and specific remodeling method may refer to " Contributions of
Viral Splice Sites and cis-Regulatory Elements to Lentivirus Vector Function,
YAN CUI, JOURNAL OF VIROLOGY, July 1999, p.6171-6176 ", specific as follows：
The transformation of 5 ' donor splicing sites：
Wild type (SEQ ID NO.3)：GGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACT
AGCGGAGGCTA；
Saltant type (SEQ ID NO.4)：GGCAAGAGGCGAGGGGCGGCGACTGCAGAGTACGCCAAAAATTTTGACT
AGCGGAGGCTA；
(2) insertion of promoter and MPS II genes：
Normal MPS II gene orders (as shown in SEQ ID NO.1) and mankind's EF1 α promoters are synthesized by full genome
(as shown in SEQ ID NO.2) sequence, it is connected through restriction enzyme site in slow virus carrier TYF, by sequencing and
Double digestion (optimum reaction condition is with reference to the suggestion of NEB genuine), 5 ' use BamHI cloning sites (ggatccacc)-AUG；3 ' use SpeI
Cloning site (actagt) identifies obtained product and starts the lower normal MPS II of carrying to obtain the hEF1 α correctly connected
Gene is inserted into slow virus carrier, and specific link position and slow virus carrier constitute as shown in Figure 2.
Nucleotide sequence is as follows shown in specific SEQ ID NO.1：
atgccgccaccccggaccggccgaggccttctctggctgggtctggttctgagctccgtctgcgtcgccctcggatc
cgaaacgcaggccaactcgaccacagatgctctgaacgttcttctcatcatcgtggatgacctgcgcccctccctgg
gctgttatggggataagctggtgaggtccccaaatattgaccaactggcatcccacagcctcctcttccagaatgcc
tttgcgcagcaagcagtgtgcgccccgagccgcgtttctttcctcactggcaggagacctgacaccacccgcctgta
cgacttcaactcctactggagggtgcacgctggaaacttctccaccatcccccagtacttcaaggagaatggctatg
tgaccatgtcggtgggaaaagtctttcaccctgggatatcttctaaccataccgatgattctccgtatagctggtct
tttccaccttatcatccttcctctgagaagtatgaaaacactaagacatgtcgagggccagatggagaactccatgc
caacctgctttgccctgtggatgtgctggatgttcccgagggcaccttgcctgacaaacagagcactgagcaagcca
tacagttgttggaaaagatgaaaacgtcagccagtcctttcttcctggccgttgggtatcataagccacacatcccc
ttcagataccccaaggaatttcagaagttgtatcccttggagaacatcaccctggcccccgatcccgaggtccctga
tggcctaccccctgtggcctacaacccctggatggacatcaggcaacgggaagacgtccaagccttaaacatcagtg
tgccgtatggtccaattcctgtggactttcaggaggaccaaagttccacaggtttcagactgaagacttcatctacc
agaaagtataagtag；
Nucleotide sequence is as follows shown in specific SEQ ID NO.2：
gcggccgctagcatgcctaggtcgaccaattctcatgtttgacagcttatcatcgataagctttggagctaagccag
caatggtagagggaagattctgcacgtcccttccaggcggcctccccgtcaccaccccccccaacccgccccgaccg
gagctgagagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaa
cgtagaacccagagatcgctgcgttcccgccccctcacccgcccgctctcgtcatcactgaggtggagaagagcatg
cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggc
aattgaaccggtgcctagagaaagtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcc
cgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaa
cacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattact
tccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggcctt
gcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctgg
tggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgct
ttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcg
gcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgg
gggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaag
gctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatgga
ggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgct
tcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtcttt
aggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggc
acttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggtt
caaagtttttttcttccatttcaggtgtcgtgaaaactctagagcggccgcggaggccgaattccgtcga.
The preparation and authentication of 2 slow virus of embodiment
1) preparation of slow virus
The slow virus carrier that embodiment 1 is prepared further is packed, purifies and is concentrated, and the slow virus is obtained, tool
Body process is as shown in figure 3, be as follows：
(1) slow virus carrier built in embodiment 1 and packaging helper plasmid pNHP and pHEF-VSV-G cotransfection are fed
24-72h is cultivated in laticiferous cell HEK293T；
(2) slow virus that culture obtains is purified and is concentrated, obtain the slow virus.
2) identification of slow virus
Neuronal cell after the normal MPS II gene slow-virus transfections of the carrying of collection and spongiocyte are subjected to protein
Expression quantity is identified, with expression of the clear MPS II genes in neuronal cell.
From result, idose -2- sulphur is not present in the neuronal cell negative control cell without slow-virus transfection
The expression of acid esters zymoprotein, and the visible significantly greater amount in neuronal cell after carrying normal MPS II gene slow-virus transfections
Iduronate-2-sulfatase protein expression.
Illustrate that the present invention can successfully pass slow virus and make neuronal cell great expression iduronate-2-sulfatase egg
In vain, there is preferable disease treatment potential.
The therapeutic effect of 3 slow virus of embodiment
It is glutinous that the slow virus for the normal MPS II genes of carrying that embodiment 2 is prepared is subjected to direct injection treatment to brain
Polysaccharide stores up disease, and treatment flow diagram is as shown in figure 4, MRI or CT by brain determine injecting lentivirus carrier
Site and the specific coordinate of brain, to carry the slow virus carriers of normal MPS II genes in such a way that encephalic carries out direct injection,
It is input to patient's brain and carries out disease treatment.
From the results, it was seen that after direct injection slow virus, transmission efficiency of the effective raising MPS II genes in brain
It lives with expression quantity.
In conclusion the MPS II genes that the application slow virus carrier can be directly damaged in repair cell, it can be effective
The MPS II genes that improve live in transmission efficiency and the expression quantity of brain, this is to ensureing that it is important that the validity of gene therapy has
Meaning, to realize that faster mucopolysaccharidosis remission and more comprehensively lasting gene therapy are laid a good foundation.
Applicant states that the present invention illustrates the method detailed of the present invention, but the present invention not office by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope.
SEQUENCE LISTING
<110>Immune-gene therapy research institute of Shenzhen
<120>A kind of mucopolysaccharidosis slow virus carrier, slow virus and its preparation method and application
<130> 2018
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 939
<212> DNA
<213>Artificial synthesized sequence
<400> 1
atgccgccac cccggaccgg ccgaggcctt ctctggctgg gtctggttct gagctccgtc 60
tgcgtcgccc tcggatccga aacgcaggcc aactcgacca cagatgctct gaacgttctt 120
ctcatcatcg tggatgacct gcgcccctcc ctgggctgtt atggggataa gctggtgagg 180
tccccaaata ttgaccaact ggcatcccac agcctcctct tccagaatgc ctttgcgcag 240
caagcagtgt gcgccccgag ccgcgtttct ttcctcactg gcaggagacc tgacaccacc 300
cgcctgtacg acttcaactc ctactggagg gtgcacgctg gaaacttctc caccatcccc 360
cagtacttca aggagaatgg ctatgtgacc atgtcggtgg gaaaagtctt tcaccctggg 420
atatcttcta accataccga tgattctccg tatagctggt cttttccacc ttatcatcct 480
tcctctgaga agtatgaaaa cactaagaca tgtcgagggc cagatggaga actccatgcc 540
aacctgcttt gccctgtgga tgtgctggat gttcccgagg gcaccttgcc tgacaaacag 600
agcactgagc aagccataca gttgttggaa aagatgaaaa cgtcagccag tcctttcttc 660
ctggccgttg ggtatcataa gccacacatc cccttcagat accccaagga atttcagaag 720
ttgtatccct tggagaacat caccctggcc cccgatcccg aggtccctga tggcctaccc 780
cctgtggcct acaacccctg gatggacatc aggcaacggg aagacgtcca agccttaaac 840
atcagtgtgc cgtatggtcc aattcctgtg gactttcagg aggaccaaag ttccacaggt 900
ttcagactga agacttcatc taccagaaag tataagtag 939
<210> 2
<211> 1533
<212> DNA
<213>Artificial synthesized sequence
<400> 2
gcggccgcta gcatgcctag gtcgaccaat tctcatgttt gacagcttat catcgataag 60
ctttggagct aagccagcaa tggtagaggg aagattctgc acgtcccttc caggcggcct 120
ccccgtcacc acccccccca acccgccccg accggagctg agagtaattc atacaaaagg 180
actcgcccct gccttgggga atcccaggga ccgtcgttaa actcccacta acgtagaacc 240
cagagatcgc tgcgttcccg ccccctcacc cgcccgctct cgtcatcact gaggtggaga 300
agagcatgcg tgaggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 360
cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaagt ggcgcggggt 420
aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 480
gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 540
acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg 600
cgtgccttga attacttcca cgcccctggc tgcagtacgt gattcttgat cccgagcttc 660
gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg 720
cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc 780
gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg 840
cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta 900
tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg 960
cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc 1020
ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc 1080
tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag 1140
ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa 1200
ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc 1260
cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg 1320
aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag 1380
cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca 1440
ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaaaac 1500
tctagagcgg ccgcggaggc cgaattccgt cga 1533
<210> 3
<211> 60
<212> DNA
<213>Artificial synthesized sequence
<400> 3
ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 60
<210> 4
<211> 60
<212> DNA
<213>Artificial synthesized sequence
<400> 4
ggcaagaggc gaggggcggc gactgcagag tacgccaaaa attttgacta gcggaggcta 60
<210> 5
<211> 60
<212> DNA
<213>Artificial synthesized sequence
<400> 5
ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 60
<210> 6
<211> 60
<212> DNA
<213>Artificial synthesized sequence
<400> 6
ggcaagaggc gaggggcggc gactggggag tacgccaaaa attttgacta gcggaggcta 60
Claims (10)
1. a kind of mucopolysaccharidosis slow virus carrier, which is characterized in that the slow virus carrier is in pTYF slow virus carriers
5 ' end donor splicing sites be transformed, specific reforming mode is as follows：
(a) donor splicing site at its 5 ' end is deleted or is transformed, and improved slow virus carrier donor splicing site is not institute
State package carrier and the potential site with reference to the homologous recombination between slow virus；
(b) it still has the function of viral packaging signal；
Wherein, the slow virus carrier further includes MPS II genes.
2. slow virus carrier according to claim 1, which is characterized in that the nucleotide sequence of the MPS II genes is such as
There is at least 80% homology, preferably at least 85% homology, further preferably at least 95% shown in SEQ ID NO.1 or with it
The nucleotide sequence of homology.
3. slow virus carrier according to claim 1 or 2, which is characterized in that further include opening before the MPS II genes
Promoter sequences；
Preferably, the promoter sequence is EF1 α and/or CMV, preferably EF1 α；
Preferably, the nucleotide sequence of the EF1 α has at least 90% homology as shown in SEQ ID NO.2 or with it, preferably
At least nucleotide sequence of 95% homology.
4. a kind of recombinant slow virus, which is characterized in that will include slow virus carrier as claimed in any one of claims 1-3
The recombinant slow virus that pTYF is obtained with packaging helper plasmid pNHP and pHEF-VSV-G cotransfection mammalian cell；
Preferably, the mammalian cell is HEK293T cells and/or TE671 cells.
5. a kind of method preparing slow virus as claimed in claim 4, which is characterized in that include the following steps：
(1) donor splicing site for holding slow virus carrier pTYF 5 ' carries out point mutation；
(2) in full genome synthetic promoter and MPS II gene orders and the slow virus carrier of inserting step (1) point mutation；
(3) slow virus carrier of structure and packaging helper plasmid cotransfection mammalian cell are obtained into recombinant slow virus.
6. according to the method described in claim 5, it is characterized in that, the site of step (2) described insertion is BamHI and SpeI enzymes
Between enzyme site.
7. method according to claim 5 or 6, which is characterized in that packaging helper plasmid described in step (3) be pNHP and
pHEF-VSV-G；
Preferably, the mammalian cell is HEK293T cells and/or TE671 cells；
Preferably, the incubation time after the cotransfection mammalian cell is 24-72h.
8. a kind of recombinant cell, which is characterized in that the recombinant cell includes slow disease as claimed in any one of claims 1-3
Poisonous carrier and/or recombinant slow virus as claimed in claim 4；
Preferably, the recombinant cell is recombination stem cell, preferably peripheral hematopoietic stem cells and/or mescenchymal stem cell.
9. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes as claimed in any one of claims 1-3
Any one in slow virus carrier, recombinant slow virus as claimed in claim 4 or recombinant cell as claimed in claim 8
Or at least two combination.
10. slow virus carrier, recombinant slow virus as claimed in claim 4, such as claim as described in claim 1-3
Recombinant cell or pharmaceutical composition as claimed in claim 9 described in 8 in the drug for preparing mucopolysaccharidosis treatment and/or
Purposes in reagent；
Preferably, the composition further includes the auxiliary material pharmaceutically received；
Preferably, the auxiliary material be excipient, diluent, carrier, flavoring agent, adhesive and filler in any one or extremely
Few two kinds of combination.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810549060.7A CN108795985A (en) | 2018-05-31 | 2018-05-31 | A kind of mucopolysaccharidosis slow virus carrier, slow virus and its preparation method and application |
| PCT/CN2019/089641 WO2019228526A1 (en) | 2018-05-31 | 2019-05-31 | Lentiviral vector used for treatment of mucopolysaccharidosis, lentivirus, and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810549060.7A CN108795985A (en) | 2018-05-31 | 2018-05-31 | A kind of mucopolysaccharidosis slow virus carrier, slow virus and its preparation method and application |
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|---|---|---|---|---|
| WO2021212279A1 (en) * | 2020-04-20 | 2021-10-28 | 广东东阳光药业有限公司 | Titer improved transfer plasmid for lentivirus |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2654800A1 (en) * | 2010-12-22 | 2013-10-30 | Fondazione Telethon | Therapeutic strategies to treat cns pathology in mucopolysaccharidoses |
| KR20160008987A (en) * | 2014-07-15 | 2016-01-25 | 주식회사 녹십자랩셀 | Intranasal composition comprising stem cells expressing a subtance for treating brain-nervous system diseases |
| EP3101125A1 (en) * | 2015-06-05 | 2016-12-07 | Laboratorios Del Dr. Esteve, S.A. | Adenoassociated virus vectors for the treatment of mucopolysaccharidoses |
| CN106480096A (en) * | 2016-09-30 | 2017-03-08 | 陕西慧康生物科技有限责任公司 | Expression vector and reconstitution cell |
| WO2017139561A1 (en) * | 2016-02-12 | 2017-08-17 | Bluebird Bio, Inc. | Vcn enhancer compositions and methods of using the same |
| CN108728495A (en) * | 2018-05-31 | 2018-11-02 | 深圳市免疫基因治疗研究院 | A kind of Sanfilippo A syndromes slow virus carrier, slow virus and its preparation method and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6207455B1 (en) * | 1997-05-01 | 2001-03-27 | Lung-Ji Chang | Lentiviral vectors |
| WO2000040741A2 (en) * | 1999-01-07 | 2000-07-13 | The Government Of The United States Of America, As Represented By The Secretary Department Of Health And Human Services, The National Institutes Of Health | Lentivirus vector system |
-
2018
- 2018-05-31 CN CN201810549060.7A patent/CN108795985A/en active Pending
-
2019
- 2019-05-31 WO PCT/CN2019/089641 patent/WO2019228526A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2654800A1 (en) * | 2010-12-22 | 2013-10-30 | Fondazione Telethon | Therapeutic strategies to treat cns pathology in mucopolysaccharidoses |
| KR20160008987A (en) * | 2014-07-15 | 2016-01-25 | 주식회사 녹십자랩셀 | Intranasal composition comprising stem cells expressing a subtance for treating brain-nervous system diseases |
| EP3101125A1 (en) * | 2015-06-05 | 2016-12-07 | Laboratorios Del Dr. Esteve, S.A. | Adenoassociated virus vectors for the treatment of mucopolysaccharidoses |
| WO2017139561A1 (en) * | 2016-02-12 | 2017-08-17 | Bluebird Bio, Inc. | Vcn enhancer compositions and methods of using the same |
| CN106480096A (en) * | 2016-09-30 | 2017-03-08 | 陕西慧康生物科技有限责任公司 | Expression vector and reconstitution cell |
| CN108728495A (en) * | 2018-05-31 | 2018-11-02 | 深圳市免疫基因治疗研究院 | A kind of Sanfilippo A syndromes slow virus carrier, slow virus and its preparation method and application |
Non-Patent Citations (5)
| Title |
|---|
| TAICHI WAKABAYASHI等: "Hematopoietic Stem Cell Gene Theray Corrects Neuropathic Phenotype in Murine Model of Mucopolysaccharidosis Type Ⅱ", 《HUMAN GENE THERAPY》 * |
| UETSUKI. T.等: "Human elongation factor EF-1-alpha gene，complete cds", 《GENBANK》 * |
| VOLLEBREGT AAM等: "Homo sapiens iduronate 2-sulfatase(IDS)，transcript variant 4，non-coding RNA", 《GENBANK》 * |
| YAN CUI等: "Contribtions of Viral Splicing Sites and cis-Regulatory Elements to Lentivirus Vector Function", 《JOURNAL OF VIROLOGY》 * |
| YONGYU WANG等: "Engineering vascular tissue with functional smooth muscle cells derived from human iPS cells and nanofibrous scaffolds", 《BIOMATERIALS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021212279A1 (en) * | 2020-04-20 | 2021-10-28 | 广东东阳光药业有限公司 | Titer improved transfer plasmid for lentivirus |
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