CN112522321A - Gene therapy vector and application thereof - Google Patents
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Abstract
The present invention relates to a gene therapy vector and its use, the gene therapy vector comprising: a) left (5') LTR; b) psi (Psi) package signal; c) a retroviral export element; d) central polypurine tract/central termination sequence; e) a promoter operably linked to a polynucleotide encoding a colony stimulating factor 1receptor polypeptide; f) a polynucleotide encoding a colony stimulating factor 1receptor polypeptide; g) a post-transcriptional regulatory element; and h) right (3') LTR. The gene therapy vector can transform cells in vitro, so that the cells express CSF1R protein, thereby treating HDLS and improving the survival state of HDLS patients.
Description
Technical Field
The invention relates to the technical field of gene therapy, in particular to a gene therapy vector and application thereof.
Background
Hereditary diffuse leukoencephalopathy combined axonometaloid changes (HDLS, hereditary diffuse leukoencephalopathy with spheres) were first described by swedish doctors in 1984. The clinical manifestations include cognitive disorders (memory decline, speech disorders, etc.), personality behavior changes (depression, apathy, anxiety, irritability, etc.), motor dysfunctions (dysbasia, dyskinesia, ataxia, etc.), susceptibility to misdiagnosis of alzheimer's disease, frontotemporal dementia, parkinson's syndrome, multiple sclerosis and leukoencephalopathy. The age of onset is 47.2 + -14.5 years, the mean course of disease is 6.0 + -3.1 years, and the age of death is 57.2 + -13.1 years. The pathological manifestations are frontal lobe, frontal lobe atrophy, ventricular dilatation, white matter deformation of the brain, myelin sheath, axonal loss and massive axonal globoid changes. At present, no effective HDLS treatment method exists in clinic, and the treatment method is mainly used for symptomatic treatment and mainly comprises general nursing, nutrition support, anti-epilepsy drugs and antibiotics. The symptomatic treatment method can not cure the disease radically and only slightly relieve the symptoms of the patient. Therefore, new treatments are urgently needed to improve the survival status of HDLS patients.
Disclosure of Invention
In view of the above, the present invention provides a gene therapy vector and its use, wherein the gene therapy vector can be used to modify cells in vitro, so that the cells can express colony stimulating factor 1receptor (CSF1R) protein, thereby treating HDLS and improving the survival status of HDLS patients.
In view of the above objects, the first aspect of the present invention provides a gene therapy vector for treating HDLS, comprising:
a) left (5') LTR;
b) psi (Psi) package signal;
c) a retroviral export element;
d) central polypurine tract/central termination sequence;
e) a promoter operably linked to a polynucleotide encoding a colony stimulating factor 1receptor polypeptide;
f) a polynucleotide encoding a colony stimulating factor 1receptor polypeptide;
g) a post-transcriptional regulatory element; and
h) right (3') LTR.
In a preferred embodiment of the present invention, wherein the gene therapy vector may be a gamma retrovirus vector, an adeno-associated virus (AAV) vector, an adenovirus vector, or a herpes virus vector;
preferably, the gene therapy vector is a lentiviral vector.
In a preferred embodiment of the invention, wherein the lentivirus is selected from the following: human Immunodeficiency Virus (HIV), visna-mei virus (VMV), caprine arthritis-encephalitis virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), or Simian Immunodeficiency Virus (SIV);
preferably, the lentivirus is Human Immunodeficiency Virus (HIV).
In a preferred embodiment of the present invention, wherein the above gene therapy vector further comprises a nucleotide sequence encoding an expression tag;
preferably, the expression tag is a truncated epidermal growth factor receptor, firefly luciferase, enhanced green fluorescent protein, streptavidin-binding peptide tag (Strep tag II), or Flag tag (Flag tag).
In a preferred embodiment of the invention, wherein the promoter of the left (5') LTR is replaced by a heterologous promoter selected from the group consisting of: a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a simian virus 40(SV40) promoter.
In a preferred embodiment of the invention, wherein said right (3') LTR comprises one or more modifications;
preferably, the right (3') LTR comprises one or more deletions that prevent viral transcription beyond the first round of viral replication;
more preferably, the right (3') LTR comprises a deletion of the TATA box and Sp1 and NF-. kappa.B transcription factor binding sites in the U3 region of the 3' LTR;
further preferably, the right (3') LTR is a self-inactivating (SIN) LTR.
In a preferred embodiment of the invention, wherein the promoter operably linked to the polynucleotide encoding the colony stimulating factor 1receptor polypeptide comprises: a short elongation factor 1 α (EF1 α) promoter or a transcriptionally active fragment thereof, a myeloproliferative sarcoma virus enhancer negative control region deleted and dl587rev primer binding site substituted (MND) promoter or a transcriptionally active fragment thereof;
preferably, the promoter operably linked to the polynucleotide encoding a colony stimulating factor 1receptor polypeptide is selected from the group consisting of: transmembrane protein 119(TMEM119), integrin subunit α M (CD11b), CD45, ionic calcium binding adaptor molecule 1(IBA1), C-X3-C motif chemokine receptor 1(CX3CR1), adhesion G protein coupled receptor E1(F4/80), CD68 or CD 40.
In a preferred embodiment of the present invention, wherein the post-transcriptional regulatory element comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
In a preferred embodiment of the invention, wherein the amino acid sequence of the colony stimulating factor 1receptor polypeptide is as set forth in SEQ ID NO: 5, respectively.
In a preferred embodiment of the invention, wherein the polynucleotide sequence encoding the colony stimulating factor 1receptor polypeptide is as set forth in SEQ ID NO: 3 is shown in the specification;
preferably, the polynucleotide encoding the colony stimulating factor 1receptor polypeptide is a codon optimised expression sequence as set forth in SEQ ID NO: 4, respectively.
In a preferred embodiment of the present invention, wherein the gene therapy vector is pMND-CSF1R-LV or pEF1 α -CSF1R-LV, respectively, the nucleotide sequence of pMND-CSF1R-LV is as shown in SEQ ID NO: 1, the nucleotide sequence of pEF1 alpha-CSF 1R-LV is shown as SEQ ID NO: 2, respectively.
Based on the same inventive concept, the second aspect of the present invention provides a cell transduced with a gene therapy vector, which includes the gene therapy vector described above.
In a preferred embodiment of the present invention, wherein said cells comprise hematopoietic stem cells, stem cells or progenitor cells;
preferably, the cell is CD34+A cell.
Based on the same inventive concept, the third aspect of the present invention provides a pharmaceutical composition comprising the above gene therapy vector or the above cell, and a pharmaceutically acceptable carrier or excipient.
Based on the same inventive concept, the fourth aspect of the present invention provides the use of the above gene therapy vector, the above cell or the above pharmaceutical composition for the preparation of a medicament for preventing, treating and alleviating hereditary diffuse leukoencephalopathy with axonal globulosis (HDLS) and related diseases; and
the use of the above gene therapy vector, the above cell or the above pharmaceutical composition for the preparation of a medicament for reducing or eliminating gene mutation of colony stimulating factor 1receptor (CSF1R) expressed by colony stimulating factor 1receptor (CSF 1R).
Drawings
FIG. 1 is a schematic diagram of CSF1R protein expression vectors of different promoters; wherein the upper panel corresponds to the structural representation of the pMND-CSF1R-LV vector (MND promoter) and the lower panel corresponds to the structural representation of the pEF1 α -CSF1R-LV vector (EF1 α -short promoter);
FIG. 2 is a graph showing the results of staining with Coomassie Brilliant blue to detect CSF1R protein expression in hematopoietic stem cells;
FIG. 3 is a flow chart showing the expression of CSF1R protein in hematopoietic stem cells (transduced with pMND-CSF1R-LV vector);
FIG. 4 shows flow-through assay of CSF1R protein expression in hematopoietic stem cells (transduced with pEF1 α -CSF1R-LV vector);
FIG. 5 is a flow chart of CSF1R protein expression in differentiated macrophages.
Detailed Description
It is to be noted that unless otherwise defined, technical or scientific terms used in one or more embodiments of the present specification should have the ordinary meaning as understood by those of ordinary skill in the art to which this disclosure belongs.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.
With the sequence information provided by the present invention, one skilled in the art can use available cloning techniques to generate vectors suitable for transduction into cells.
"Polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA/RNA hybrids.
By "patient" is meant a subject who has been diagnosed with a particular disease, disorder, or condition that can be treated with gene therapy vectors, cell-based therapies, and methods disclosed elsewhere herein.
"treatment" includes any beneficial or desired effect on a disease or pathological condition and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may involve, optionally, reduction of the disease or condition or delay in progression of the disease or condition. "treatment" does not necessarily indicate complete eradication or cure of the disease or condition or symptoms associated therewith.
"prevent" and words like "prevent" and the like indicate the possibility of preventing, inhibiting or reducing the occurrence or recurrence of a disease or condition. "preventing" also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "preventing" and similar words also include reducing the strength, effect, symptoms, and/or burden of a disease or condition prior to the onset or recurrence of the disease or condition.
Hereditary diffuse white matter encephalopathy (HDLS) is a rare autosomal dominant hereditary white matter disease, which is clinically mainly manifested by progressive cognitive impairment, Parkinson's disease, epilepsy, personality and behavior changes and the like, and is asymmetric white matter impairment on nuclear magnetic resonance imaging.
At present, no effective HDLS treatment method exists in clinic, and the treatment method is mainly used for symptomatic treatment and mainly comprises general nursing, nutrition support, anti-epilepsy drugs and antibiotics. The method specifically comprises the following steps: the effectiveness of L-dopa (levodopa) or other dopamine therapy for HDLS or HDLS patients with atypical Parkinson's phenotype has not been demonstrated. Antidepressants may be used to treat the symptoms of depression, but there are no long-term effective reports. Antipsychotic drugs are generally not recommended for routine use due to extrapyramidal side effects, but may be used for a short period in HDLS patients with significant psychiatric symptoms. As the condition of HDLS progresses, social problems (unemployment, divorce, financial difficulties, alcoholism) and suicidal tendency are often accompanied. Periodically performing clinical assessment and monitoring changes in HDLS patient behavior and communication, changing care and support systems (wheelchairs or personal assistance) in time; appropriate physical rehabilitation therapy helps to improve the quality of life by reducing muscle contracture, gastrostomy when dysphagia is severe, and the like.
The only causative gene has been identified to date as the Colony Stimulating Factor 1Receptor (CSF1R, Colony Stimulating Factor 1Receptor) gene. CSF1R gene encodes type III tyrosine kinase receptor, belong to platelet-derived growth factor receptor family, it is 972 amino acids (19 amino acid signal peptide, 493 amino acid extracellular region, 25 amino acid transmembrane region, 435 amino acid intracellular tyrosine kinase catalytic region), mainly express mononuclear phagocyte (also called microglia) in the brain, the microglia has important roles in neuronal and brain development; also expressed in osteoclasts, which play an important role in bone mineralization. CSF1R forms a dimer with ligands (CSF1, IL-34) and activates multiple downstream kinase signaling pathways, including phosphatidylinositol 3-kinase (PI3K), extracellular regulatory protein kinase (ERK1/2), and c-Jun amino terminal kinase (JNK), via autophosphorylation.
In 2012, genome-wide association analysis and exon sequencing confirmed that HDLS is an autosomal dominant genetic disease (disease cases show that HDLS can sporadically develop diseases and incomplete exons), CSF1R is a pathogenic gene of HDLS, and influence of CSF1R mutation on a tyrosine kinase domain may be the pathological basis of HDLS. By 1 month of 2018, 71 HDLS pathogenic mutations (56 missense mutations, 8 splice site mutations, 3 frameshift mutations, 2 nonsense mutations, 2 small deletions) were found. Almost all pathogenic mutations occur in the intracellular tyrosine kinase domain, mainly in exons 18 and 19, while mutations in the extracellular domain or increased CSF1R copy number may lead to the development of hematologic tumors or renal cell carcinoma. Whether HDLS pathogenic mutations affect CSF1R autophosphorylation has not been a uniform result. Whether the pathogenic mechanism of HDLS is dominant negative or haploidentical insufficiency cannot be determined.
The pathogenic mechanism of HDLS has been studied for a short time and has not been clearly concluded. With the continuous development and improvement of cell therapy and gene therapy technology, the substitution and treatment of diseased tissues by using genetically engineered cells has become a research hotspot in the medical field and even in the whole life science field. Hematopoietic stem cells can enter the brain and differentiate into microglia have been demonstrated in mice. Currently, there is no effective treatment for HDLS in clinical practice, and new treatments are urgently needed to improve the survival status of HDLS patients. The technical solution provided by the present invention addresses this need.
Gene therapy refers to the introduction of foreign genes into target cells to correct or compensate for diseases caused by gene defects or abnormal expression of genes. The invention aims to modify hematopoietic stem cells in vitro by a gene therapy vector so as to treat hereditary diffuse leukoencephalopathy with axonal spheroidicity (HDLS).
1. Gene therapy vector
In a first aspect the present invention provides a gene therapy vector for the treatment of HDLS comprising:
a) left (5') LTR;
b) psi (Psi) package signal;
c) a retroviral export element;
d) central polypurine tract/central termination sequence;
e) a promoter operably linked to a polynucleotide encoding a colony stimulating factor 1receptor polypeptide;
f) a polynucleotide encoding a colony stimulating factor 1receptor polypeptide;
g) a post-transcriptional regulatory element; and
h) right (3') LTR.
In the present invention, the gene therapy vector includes: a gamma retroviral vector, an adeno-associated virus (AAV) vector, an adenoviral vector, or a herpes viral vector; preferably, the gene therapy vector is a lentiviral vector, which is a broad class of retroviral vectors, preferably a Human Immunodeficiency Virus (HIV).
The Long Terminal Repeat (LTR) is a base pair domain located at the end of the retroviral DNA, which in the case of its native sequence is a direct repeat and contains the U3, R and U5 regions. LTRs typically provide essential functions for retroviral gene expression (e.g., initiation, and polyadenylation of gene transcripts) and viral replication. The LTRs contain numerous regulatory signals including transcriptional control elements, polyadenylation signals, and sequences required for replication and integration of the viral genome. The viral LTR is divided into three regions called U3, R and U5. The U3 region contains enhancer and promoter elements. The U5 region is a sequence between the primer binding site and the R region and contains a polyadenylation sequence. The R (repeat sequence) region is flanked by the U3 and U5 regions. The LTRs are composed of the U3, R, and U5 regions, and occur at the 5 'and 3' ends of the viral genome.
"packaging signal" refers to a sequence located within the retroviral genome required for insertion of viral RNA into the viral capsid or particle. Several retroviral vectors use a minimal packaging signal (also known as a psi [ psi ] sequence) required for encapsidation of the viral genome.
"retroviral export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm. Retroviral export elements include, but are not limited to, the Human Immunodeficiency Virus (HIV) Rev Response Element (RRE) and the hepatitis B virus post-transcriptional regulatory element (HPRE).
The gene therapy vector contains cis-acting elements, namely a central polypurine tract (cPPT) and a Central Termination Sequence (CTS), wherein the cPPT/CTS sequence can be the cPPT/CTS of HIV1, and the integration and transduction efficiency of the vector can be improved.
By "operably linked" is meant that the nucleic acid sequences are functionally related to the sequences to which they are operably linked such that they are linked in a manner such that they affect the expression or function of each other. For example, a nucleic acid sequence operably linked to a promoter will have an expression pattern that is affected by the promoter.
Promoters mediate the expression of nucleic acid sequences to which they are linked. Promoters may be constitutive or may be inducible. The promoter may direct expression prevalent in hematopoietic stem cells, or progenitor cells. A promoter operably linked to a polynucleotide encoding a colony stimulating factor 1receptor polypeptide comprises: short elongation factor 1 alpha (EF1 alpha) promoter or a transcriptionally active fragment thereof, a MND promoter or a transcriptionally active fragment thereof in which the myeloproliferative sarcoma virus enhancer negative control region is deleted and the dl587rev primer binding site is substituted (see Challita et al, J Virol., 69(2):748-55 (1995)); suitable promoters may be selected from the following: transmembrane protein 119(TMEM119), integrin subunit α M (CD11B), CD45 (expressed by hematopoietic cells), ionic calcium binding adaptor 1(IBA1), C-X3-C motif chemokine receptor 1(CX3CR1), adhesion G protein-coupled receptor E1(F4/80), CD68 (expressed by macrophages), or CD40 (expressed by T and B cells).
The short elongation factor 1 α (EF1 α) promoter refers to the original EF1 α promoter that lacks the first intron of the human EF1a gene.
The post-transcriptional regulatory element may improve transgene expression by promoting mRNA transcript maturation, and may be woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
In a preferred embodiment of the present invention, the above gene therapy vector further comprises a nucleotide sequence encoding an expression tag;
preferably, the expression tags are truncated epidermal growth factor receptor (EGFRt), firefly luciferase, Enhanced Green Fluorescent Protein (EGFP), streptavidin-binding peptide tag Strep tag II, or Flag tag, which can be attached to the C-terminus or N-terminus of a protein of interest (e.g., colony stimulating factor 1receptor polypeptide) to measure the expression of the protein of interest in a particular cell in an assay. EGFRt is a truncated form of EGFRt in which domain III and domain IV of a wild-type EGFR are truncated, and this truncated EGFRt does not transmit an intracellular signal and does not transmit other signals into cells. The Enhanced Green Fluorescent Protein (EGFP) is a mutant of the Green Fluorescent Protein (GFP), emits fluorescence intensity which is more than 6 times that of the GFP, and is expressed by escherichia coli, and the non-glycosylated homodimeric protein contains 239 amino acids and has the molecular weight of 26.9 kDa. The streptavidin-binding peptide tag (Strep tag II) consists of 8 amino acid residues, the sequence being Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (W-S-H-P-Q-F-E-K). The Flag tag (Flag tag) consists of 8 amino acid residues, and the sequence is Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (D-Y-K-D-D-D-D-K)
In a preferred embodiment of the invention, the promoter of the left (5') LTR is replaced by a heterologous promoter selected from the group consisting of: cytomegalovirus (CMV) promoter, Rous Sarcoma Virus (RSV) promoter, or simian virus 40(SV40) promoter, i.e., by replacing the U3 region of the (5') LTR with a heterologous promoter to provide additional safety enhancements to drive transcription of the viral genome during viral particle production. This substitution reduces the likelihood of recombination to generate a replication competent virus because the complete U3 sequence is not present in the virus production system.
The safety of gene therapy vectors is crucial for any possible gene therapy. The gene therapy vectors of the invention include one or more modifications, including but not limited to modifications to one or more LTRs, to render the vector replication deficient. For example, the gene therapy vector includes a modified 3'LTR, wherein the modification includes deletion of viral promoters and enhancers in the U3 region of the 3' LTR to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication, and thus, without the U3 enhancer-promoter, the viral transcription cannot proceed.
The self-inactivating (SIN) LTR is a fragment in which the enhancer and promoter sequences of the U3 region of the viral 3' LTR are deleted from the proviral vector, so that the HIV-1 enhancer and promoter sequences are lost from the vector and RNA cannot be transcribed even if all viral proteins are present.
In a preferred embodiment of the invention, wherein the polynucleotide sequence encoding a colony stimulating factor 1receptor (CSF1R) polypeptide is as set forth in SEQ ID NO: 3, namely the original sequence of the CSF1R polypeptide;
preferably, the polynucleotide encoding the colony stimulating factor 1receptor polypeptide is a codon optimised expression sequence as set forth in SEQ ID NO: 4 to enhance its expression in a host cell. The main process of codon optimization: preferentially selecting codons preferred by the expression host cells according to the codon preference, and eliminating rare codons; the GC content of the whole encoding gene is adjusted to a suitable range by the principle that the codon having the highest AT content is selected preferentially among a plurality of synonymous codons corresponding to the same amino acid. In addition, the codon optimization process also needs to consider: stability of secondary structure of mRNA, elimination of tandem rare codons, avoidance of codon duplication, adjustment of ribosome binding sites, attention to the environment of the initiation termination codon, and the like. In a preferred embodiment of the invention, wherein the amino acid sequence of the colony stimulating factor 1receptor polypeptide is as set forth in SEQ ID NO: 5, respectively.
The invention constructs two gene therapy vectors which are respectively pMND-CSF1R-LV or pEF1 alpha-CSF 1R-LV, and the pMND-CSF1R-LV vector mainly comprises: a chimeric 5 'LTR, Psi (Psi) packaging signal, Human Immunodeficiency Virus (HIV) Rev Response Element (RRE), cPPT/FLAP, a myeloproliferative sarcoma virus enhancer negative control region deleted and dl587rev primer binding site substituted (MND) promoter, a polynucleotide encoding a CSF1R polypeptide, a truncated epidermal growth factor receptor (EGFRt) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a self-inactivating (SIN)3' LTR, the nucleotide sequence of which is as set forth in SEQ ID NO: 1 is shown in the specification; pEF1 alpha-CSF 1R-LV vector mainly comprises: a chimeric 5 'LTR, Psi (Psi) packaging signal, Human Immunodeficiency Virus (HIV) Rev Response Element (RRE), cPPT/FLAP, short elongation factor 1 alpha (EF1 alpha) promoter, polynucleotide encoding CSF1R polypeptide, truncated epidermal growth factor receptor (EGFRt) tag, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and self-inactivating (SIN)3' LTR, the nucleotide sequence of which is as set forth in SEQ ID NO: 2, respectively.
The term "cPPT/FLAP" as used herein refers to a nucleic acid sequence comprising the central polypurine tract (cPPT) and Central Termination Sequence (CTS) of a retrovirus (e.g., HIV-1 and HIV-2) (see U.S. Pat. No. 6,682,907)
A "chimeric 5 'LTR" as referred to herein refers to a 5' LTR in which the U3 region has been replaced by a heterologous promoter (e.g., RSV promoter) to drive transcription of the viral genome during production of the viral particle. Because the complete U3 sequence is not present in the virus production system, this substitution reduces the likelihood of recombination to generate replication competent virus.
The gene therapy vector can generate higher virus titer when being transduced into cells and has good safety. The gene therapy vector can transform cells in vitro, so that the cells express CSF1R protein, and are differentiated into microglia after being infused back into a patient body, so that the microglia with normal functions is obtained, and the purpose of treating HDLS diseases is achieved.
2. Cells
In a second aspect, the present invention provides a cell transduced with a gene therapy vector, comprising the gene therapy vector described above, capable of expressing CSF1R protein. The cells may be autologous ("self") cells, i.e., cells from the same subject.
In a preferred embodiment of the present invention, wherein said cells comprise hematopoietic stem cells, stem cells or progenitor cells; preferably, the cell is CD34+And (3) the cells increase the expression level of CSF1R protein.
"transduction" refers to the process of introducing foreign DNA into the genome of a cell using a viral vector.
The present invention relates to "stem cells" which refer to undifferentiated cells that are capable of (1) long-term self-renewal, or are capable of producing at least one identical copy of the original cell, (2) differentiation into multiple, and in some cases, only one particular cell type at the single cell level; and (3) effecting functional regeneration of the tissue in vivo. The stem cell fine is divided into totipotent, sub-totipotent, pluripotent and oligo/unipotent according to its developmental potential.
The present invention relates to "progenitor cells" refers to cells that have the ability to self-renew and differentiate into more mature cells. Many progenitor cells differentiate along a single lineage, but can have a fairly broad proliferative capacity.
The term "Hematopoietic Stem Cells" as used herein refers to pluripotent Stem Cells that are capable of producing all blood cell types of an organism, including bone marrow (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic Cells) and lymphoid lineages (e.g., T Cells, B Cells, NK Cells).
3. Pharmaceutical composition
In a third aspect, the present invention provides a pharmaceutical composition comprising the above gene therapy vector or the above cell, and a pharmaceutically acceptable carrier or excipient.
The "active ingredient" in the pharmaceutical composition of the present invention refers to the gene therapy vector or the genetically modified cell (e.g., hematopoietic stem cell or progenitor cell transduced by the gene therapy vector, such as CD 34)+A cell). "pharmaceutically acceptable carrier or excipient" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being combined with the active ingredients of the present invention and with each other without significantly diminishing the efficacy of the active ingredient.
The composition may be a liquid or a solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.
Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
The compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
4. Method of treatment
The fourth aspect of the invention provides the use of the above gene therapy vector, the above cell or the above pharmaceutical composition for the preparation of a medicament for the prevention, treatment and alleviation of hereditary diffuse leukoencephalopathy associated axono-globoid changes (HDLS) and related diseases; and
the use of the above gene therapy vector, the above cell or the above pharmaceutical composition for the preparation of a medicament for reducing or eliminating gene mutation of colony stimulating factor 1receptor (CSF1R) expressed by colony stimulating factor 1receptor (CSF 1R).
The above gene therapy vector, the above cell or the above pharmaceutical composition provides a therapeutic, prophylactic or ameliorating effect to a subject diagnosed with or suspected of having hereditary diffuse leukoencephalopathy combined with axonomethylactic changes (HDLS) or a subject having a CSF1R gene comprising one or more mutations that reduce the expression of CSF 1R. The above gene therapy vector, the above cell or the above pharmaceutical composition is administered in vivo by direct injection to a cell, tissue or organ of a subject in need of gene therapy, for example, can be administered by direct brain injection. Alternatively, the gene therapy vector transduces cells in vitro or ex vivo, and optionally expands the cells ex vivo, and then the transduced cells are administered to a subject in need of gene therapy.
Without wishing to be bound by any particular theory, increasing the amount of CSF1R gene expression in a pharmaceutical composition (e.g., increasing the amount of CSF1R protein expression of a cell) may allow for treatment of subjects with no or minimal expression of the corresponding gene in vivo, thereby significantly increasing the chance of administering gene therapy to the subject, which was not previously a viable treatment option.
Without wishing to be bound by any particular theory, the gene therapy vectors, cells, pharmaceutical compositions, and methods of treatment of the present invention may achieve gene therapy efficacy by administering a cell population that includes transduced cells with high CSF1R expression levels, as compared to existing methods.
The technical solution provided by the present invention is further described with reference to specific embodiments. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.
The reagents referred to in the following examples are commercially available, for example, from Biotechnology Inc. of Beijing Noro.
Example 1: construction of CSF1R Lentiviral vectors
In this example, vectors pMND-CSF1R-LV and pEF1 α -CSF1R-LV were constructed, respectively, as follows: a third generation lentiviral vector containing a chimeric 5' LTR; a (MND) promoter or a short elongation factor 1 α (EF1 α) promoter deleted from the myeloproliferative sarcoma virus enhancer negative control region and substituted for the dl587rev primer binding site; a polynucleotide encoding a CSF1R polypeptide; and self-inactivating (SIN)3' LTR (see FIG. 1). Specifically, the pMND-CSF1R-LV vector mainly comprises: a chimeric 5 'LTR, Psi (Psi) packaging signal, Human Immunodeficiency Virus (HIV) Rev Response Element (RRE), cPPT/FLAP, a myeloproliferative sarcoma virus enhancer negative control region deleted and dl587rev primer binding site substituted (MND) promoter, a polynucleotide encoding a CSF1R polypeptide, a truncated epidermal growth factor receptor (EGFRt) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a self-inactivating (SIN)3' LTR, the nucleotide sequence of which is as set forth in SEQ ID NO: 1 is shown.
pEF1 alpha-CSF 1R-LV vector mainly comprises: a chimeric 5 'LTR, Psi (Psi) packaging signal, Human Immunodeficiency Virus (HIV) Rev Response Element (RRE), cPPT/FLAP, short elongation factor 1 alpha (EF1 alpha) promoter, polynucleotide encoding CSF1R polypeptide, truncated epidermal growth factor receptor (EGFRt) tag, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and self-inactivating (SIN)3' LTR, the nucleotide sequence of which is as set forth in SEQ ID NO: 2, respectively.
Construction of pEF1 α -CSF 1R-LV: firstly amplifying fragments CSF1R and EGFRT, and then splicing with a linear vector pEF1 alpha to obtain the EGFRT:
1) amplification fragments CSF1R and EGFRT
Primers were designed as follows:
EF-L-CSF1R-F1：
5’-CAGGTGTCGTGACGCGTCTAGAATGGGCCCAGGAGTTC-3’
WF-2A-CSF1R-R：
5’-GAAGTTAGTAGCTCCGCTTCCGCAGAACTGATAGTTG-3’
WF-2A-CSF1R-F：
5’-CAACTATCAGTTCTGCGGAAGCGGAGCTACTAACTTC-3’
WF-WPRE-SalⅠ-R：
5’-CCAGAGGTTGATTGTCGACTGGAATTCTCAGTCCTG-3’
the cDNA of a human myelogenous leukemia mononuclear cell line (THP-1) is taken as a template, upstream and downstream primers EF-L-CSF1R-F1 and WF-2A-CSF1R-R are subjected to PCR amplification according to the PrimeSTAR HS enzyme (Takara, Cat: R044A) operation instruction to obtain a CSF1R fragment, the size is 2959bp detected by agarose gel electrophoresis, and the gel is cut and recovered.
The EGFRT sequence-containing plasmid is synthesized by Shanghai Czeri Bio-Inc, the EGFRT plasmid is used as a template, upstream and downstream primers WF-2A-CSF1R-F and WF-WPRE-SalI-R are subjected to PCR amplification according to the PrimeSTAR HS enzyme (Takara, Cat: R044A) operation instruction to obtain an EGFRT fragment, the size is 1223bp through agarose gel electrophoresis detection, and gel cutting and recovery are carried out.
2) Linearized vector pEF1 alpha
The vector pEF 1. alpha. was digested simultaneously with restriction enzymes Xba I (Thermo, Cat: FD0684) and SalI (Thermo, Cat: FD0644), and the fragments of 6340bp were recovered by agarose gel electrophoresis.
3) pEF1 alpha-CSF 1R-LV vector splice
The agarose gel recovery products from step 1) and step 2) were ligated using the Seamless Assembly Cloning Kit (Zhongmei and, Cat: C5891) according to the protocol, E.coli Stbl3 competent cells were transformed, and after the single clone was sequenced correctly, pEF 1. alpha. -CSF1R-LV vector was obtained.
Construction of pMND-CSF 1R-LV: firstly, the MND fragment is amplified and then spliced with a linearized vector pEF1 alpha-CSF 1R-LV, and the MND fragment is specifically prepared by the following steps:
1) amplified fragment MND
Primers were designed as follows:
WF-MND-F：
5’-ACAAAATTCAAAATTTTATCGATTGGCTCCGGTGCCCGTC-3’
WF-MND-R：
5’-AGAACTCCTGGGCCCATTCTAGACGCGTCACGACACCG-3’
MND plasmid containing MND sequence is synthesized by Shanghai Czeri biological company, MND plasmid is used as template, upstream and downstream primers WF-MND-F and WF-MND-R are subjected to PCR amplification according to PrimeSTAR HS enzyme (Takara, Cat: R044A) operation instruction to obtain MND fragment, the size is 497bp through agarose gel electrophoresis detection, and gel cutting and recovery are carried out.
2) Linear vector pEF1 alpha-CSF 1R-LV
The vector pEF1 alpha-CSF 1R-LV was double-digested with restriction enzymes Bsu 15I (Thermo, Cat: FD0143) and Xba I (Thermo, Cat: FD0684), checked by agarose gel electrophoresis, and a 10199bp fragment was recovered by gel cutting.
3) pMND-CSF1R-LV vector splice
The agarose gel recovery products from step 1) and step 2) were ligated using the Seamless Assembly Cloning Kit (Zhongmei and, Cat: C5891) according to the protocol, E.coli Stbl3 competent cells were transformed, and the single clone was selected for correct sequencing to obtain the pMND-CSF1R-LV vector.
Example 2: transduction of human CD34 with Lentiviral vectors encoding CSF1R+Hematopoietic stem cells
(1) Treatment of cell plates with recombinant human fibronectin (Retronectin)
The recombinant human fibronectin solution was diluted to 60. mu.g/ml with buffer. Recombinant human fibronectin dilutions were added to cell culture plates (0.2 mL in 48-well plates). Standing at room temperature for 4h, or at 4 deg.C, and keeping away from light overnight. The recombinant human fibronectin solution was aspirated and blocked with an appropriate amount of DPBS (dunker's phosphate buffer, calcium and magnesium free) containing 2% BSA (bovine serum albumin). Standing at room temperature for 30 min. The BSA blocking solution was aspirated and the cell plates were washed twice with DPBS. Adding proper amount of DPBS for standby.
(2) HSCs Pre-stimulation
Culturing the separated hematopoietic stem cells in a cell plate pretreated by recombinant human fibronectin, wherein the cell plating density is as follows: 1X 106Culture Medium StemSpan Serum-Free Expansion Medium II (Serum-Free Expansion Medium II) was used, and the cytokine StemSpan CD34 was added+Amplification Supplement (Expansion Supplement, 10 ×) and auxiliary reagents: protamine sulfate (10. mu.g/mL), stimulation time: and (5) 24 h.
(3) Lentivirus infected HSCs
After 24 hours, lentiviral infections of HSCs were performed, lentivirus titers: not less than 1 x 108IU/mL. Protamine sulfate: 10 μ g/mL, lentiviral transduction enhancer (LentiBoost): 1mg/mL, number of infections: cell fluid changes were performed 24h after 2 times, 4h intervals, at a multiplicity of infection (MOI) of 100/time, and lentivirus infection. And continuously culturing after the liquid is changed.
Example 3: detection of protein expression in cells transduced with lentiviral vector encoding CSF1R (Coomassie blue staining assay)
1. 3 days after infecting hematopoietic stem cells with lentivirus encoding CSF1R, the cells were harvested and assayed for expression of the relevant protein.
2. Preparing the separation gel with the concentration of 8 percent and the volume of 15ml
H2O：6.9ml
30% gel stock solution: 4.0ml
1.5mol/L Tris-HCl(pH8.8)：3.8ml
10% SDS (sodium dodecyl sulfate): 0.15ml
10% ammonium persulfate: 0.15ml
TEMED (tetramethylethylenediamine): 0.01ml
3. Once TEMED was added, the separation gel was carefully injected into the prepared gap of the glass plate immediately after mixing, leaving enough room for the layering gel. A few ml of n-butanol was added gently to the top layer by capillary tubing to prevent the inhibition of gel polymerization by oxygen in the air.
4. After completion of the polymerization, the covering liquid was poured off, and the upper part of the gel was washed several times with deionized water to suck up the remaining liquid at the tip with absorbent paper as much as possible.
5. 5ml of 5% of the upper layer glue is prepared, a comb is inserted, the air bubbles are carefully avoided from being mixed, and the mixture is vertically placed at room temperature.
H2O：3.4ml
30% gel stock solution: 0.83ml
1mol/L Tris-HCl(pH6.8)：0.63ml
10％SDS：0.05ml
10% ammonium persulfate: 0.05ml
TEMED：0.01ml
6. When the upper layer glue is polymerized, the collected cells are uniformly mixed with the sample adding buffer solution and heated for 15min at 100 ℃.
7. After the polymerization of the upper layer gel was completed, the comb holes were washed with deionized water to remove unpolymerized acrylamide, and the gel was placed on an electrophoresis tank.
8. And (4) sample adding.
9. Electrophoresis: the voltage is 8V/cm gel at the beginning, after the dye enters the separation gel, the voltage is increased to 15V/cm gel, electrophoresis is continued until the dye reaches the bottom of the separation gel, and the power supply is cut off.
10. The gel is removed, fixed, stained or analyzed.
11. The polyacrylamide gel was placed in a plastic container and covered with a 3-5-fold volume of a fixing solution, and slowly rocked in a rotary shaker for 2 hours.
12. The fixative was decanted, the gel covered with Coomassie Brilliant blue stain and shaken slowly for 4 h.
13. The staining solution was decanted and the gel was rinsed with approximately 50ml of fixative.
14. Pouring out the fixed solution, covering the gel with the destaining solution, slowly shaking for 2h, pouring out the destaining solution, and simultaneously adding new destaining solution until a blue strip and a clean background are obtained. The gel was photographed. The gel can be stored in water.
As shown in FIG. 2, after infecting hematopoietic stem cells with pMND-CSF1R-LV and pEF1 α -CSF1R-LV lentiviral vectors, the expression level of CSF1R protein was significantly increased compared with wild type (hematopoietic stem cells not transduced with lentiviral vectors).
Example 4: CSF1R protein expression (flow assay) in hematopoietic Stem cells transduced with Lentiviral vector encoding CSF1R
1. After 3 days of infecting hematopoietic stem cells with lentivirus, corresponding cells were taken to detect the expression of the relevant protein.
2. Take 1X 106The following protein antibodies LIN, CD38, CD34, CD45RA, CD90 and CSF1R are added into each 100 mu l of cells and mixed uniformly (Lin-CD34+ CD38-CD90+ CD45 RA-is hematopoietic stem cells), and the cells are placed at room temperature and protected from light for reaction for 30 min.
3. The cells were washed 2 times with PBS and the supernatant was discarded by centrifugation (1000 rpm, 5 min).
4. The cells were washed 2 more times with PBS, resuspended in a single cell suspension by adding 200. mu.l PBS, and tested on the machine (flow assay).
As shown in FIGS. 3 and 4, after infecting hematopoietic stem cells with pMND-CSF1R-LV and pEF1 α -CSF1R-LV lentiviral vectors, the expression levels of CSF1R protein were significantly increased, 24.08% and 44.55%, respectively, compared with wild type (hematopoietic stem cells not transduced with lentiviral vectors).
Example 5: CSF1R protein expression in macrophages following differentiation of hematopoietic stem cells transduced with lentiviral vector encoding CSF1R (flow assay)
1. Lentivirus is used to infect hemopoietic stem cell, GM-CSF (granulocyte-macrophage colony stimulating factor) is added to induce culture for 10-15 days, and corresponding cell is taken to detect expression of related protein.
2. Take 1X 106Each cell was added to 100. mu.l of the mixture, and the following protein antibodies CSF1R and F4/80 were mixed and reacted at room temperature in the absence of light for 30 min.
3. The cells were washed 2 times with PBS and the supernatant was discarded by centrifugation (1000 rpm, 5 min).
4. The cells were washed 2 more times with PBS, resuspended in a single cell suspension by adding 200. mu.l PBS, and tested on the machine (flow assay).
As shown in FIG. 5, after pMND-CSF1R-LV and pEF1 alpha-CSF 1R-LV lentivirus infected hematopoietic stem cells, differentiation of macrophages was shown by expression of F4/80 protein, and the expression levels of CSF1R protein in macrophages were 14.94% and 39.97%, respectively.
Sequence listing
<110> Beijing Art Shenzhou pharmaceutical science and technology, Inc.; beijing Art Miao medical science and technology Co., Ltd
<120> a gene therapy vector and use thereof
<130> CP1200804/CB
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 10650
<212> DNA
<213> Artificial sequence
<223> pMND-CSF1R-LV
<400> 1
gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60
ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120
tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180
agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca 240
ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 300
caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 360
agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa 420
aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc 480
ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc 540
ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg 600
tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc 660
aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga 720
gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 780
ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 840
ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 900
tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 960
aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1020
aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1080
gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1140
tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 1200
gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 1260
gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 1320
aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 1380
ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 1440
ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata 1500
gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatctcga 1560
cggtatcggt taacttttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga 1620
atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa 1680
ttcaaaattt tatcgattgg ctccggtgcc cgtcagtttt atttagtctc cagaaaaagg 1740
ggggaatgaa agaccccacc tgtaggtttg gcaagctagg atcaaggtta ggaacagaga 1800
gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg 1860
gccaagaaca gttggaacag cagaatatgg gccaaacagg atatctgtgg taagcagttc 1920
ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt 1980
tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt 2040
atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc 2100
tcaataaaag agcccacaac ccctcactcg gcgcggtgtc gtgacgcgtc tagaatgggc 2160
ccaggagttc tgctgctcct gctggtggcc acagcttggc atggtcaggg aatcccagtg 2220
atagagccca gtgtccctga gctggtcgtg aagccaggag caacggtgac cttgcgatgt 2280
gtgggcaatg gcagcgtgga atgggatggc cccccatcac ctcactggac cctgtactct 2340
gatggctcca gcagcatcct cagcaccaac aacgctacct tccaaaacac ggggacctat 2400
cgctgcactg agcctggaga ccccctggga ggcagcgccg ccatccacct ctatgtcaaa 2460
gaccctgccc ggccctggaa cgtgctagca caggaggtgg tcgtgttcga ggaccaggac 2520
gcactactgc cctgtctgct cacagacccg gtgctggaag caggcgtctc gctggtgcgt 2580
gtgcgtggcc ggcccctcat gcgccacacc aactactcct tctcgccctg gcatggcttc 2640
accatccaca gggccaagtt cattcagagc caggactatc aatgcagtgc cctgatgggt 2700
ggcaggaagg tgatgtccat cagcatccgg ctgaaagtgc agaaagtcat cccagggccc 2760
ccagccttga cactggtgcc tgcagagctg gtgcggattc gaggggaggc tgcccagatc 2820
gtgtgctcag ccagcagcgt tgatgttaac tttgatgtct tcctccaaca caacaacacc 2880
aagctcgcaa tccctcaaca atctgacttt cataataacc gttaccaaaa agtcctgacc 2940
ctcaacctcg atcaagtaga tttccaacat gccggcaact actcctgcgt ggccagcaac 3000
gtgcagggca agcactccac ctccatgttc ttccgggtgg tagagagtgc ctacttgaac 3060
ttgagctctg agcagaacct catccaggag gtgaccgtgg gggaggggct caacctcaaa 3120
gtcatggtgg aggcctaccc aggcctgcaa ggttttaact ggacctacct gggacccttt 3180
tctgaccacc agcctgagcc caagcttgct aatgctacca ccaaggacac atacaggcac 3240
accttcaccc tctctctgcc ccgcctgaag ccctctgagg ctggccgcta ctccttcctg 3300
gccagaaacc caggaggctg gagagctctg acgtttgagc tcacccttcg atacccccca 3360
gaggtaagcg tcatatggac attcatcaac ggctctggca cccttttgtg tgctgcctct 3420
gggtaccccc agcccaacgt gacatggctg cagtgcagtg gccacactga taggtgtgat 3480
gaggcccaag tgctgcaggt ctgggatgac ccataccctg aggtcctgag ccaggagccc 3540
ttccacaagg tgacggtgca gagcctgctg actgttgaga ccttagagca caaccaaacc 3600
tacgagtgca gggcccacaa cagcgtgggg agtggctcct gggccttcat acccatctct 3660
gcaggagccc acacgcatcc cccggatgag ttcctcttca caccagtggt ggtcgcctgc 3720
atgtccatca tggccttgct gctgctgctg ctcctgctgc tattgtacaa gtataagcag 3780
aagcccaagt accaggtccg ctggaagatc atcgagagct atgagggcaa cagttatact 3840
ttcatcgacc ccacgcagct gccttacaac gagaagtggg agttcccccg gaacaacctg 3900
cagtttggta agaccctcgg agctggagcc tttgggaagg tggtggaggc cacggccttt 3960
ggtctgggca aggaggatgc tgtcctgaag gtggctgtga agatgctgaa gtccacggcc 4020
catgctgatg agaaggaggc cctcatgtcc gagctgaaga tcatgagcca cctgggccag 4080
cacgagaaca tcgtcaacct tctgggagcc tgtacccatg gaggccctgt actggtcatc 4140
acggagtact gttgctatgg cgacctgctc aactttctgc gaaggaaggc tgaggccatg 4200
ctgggaccca gcctgagccc cggccaggac cccgagggag gcgtcgacta taagaacatc 4260
cacctcgaga agaaatatgt ccgcagggac agtggcttct ccagccaggg tgtggacacc 4320
tatgtggaga tgaggcctgt ctccacttct tcaaatgact ccttctctga gcaagacctg 4380
gacaaggagg atggacggcc cctggagctc cgggacctgc ttcacttctc cagccaagta 4440
gcccagggca tggccttcct cgcttccaag aattgcatcc accgggacgt ggcagcgcgt 4500
aacgtgctgt tgaccaatgg tcatgtggcc aagattgggg acttcgggct ggctagggac 4560
atcatgaatg actccaacta cattgtcaag ggcaatgccc gcctgcctgt gaagtggatg 4620
gccccagaga gcatctttga ctgtgtctac acggttcaga gcgacgtctg gtcctatggc 4680
atcctcctct gggagatctt ctcacttggg ctgaatccct accctggcat cctggtgaac 4740
agcaagttct ataaactggt gaaggatgga taccaaatgg cccagcctgc atttgcccca 4800
aagaatatat acagcatcat gcaggcctgc tgggccttgg agcccaccca cagacccacc 4860
ttccagcaga tctgctcctt ccttcaggag caggcccaag aggacaggag agagcgggac 4920
tataccaatc tgccgagcag cagcagaagc ggtggcagcg gcagcagcag cagtgagctg 4980
gaggaggaga gctctagtga gcacctgacc tgctgcgagc aaggggatat cgcccagccc 5040
ttgctgcagc ccaacaacta tcagttctgc ggaagcggag ctactaactt cagcctgctg 5100
aagcaggctg gagacgtgga ggagaaccct ggacctagga tgcttctcct ggtgacaagc 5160
cttctgctct gtgagttacc acacccagca ttcctcctga tcccacgcaa agtgtgtaac 5220
ggaataggta ttggtgaatt taaagactca ctctccataa atgctacgaa tattaaacac 5280
ttcaaaaact gcacctccat cagtggcgat ctccacatcc tgccggtggc atttaggggt 5340
gactccttca cacatactcc tcctctggat ccacaggaac tggatattct gaaaaccgta 5400
aaggaaatca cagggttttt gctgattcag gcttggcctg aaaacaggac ggacctccat 5460
gcctttgaga acctagaaat catacgcggc aggaccaagc aacatggtca gttttctctt 5520
gcagtcgtca gcctgaacat aacatccttg ggattacgct ccctcaagga gataagtgat 5580
ggagatgtga taatttcagg aaacaaaaat ttgtgctatg caaatacaat aaactggaaa 5640
aaactgtttg ggacctccgg tcagaaaacc aaaattataa gcaacagagg tgaaaacagc 5700
tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg 5760
gagcccaggg actgcgtctc ttgccggaat gtcagccgag gcagggaatg cgtggacaag 5820
tgcaaccttc tggagggtga gccaagggag tttgtggaga actctgagtg catacagtgc 5880
cacccagagt gcctgcctca ggccatgaac atcacctgca caggacgggg accagacaac 5940
tgtatccagt gtgcccacta cattgacggc ccccactgcg tcaagacctg cccggcagga 6000
gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca tgtgtgccac 6060
ctgtgccatc caaactgcac ctacggatgc actgggccag gtcttgaagg ctgtccaacg 6120
aatgggccta agatcccgtc catcgccact gggatggtgg gggccctcct cttgctgctg 6180
gtggtggccc tggggatcgg cctcttcatg tgataaacgc gtgcacttcg tggccgagga 6240
gcaggactga gaattccagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg 6300
actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct 6360
ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg 6420
ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact 6480
gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc 6540
gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc 6600
cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag 6660
ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc 6720
ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg 6780
gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg 6840
gccgcctccc cgcctggaat tcgagctcgg tacctttaag accaatgact tacaaggcag 6900
ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 6960
aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag 7020
cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt 7080
gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca 7140
gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc 7200
agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag 7260
cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 7320
cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct 7380
agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc 7440
tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc 7500
tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg 7560
ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg 7620
gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 7680
cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 7740
gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 7800
gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 7860
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 7920
cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 7980
agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 8040
aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 8100
gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 8160
aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg 8220
gaaatgtgcg cggaacccct atttgtttat ttttctaaat acaccggaat tgccagctgg 8280
ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tctcgccgcc 8340
aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag gatcgtttcg 8400
catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt 8460
cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc 8520
agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact 8580
gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt 8640
gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca 8700
ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat 8760
gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg 8820
catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga 8880
agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga 8940
cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa 9000
tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga 9060
catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt 9120
cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct 9180
tgacgagttc ttctgactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 9240
acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 9300
aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 9360
atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 9420
gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 9480
tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 9540
ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 9600
ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 9660
ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 9720
aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 9780
cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 9840
gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 9900
ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 9960
cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 10020
tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 10080
cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 10140
cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga 10200
caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac 10260
tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt 10320
gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat 10380
taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata 10440
ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa 10500
aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg 10560
caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt 10620
gtatttaagt gcctagctcg atacataaac 10650
<210> 2
<211> 10444
<212> DNA
<213> Artificial sequence
<223> pEF1α-CSF1R-LV
<400> 2
gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60
ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120
tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180
agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca 240
ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 300
caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 360
agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa 420
aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc 480
ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc 540
ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg 600
tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc 660
aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga 720
gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 780
ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 840
ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 900
tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 960
aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1020
aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1080
gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1140
tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 1200
gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 1260
gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 1320
aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 1380
ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 1440
ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata 1500
gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatctcga 1560
cggtatcggt taacttttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga 1620
atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa 1680
ttcaaaattt tatcgattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca 1740
gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc 1800
ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga 1860
gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc 1920
agaacacagg tgtcgtgacg cgtctagaat gggcccagga gttctgctgc tcctgctggt 1980
ggccacagct tggcatggtc agggaatccc agtgatagag cccagtgtcc ctgagctggt 2040
cgtgaagcca ggagcaacgg tgaccttgcg atgtgtgggc aatggcagcg tggaatggga 2100
tggcccccca tcacctcact ggaccctgta ctctgatggc tccagcagca tcctcagcac 2160
caacaacgct accttccaaa acacggggac ctatcgctgc actgagcctg gagaccccct 2220
gggaggcagc gccgccatcc acctctatgt caaagaccct gcccggccct ggaacgtgct 2280
agcacaggag gtggtcgtgt tcgaggacca ggacgcacta ctgccctgtc tgctcacaga 2340
cccggtgctg gaagcaggcg tctcgctggt gcgtgtgcgt ggccggcccc tcatgcgcca 2400
caccaactac tccttctcgc cctggcatgg cttcaccatc cacagggcca agttcattca 2460
gagccaggac tatcaatgca gtgccctgat gggtggcagg aaggtgatgt ccatcagcat 2520
ccggctgaaa gtgcagaaag tcatcccagg gcccccagcc ttgacactgg tgcctgcaga 2580
gctggtgcgg attcgagggg aggctgccca gatcgtgtgc tcagccagca gcgttgatgt 2640
taactttgat gtcttcctcc aacacaacaa caccaagctc gcaatccctc aacaatctga 2700
ctttcataat aaccgttacc aaaaagtcct gaccctcaac ctcgatcaag tagatttcca 2760
acatgccggc aactactcct gcgtggccag caacgtgcag ggcaagcact ccacctccat 2820
gttcttccgg gtggtagaga gtgcctactt gaacttgagc tctgagcaga acctcatcca 2880
ggaggtgacc gtgggggagg ggctcaacct caaagtcatg gtggaggcct acccaggcct 2940
gcaaggtttt aactggacct acctgggacc cttttctgac caccagcctg agcccaagct 3000
tgctaatgct accaccaagg acacatacag gcacaccttc accctctctc tgccccgcct 3060
gaagccctct gaggctggcc gctactcctt cctggccaga aacccaggag gctggagagc 3120
tctgacgttt gagctcaccc ttcgataccc cccagaggta agcgtcatat ggacattcat 3180
caacggctct ggcacccttt tgtgtgctgc ctctgggtac ccccagccca acgtgacatg 3240
gctgcagtgc agtggccaca ctgataggtg tgatgaggcc caagtgctgc aggtctggga 3300
tgacccatac cctgaggtcc tgagccagga gcccttccac aaggtgacgg tgcagagcct 3360
gctgactgtt gagaccttag agcacaacca aacctacgag tgcagggccc acaacagcgt 3420
ggggagtggc tcctgggcct tcatacccat ctctgcagga gcccacacgc atcccccgga 3480
tgagttcctc ttcacaccag tggtggtcgc ctgcatgtcc atcatggcct tgctgctgct 3540
gctgctcctg ctgctattgt acaagtataa gcagaagccc aagtaccagg tccgctggaa 3600
gatcatcgag agctatgagg gcaacagtta tactttcatc gaccccacgc agctgcctta 3660
caacgagaag tgggagttcc cccggaacaa cctgcagttt ggtaagaccc tcggagctgg 3720
agcctttggg aaggtggtgg aggccacggc ctttggtctg ggcaaggagg atgctgtcct 3780
gaaggtggct gtgaagatgc tgaagtccac ggcccatgct gatgagaagg aggccctcat 3840
gtccgagctg aagatcatga gccacctggg ccagcacgag aacatcgtca accttctggg 3900
agcctgtacc catggaggcc ctgtactggt catcacggag tactgttgct atggcgacct 3960
gctcaacttt ctgcgaagga aggctgaggc catgctggga cccagcctga gccccggcca 4020
ggaccccgag ggaggcgtcg actataagaa catccacctc gagaagaaat atgtccgcag 4080
ggacagtggc ttctccagcc agggtgtgga cacctatgtg gagatgaggc ctgtctccac 4140
ttcttcaaat gactccttct ctgagcaaga cctggacaag gaggatggac ggcccctgga 4200
gctccgggac ctgcttcact tctccagcca agtagcccag ggcatggcct tcctcgcttc 4260
caagaattgc atccaccggg acgtggcagc gcgtaacgtg ctgttgacca atggtcatgt 4320
ggccaagatt ggggacttcg ggctggctag ggacatcatg aatgactcca actacattgt 4380
caagggcaat gcccgcctgc ctgtgaagtg gatggcccca gagagcatct ttgactgtgt 4440
ctacacggtt cagagcgacg tctggtccta tggcatcctc ctctgggaga tcttctcact 4500
tgggctgaat ccctaccctg gcatcctggt gaacagcaag ttctataaac tggtgaagga 4560
tggataccaa atggcccagc ctgcatttgc cccaaagaat atatacagca tcatgcaggc 4620
ctgctgggcc ttggagccca cccacagacc caccttccag cagatctgct ccttccttca 4680
ggagcaggcc caagaggaca ggagagagcg ggactatacc aatctgccga gcagcagcag 4740
aagcggtggc agcggcagca gcagcagtga gctggaggag gagagctcta gtgagcacct 4800
gacctgctgc gagcaagggg atatcgccca gcccttgctg cagcccaaca actatcagtt 4860
ctgcggaagc ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa 4920
ccctggacct aggatgcttc tcctggtgac aagccttctg ctctgtgagt taccacaccc 4980
agcattcctc ctgatcccac gcaaagtgtg taacggaata ggtattggtg aatttaaaga 5040
ctcactctcc ataaatgcta cgaatattaa acacttcaaa aactgcacct ccatcagtgg 5100
cgatctccac atcctgccgg tggcatttag gggtgactcc ttcacacata ctcctcctct 5160
ggatccacag gaactggata ttctgaaaac cgtaaaggaa atcacagggt ttttgctgat 5220
tcaggcttgg cctgaaaaca ggacggacct ccatgccttt gagaacctag aaatcatacg 5280
cggcaggacc aagcaacatg gtcagttttc tcttgcagtc gtcagcctga acataacatc 5340
cttgggatta cgctccctca aggagataag tgatggagat gtgataattt caggaaacaa 5400
aaatttgtgc tatgcaaata caataaactg gaaaaaactg tttgggacct ccggtcagaa 5460
aaccaaaatt ataagcaaca gaggtgaaaa cagctgcaag gccacaggcc aggtctgcca 5520
tgccttgtgc tcccccgagg gctgctgggg cccggagccc agggactgcg tctcttgccg 5580
gaatgtcagc cgaggcaggg aatgcgtgga caagtgcaac cttctggagg gtgagccaag 5640
ggagtttgtg gagaactctg agtgcataca gtgccaccca gagtgcctgc ctcaggccat 5700
gaacatcacc tgcacaggac ggggaccaga caactgtatc cagtgtgccc actacattga 5760
cggcccccac tgcgtcaaga cctgcccggc aggagtcatg ggagaaaaca acaccctggt 5820
ctggaagtac gcagacgccg gccatgtgtg ccacctgtgc catccaaact gcacctacgg 5880
atgcactggg ccaggtcttg aaggctgtcc aacgaatggg cctaagatcc cgtccatcgc 5940
cactgggatg gtgggggccc tcctcttgct gctggtggtg gccctgggga tcggcctctt 6000
catgtgataa acgcgtgcac ttcgtggccg aggagcagga ctgagaattc cagtcgacaa 6060
tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 6120
ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 6180
ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 6240
gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 6300
ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 6360
tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 6420
gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc 6480
ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 6540
tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 6600
ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaattcgagc 6660
tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa 6720
gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct 6780
tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg 6840
aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt 6900
ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc 6960
tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga 7020
atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat 7080
agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 7140
aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca 7200
tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 7260
ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag 7320
gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg 7380
ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 7440
tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 7500
tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 7560
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 7620
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 7680
tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 7740
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 7800
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 7860
aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 7920
ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 7980
attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 8040
ttatttttct aaatacaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc 8100
cctgcaaagt aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa 8160
gctctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 8220
caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 8280
tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 8340
tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt 8400
ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 8460
gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 8520
ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 8580
ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 8640
aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 8700
aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg 8760
gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 8820
gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 8880
ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 8940
ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ctgtcagacc 9000
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 9060
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 9120
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 9180
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 9240
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 9300
atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 9360
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 9420
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 9480
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 9540
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 9600
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 9660
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 9720
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 9780
tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 9840
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 9900
gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 9960
cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 10020
gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 10080
ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 10140
acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc 10200
tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac 10260
gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga 10320
agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg 10380
acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat 10440
aaac 10444
<210> 3
<211> 2916
<212> DNA
<213> Artificial sequence
<223> CSF1R original sequence
<400> 3
atgggcccag gagttctgct gctcctgctg gtggccacag cttggcatgg tcagggaatc 60
ccagtgatag agcccagtgt ccctgagctg gtcgtgaagc caggagcaac ggtgaccttg 120
cgatgtgtgg gcaatggcag cgtggaatgg gatggccccc catcacctca ctggaccctg 180
tactctgatg gctccagcag catcctcagc accaacaacg ctaccttcca aaacacgggg 240
acctatcgct gcactgagcc tggagacccc ctgggaggca gcgccgccat ccacctctat 300
gtcaaagacc ctgcccggcc ctggaacgtg ctagcacagg aggtggtcgt gttcgaggac 360
caggacgcac tactgccctg tctgctcaca gacccggtgc tggaagcagg cgtctcgctg 420
gtgcgtgtgc gtggccggcc cctcatgcgc cacaccaact actccttctc gccctggcat 480
ggcttcacca tccacagggc caagttcatt cagagccagg actatcaatg cagtgccctg 540
atgggtggca ggaaggtgat gtccatcagc atccggctga aagtgcagaa agtcatccca 600
gggcccccag ccttgacact ggtgcctgca gagctggtgc ggattcgagg ggaggctgcc 660
cagatcgtgt gctcagccag cagcgttgat gttaactttg atgtcttcct ccaacacaac 720
aacaccaagc tcgcaatccc tcaacaatct gactttcata ataaccgtta ccaaaaagtc 780
ctgaccctca acctcgatca agtagatttc caacatgccg gcaactactc ctgcgtggcc 840
agcaacgtgc agggcaagca ctccacctcc atgttcttcc gggtggtaga gagtgcctac 900
ttgaacttga gctctgagca gaacctcatc caggaggtga ccgtggggga ggggctcaac 960
ctcaaagtca tggtggaggc ctacccaggc ctgcaaggtt ttaactggac ctacctggga 1020
cccttttctg accaccagcc tgagcccaag cttgctaatg ctaccaccaa ggacacatac 1080
aggcacacct tcaccctctc tctgccccgc ctgaagccct ctgaggctgg ccgctactcc 1140
ttcctggcca gaaacccagg aggctggaga gctctgacgt ttgagctcac ccttcgatac 1200
cccccagagg taagcgtcat atggacattc atcaacggct ctggcaccct tttgtgtgct 1260
gcctctgggt acccccagcc caacgtgaca tggctgcagt gcagtggcca cactgatagg 1320
tgtgatgagg cccaagtgct gcaggtctgg gatgacccat accctgaggt cctgagccag 1380
gagcccttcc acaaggtgac ggtgcagagc ctgctgactg ttgagacctt agagcacaac 1440
caaacctacg agtgcagggc ccacaacagc gtggggagtg gctcctgggc cttcataccc 1500
atctctgcag gagcccacac gcatcccccg gatgagttcc tcttcacacc agtggtggtc 1560
gcctgcatgt ccatcatggc cttgctgctg ctgctgctcc tgctgctatt gtacaagtat 1620
aagcagaagc ccaagtacca ggtccgctgg aagatcatcg agagctatga gggcaacagt 1680
tatactttca tcgaccccac gcagctgcct tacaacgaga agtgggagtt cccccggaac 1740
aacctgcagt ttggtaagac cctcggagct ggagcctttg ggaaggtggt ggaggccacg 1800
gcctttggtc tgggcaagga ggatgctgtc ctgaaggtgg ctgtgaagat gctgaagtcc 1860
acggcccatg ctgatgagaa ggaggccctc atgtccgagc tgaagatcat gagccacctg 1920
ggccagcacg agaacatcgt caaccttctg ggagcctgta cccatggagg ccctgtactg 1980
gtcatcacgg agtactgttg ctatggcgac ctgctcaact ttctgcgaag gaaggctgag 2040
gccatgctgg gacccagcct gagccccggc caggaccccg agggaggcgt cgactataag 2100
aacatccacc tcgagaagaa atatgtccgc agggacagtg gcttctccag ccagggtgtg 2160
gacacctatg tggagatgag gcctgtctcc acttcttcaa atgactcctt ctctgagcaa 2220
gacctggaca aggaggatgg acggcccctg gagctccggg acctgcttca cttctccagc 2280
caagtagccc agggcatggc cttcctcgct tccaagaatt gcatccaccg ggacgtggca 2340
gcgcgtaacg tgctgttgac caatggtcat gtggccaaga ttggggactt cgggctggct 2400
agggacatca tgaatgactc caactacatt gtcaagggca atgcccgcct gcctgtgaag 2460
tggatggccc cagagagcat ctttgactgt gtctacacgg ttcagagcga cgtctggtcc 2520
tatggcatcc tcctctggga gatcttctca cttgggctga atccctaccc tggcatcctg 2580
gtgaacagca agttctataa actggtgaag gatggatacc aaatggccca gcctgcattt 2640
gccccaaaga atatatacag catcatgcag gcctgctggg ccttggagcc cacccacaga 2700
cccaccttcc agcagatctg ctccttcctt caggagcagg cccaagagga caggagagag 2760
cgggactata ccaatctgcc gagcagcagc agaagcggtg gcagcggcag cagcagcagt 2820
gagctggagg aggagagctc tagtgagcac ctgacctgct gcgagcaagg ggatatcgcc 2880
cagcccttgc tgcagcccaa caactatcag ttctgc 2916
<210> 4
<211> 2916
<212> DNA
<213> Artificial sequence
<223> CSF1R codon optimized expression sequence
<400> 4
atgggccccg gcgtgctgct gctgctgctg gtggccaccg cctggcacgg ccagggcatc 60
cccgtgatcg agcccagcgt gcccgagctg gtggtgaagc ccggcgccac cgtgaccctg 120
cgctgcgtgg gcaacggcag cgtggagtgg gacggccccc ccagccccca ctggaccctg 180
tacagcgacg gcagcagcag catcctgagc accaacaacg ccaccttcca gaacaccggc 240
acctaccgct gcaccgagcc cggcgacccc ctgggcggca gcgccgccat ccacctgtac 300
gtgaaggacc ccgcccgccc ctggaacgtg ctggcccagg aggtggtggt gttcgaggac 360
caggacgccc tgctgccctg cctgctgacc gaccccgtgc tggaggccgg cgtgagcctg 420
gtgcgcgtgc gcggccgccc cctgatgcgc cacaccaact acagcttcag cccctggcac 480
ggcttcacca tccaccgcgc caagttcatc cagagccagg actaccagtg cagcgccctg 540
atgggcggcc gcaaggtgat gagcatcagc atccgcctga aggtgcagaa ggtgatcccc 600
ggcccccccg ccctgaccct ggtgcccgcc gagctggtgc gcatccgcgg cgaggccgcc 660
cagatcgtgt gcagcgccag cagcgtggac gtgaacttcg acgtgttcct gcagcacaac 720
aacaccaagc tggccatccc ccagcagagc gacttccaca acaaccgcta ccagaaggtg 780
ctgaccctga acctggacca ggtggacttc cagcacgccg gcaactacag ctgcgtggcc 840
agcaacgtgc agggcaagca cagcaccagc atgttcttcc gcgtggtgga gagcgcctac 900
ctgaacctga gcagcgagca gaacctgatc caggaggtga ccgtgggcga gggcctgaac 960
ctgaaggtga tggtggaggc ctaccccggc ctgcagggct tcaactggac ctacctgggc 1020
cccttcagcg accaccagcc cgagcccaag ctggccaacg ccaccaccaa ggacacctac 1080
cgccacacct tcaccctgag cctgccccgc ctgaagccca gcgaggccgg ccgctacagc 1140
ttcctggccc gcaaccccgg cggctggcgc gccctgacct tcgagctgac cctgcgctac 1200
ccccccgagg tgagcgtgat ctggaccttc atcaacggca gcggcaccct gctgtgcgcc 1260
gccagcggct acccccagcc caacgtgacc tggctgcagt gcagcggcca caccgaccgc 1320
tgcgacgagg cccaggtgct gcaggtgtgg gacgacccct accccgaggt gctgagccag 1380
gagcccttcc acaaggtgac cgtgcagagc ctgctgaccg tggagaccct ggagcacaac 1440
cagacctacg agtgccgcgc ccacaacagc gtgggcagcg gcagctgggc cttcatcccc 1500
atcagcgccg gcgcccacac ccaccccccc gacgagttcc tgttcacccc cgtggtggtg 1560
gcctgcatga gcatcatggc cctgctgctg ctgctgctgc tgctgctgct gtacaagtac 1620
aagcagaagc ccaagtacca ggtgcgctgg aagatcatcg agagctacga gggcaacagc 1680
tacaccttca tcgaccccac ccagctgccc tacaacgaga agtgggagtt cccccgcaac 1740
aacctgcagt tcggcaagac cctgggcgcc ggcgccttcg gcaaggtggt ggaggccacc 1800
gccttcggcc tgggcaagga ggacgccgtg ctgaaggtgg ccgtgaagat gctgaagagc 1860
accgcccacg ccgacgagaa ggaggccctg atgagcgagc tgaagatcat gagccacctg 1920
ggccagcacg agaacatcgt gaacctgctg ggcgcctgca cccacggcgg ccccgtgctg 1980
gtgatcaccg agtactgctg ctacggcgac ctgctgaact tcctgcgccg caaggccgag 2040
gccatgctgg gccccagcct gagccccggc caggaccccg agggcggcgt ggactacaag 2100
aacatccacc tggagaagaa gtacgtgcgc cgcgacagcg gcttcagcag ccagggcgtg 2160
gacacctacg tggagatgcg ccccgtgagc accagcagca acgacagctt cagcgagcag 2220
gacctggaca aggaggacgg ccgccccctg gagctgcgcg acctgctgca cttcagcagc 2280
caggtggccc agggcatggc cttcctggcc agcaagaact gcatccaccg cgacgtggcc 2340
gcccgcaacg tgctgctgac caacggccac gtggccaaga tcggcgactt cggcctggcc 2400
cgcgacatca tgaacgacag caactacatc gtgaagggca acgcccgcct gcccgtgaag 2460
tggatggccc ccgagagcat cttcgactgc gtgtacaccg tgcagagcga cgtgtggagc 2520
tacggcatcc tgctgtggga gatcttcagc ctgggcctga acccctaccc cggcatcctg 2580
gtgaacagca agttctacaa gctggtgaag gacggctacc agatggccca gcccgccttc 2640
gcccccaaga acatctacag catcatgcag gcctgctggg ccctggagcc cacccaccgc 2700
cccaccttcc agcagatctg cagcttcctg caggagcagg cccaggagga ccgccgcgag 2760
cgcgactaca ccaacctgcc cagcagcagc cgcagcggcg gcagcggcag cagcagcagc 2820
gagctggagg aggagagcag cagcgagcac ctgacctgct gcgagcaggg cgacatcgcc 2880
cagcccctgc tgcagcccaa caactaccag ttctgc 2916
<210> 5
<211> 972
<212> PRT
<213> Artificial sequence
<223> CSF1R polypeptide sequence
<400> 5
Met Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr Ala Trp His
1 5 10 15
Gly Gln Gly Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val
20 25 30
Lys Pro Gly Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val
35 40 45
Glu Trp Asp Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly
50 55 60
Ser Ser Ser Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly
65 70 75 80
Thr Tyr Arg Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala
85 90 95
Ile His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala
100 105 110
Gln Glu Val Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu
115 120 125
Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg
130 135 140
Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His
145 150 155 160
Gly Phe Thr Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln
165 170 175
Cys Ser Ala Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg
180 185 190
Leu Lys Val Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val
195 200 205
Pro Ala Glu Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys
210 215 220
Ser Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn
225 230 235 240
Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg
245 250 255
Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His
260 265 270
Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser
275 280 285
Thr Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser
290 295 300
Ser Glu Gln Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn
305 310 315 320
Leu Lys Val Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp
325 330 335
Thr Tyr Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala
340 345 350
Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu
355 360 365
Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg
370 375 380
Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr
385 390 395 400
Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr
405 410 415
Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu
420 425 430
Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln
435 440 445
Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His
450 455 460
Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn
465 470 475 480
Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp
485 490 495
Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu
500 505 510
Phe Leu Phe Thr Pro Val Val Val Ala Cys Met Ser Ile Met Ala Leu
515 520 525
Leu Leu Leu Leu Leu Leu Leu Leu Leu Tyr Lys Tyr Lys Gln Lys Pro
530 535 540
Lys Tyr Gln Val Arg Trp Lys Ile Ile Glu Ser Tyr Glu Gly Asn Ser
545 550 555 560
Tyr Thr Phe Ile Asp Pro Thr Gln Leu Pro Tyr Asn Glu Lys Trp Glu
565 570 575
Phe Pro Arg Asn Asn Leu Gln Phe Gly Lys Thr Leu Gly Ala Gly Ala
580 585 590
Phe Gly Lys Val Val Glu Ala Thr Ala Phe Gly Leu Gly Lys Glu Asp
595 600 605
Ala Val Leu Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala His Ala
610 615 620
Asp Glu Lys Glu Ala Leu Met Ser Glu Leu Lys Ile Met Ser His Leu
625 630 635 640
Gly Gln His Glu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr His Gly
645 650 655
Gly Pro Val Leu Val Ile Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu
660 665 670
Asn Phe Leu Arg Arg Lys Ala Glu Ala Met Leu Gly Pro Ser Leu Ser
675 680 685
Pro Gly Gln Asp Pro Glu Gly Gly Val Asp Tyr Lys Asn Ile His Leu
690 695 700
Glu Lys Lys Tyr Val Arg Arg Asp Ser Gly Phe Ser Ser Gln Gly Val
705 710 715 720
Asp Thr Tyr Val Glu Met Arg Pro Val Ser Thr Ser Ser Asn Asp Ser
725 730 735
Phe Ser Glu Gln Asp Leu Asp Lys Glu Asp Gly Arg Pro Leu Glu Leu
740 745 750
Arg Asp Leu Leu His Phe Ser Ser Gln Val Ala Gln Gly Met Ala Phe
755 760 765
Leu Ala Ser Lys Asn Cys Ile His Arg Asp Val Ala Ala Arg Asn Val
770 775 780
Leu Leu Thr Asn Gly His Val Ala Lys Ile Gly Asp Phe Gly Leu Ala
785 790 795 800
Arg Asp Ile Met Asn Asp Ser Asn Tyr Ile Val Lys Gly Asn Ala Arg
805 810 815
Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Cys Val Tyr
820 825 830
Thr Val Gln Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile
835 840 845
Phe Ser Leu Gly Leu Asn Pro Tyr Pro Gly Ile Leu Val Asn Ser Lys
850 855 860
Phe Tyr Lys Leu Val Lys Asp Gly Tyr Gln Met Ala Gln Pro Ala Phe
865 870 875 880
Ala Pro Lys Asn Ile Tyr Ser Ile Met Gln Ala Cys Trp Ala Leu Glu
885 890 895
Pro Thr His Arg Pro Thr Phe Gln Gln Ile Cys Ser Phe Leu Gln Glu
900 905 910
Gln Ala Gln Glu Asp Arg Arg Glu Arg Asp Tyr Thr Asn Leu Pro Ser
915 920 925
Ser Ser Arg Ser Gly Gly Ser Gly Ser Ser Ser Ser Glu Leu Glu Glu
930 935 940
Glu Ser Ser Ser Glu His Leu Thr Cys Cys Glu Gln Gly Asp Ile Ala
945 950 955 960
Gln Pro Leu Leu Gln Pro Asn Asn Tyr Gln Phe Cys
965 970
Claims (16)
1. A gene therapy vector comprising:
a) left (5') LTR;
b) psi (Psi) package signal;
c) a retroviral export element;
d) central polypurine tract/central termination sequence;
e) a promoter operably linked to a polynucleotide encoding a colony stimulating factor 1receptor polypeptide;
f) a polynucleotide encoding a colony stimulating factor 1receptor polypeptide;
g) a post-transcriptional regulatory element; and
h) right (3') LTR.
2. The gene therapy vector of claim 1, wherein the gene therapy vector is a gamma retrovirus vector, an adeno-associated virus vector, an adenovirus vector, or a herpes virus vector;
preferably, the gene therapy vector is a lentiviral vector.
3. The gene therapy vector of claim 2, wherein the lentivirus is selected from the group consisting of: human immunodeficiency virus, visna-meidu virus, caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, bovine immunodeficiency virus, or simian immunodeficiency virus;
preferably, the lentivirus is a human immunodeficiency virus.
4. The gene therapy vector according to any one of claims 1 to 3, further comprising a nucleotide sequence encoding an expression tag;
preferably, the expression tag is a truncated epidermal growth factor receptor, firefly luciferase, enhanced green fluorescent protein, streptavidin-binding peptide tag, or Flag tag.
5. The gene therapy vector of one of claims 1-4, wherein the promoter of the left (5') LTR is replaced by a heterologous promoter selected from the group consisting of: a cytomegalovirus promoter, a rous sarcoma virus promoter, or a simian virus 40 promoter.
6. The gene therapy vector of one of claims 1-5, wherein said right (3') LTR comprises one or more modifications;
preferably, the right (3') LTR comprises one or more deletions that prevent viral transcription beyond the first round of viral replication;
more preferably, the right (3') LTR comprises a deletion of the TATA box and Sp1 and NF-. kappa.B transcription factor binding sites in the U3 region of the 3' LTR;
further preferably, the right (3') LTR is a self-inactivating (SIN) LTR.
7. A gene therapy vector according to any one of claims 1 to 6, wherein the promoter operably linked to the polynucleotide encoding the colony stimulating factor 1receptor polypeptide comprises: a short elongation factor 1 alpha promoter or a transcriptionally active fragment thereof, a promoter in which the myeloproliferative sarcoma virus enhancer negative control region is deleted and the dl587rev primer binding site is substituted, or a transcriptionally active fragment thereof;
preferably, the promoter operably linked to the polynucleotide encoding a colony stimulating factor 1receptor polypeptide is selected from the group consisting of: transmembrane protein 119, integrin subunit α M, CD45, ionic calcium binding adaptor molecule 1, C-X3-C motif chemokine receptor 1, adhesion G protein coupled receptor E1, CD68, or CD 40.
8. The gene therapy vector of any one of claims 1-7, wherein said post-transcriptional regulatory element comprises a woodchuck hepatitis virus post-transcriptional regulatory element.
9. The gene therapy vector of any one of claims 1-8, wherein the amino acid sequence of the colony stimulating factor 1receptor polypeptide is as set forth in SEQ ID NO: 5, respectively.
10. The gene therapy vector of any one of claims 1-9, wherein the polynucleotide sequence encoding the colony stimulating factor 1receptor polypeptide is as set forth in SEQ ID NO: 3 is shown in the specification;
preferably, the polynucleotide encoding the colony stimulating factor 1receptor polypeptide is a codon optimised expression sequence as set forth in SEQ ID NO: 4, respectively.
11. The gene therapy vector according to one of claims 1 to 10, wherein said gene therapy vector is pMND-CSF1R-LV or pEF1 α -CSF1R-LV, respectively, said pMND-CSF1R-LV having the nucleotide sequence as set forth in SEQ ID NO: 1, the nucleotide sequence of pEF1 alpha-CSF 1R-LV is shown as SEQ ID NO: 2, respectively.
12. A cell transduced with a gene therapy vector comprising the gene therapy vector according to any one of claims 1-11.
13. The cell of claim 12, wherein the cell comprises a hematopoietic stem cell, or progenitor cell;
preferably, the cell is CD34+A cell.
14. A pharmaceutical composition comprising the gene therapy vector of any one of claims 1-11 or the cell of claim 12 or 13, and a pharmaceutically acceptable carrier or excipient.
15. Use of the gene therapy vector of any one of claims 1-11, the cell of claim 12 or 13, or the pharmaceutical composition of claim 14 for the preparation of a medicament for the prevention, treatment, and amelioration of hereditary diffuse leukoencephalopathy associated with axono-globulosis and related diseases.
16. Use of a gene therapy vector according to any one of claims 1 to 11, a cell according to claim 12 or 13 or a pharmaceutical composition according to claim 14 for the manufacture of a medicament for reducing or eliminating mutations in the colony stimulating factor 1receptor gene resulting in expression of the colony stimulating factor 1 receptor.
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| CN202011486921.5A CN112522321B (en) | 2020-12-16 | 2020-12-16 | Gene therapy vector and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011486921.5A CN112522321B (en) | 2020-12-16 | 2020-12-16 | Gene therapy vector and application thereof |
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| CN112522321B CN112522321B (en) | 2022-04-08 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107396A (en) * | 2021-11-26 | 2022-03-01 | 和元智造(上海)基因技术有限公司 | Lentiviral vectors, systems and uses thereof |
| CN116463353A (en) * | 2022-12-23 | 2023-07-21 | 首都医科大学附属北京天坛医院 | Construction method and application of Csf1r gene mutation mouse model |
Citations (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114107396A (en) * | 2021-11-26 | 2022-03-01 | 和元智造(上海)基因技术有限公司 | Lentiviral vectors, systems and uses thereof |
| CN114107396B (en) * | 2021-11-26 | 2024-02-02 | 和元智造(上海)基因技术有限公司 | Lentiviral vector, system and application thereof |
| CN116463353A (en) * | 2022-12-23 | 2023-07-21 | 首都医科大学附属北京天坛医院 | Construction method and application of Csf1r gene mutation mouse model |
| CN116463353B (en) * | 2022-12-23 | 2023-09-19 | 首都医科大学附属北京天坛医院 | Construction method and application of Csf1r gene mutation mouse model |
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| CN112522321B (en) | 2022-04-08 |
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