CN115772503B - Gene modified cell medicine for expressing PAH and preparation method and application thereof - Google Patents
Gene modified cell medicine for expressing PAH and preparation method and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/867—Retroviral vectors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
Abstract
The invention belongs to the technical field of biological medicine, and discloses a gene modified cell medicine for expressing PAH, and a preparation method and application thereof. The invention provides a genetically modified cell medicament for expressing PAH, which comprises a cell modified by utilizing a human normal phenylalanine hydroxylase (PAH) gene or a nucleic acid molecule with at least 75% homology with the nucleotide sequence of the human normal PAH gene, wherein the cell can continuously and stably express the PAH in vivo/in vitro. When the gene modified cell medicine for expressing PAH provided by the invention is used as a cell therapeutic medicine for treating phenylketonuria, the gene modified cell medicine can be used as a 'medicine factory' for producing PAH in vivo, and the PAH can be continuously and stably expressed for a long time, so that the defects of short half-life period and frequent medicine use for lifetime of PAH protein in the existing phenylketonuria treatment method can be effectively overcome.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a genetically modified cell medicine for expressing phenylalanine hydroxylase (PAH), and a preparation method and application thereof.
Background
Phenylketonuria (PKU) is an autosomal recessive inherited disease mainly caused by mutations in the phenylalanine hydroxylase (PAH) gene, characterized by the accumulation of high concentrations of phenylalanine (Phe) in organs such as the blood and brain of patients, resulting in irreversible and severe mental retardation.
The therapeutic strategy for PKU is to restore and increase PAH activity and reduce phenylalanine concentration in the blood. Currently, PKU treatment methods mainly include diet restriction therapy, sapropterin, and enzyme replacement therapy (Pegvaliase), the latter two of which are the only two drugs approved by the FDA for treatment of PKU. Clinically, a Phe-restricted diet is effective in maintaining plasma Phe levels within normal ranges, but during puberty and adulthood, patients have difficulty adhering to the Phe-restricted diet, thereby increasing the risk of unexpected peaks in plasma Phe levels. Sapropterin is a synthetic form of cofactor BH4, which is very effective in mild PKU patients, but in most patients, a Phe-restricted diet is required to completely normalize Phe levels while Sha Bingdie is used. The first enzyme replacement therapy for the treatment of PKU, pegvaliase, an oral form of polyethylene glycol derivatives of phenylalanine ammonia lyase (PEG-PAL), was approved by the FDA in 2018, which metabolizes Phe to ammonia and trans-cinnamic acid. Phase III clinical trial data indicate that 60.7% of patients are able to reach blood Phe levels below 360 μmol/L recommended by the guidelines at 24 months of Pegvaliase use. Pegvaliase is a promising new treatment option for adult patients with PKU. However, the action of PEG-PAL cannot last for a long time and may induce severe allergic reactions in some patients.
Gene therapy is based on changing or correcting genetic material of human, and the normal or therapeutic gene is introduced into target cell of human body via corresponding carrier to correct or compensate defective gene, so as to treat corresponding diseases. The gene therapy targeting the somatic cells can be realized by directly injecting and administering the virus (In vivo therapy), or can be realized by separating the somatic cells, culturing the somatic cells In vitro, infecting and modifying the somatic cells by a virus vector carrying normal genes and then reinjecting the somatic cells into a host (Ex vivo therapy). PKU is a typical genetic disorder of abnormal liver metabolism, mainly due to missense mutations in the gene encoding phenylalanine hydroxylase (Phenylalanine hydroxylase, PAH). Liver cells cannot be treated in vitro, and with the development of vector technology (such as AAV), targeted modification can be achieved, so in vivo gene therapy using viral vectors is preferred.
PKU gene therapies based on a variety of mechanisms are being developed. In 2020, phenylketonuria gene therapy BMN307 (AAV 5-PAH) has obtained rapid channel assignment (FTD) for the treatment of PKU in the united states. In a mouse model of the disease, the therapy was demonstrated to lead to a lifetime normalization of Phe levels. HMI-102 is also an AAV gene therapy for PKU, granted orphan drug qualification (ODD) and fast-path qualification (FTD) by the U.S. FDA. HMI-102 treatment restores a PKU model Phe metabolic pathway to normalize blood phenylalanine levels. However, clinical studies of these two drugs are called out by the FDA for potential serious safety risks such as tumorigenicity and liver injury. With intensive research into AAV vectors, it was found that there are safety problems including immune response, induction of insertional mutagenesis and induction of cancer, hepatotoxicity (elevation of transaminase, liver failure), and the like.
Although PKU treatments have been explored for decades, existing treatments for PKU have certain clinical limitations, including long-term repeated dosing or diet restriction, poor compliance, high safety risks, and heavy economic burden. Thus, research into new methods for treating PKU is urgent, and it is an urgent need to solve the problem of PKU treatment to extend the half-life of PAH proteins to reduce the frequency of administration and improve the safety of treatment.
Disclosure of Invention
The invention aims to provide a genetically modified cell medicament for expressing PAH, which aims to solve at least one of the technical problems.
Another object of the present invention is to provide a method for preparing the above-mentioned PAH-expressing genetically modified cell drug.
It is still another object of the present invention to provide the use of the above-described genetically modified cell drug expressing PAH in the preparation of a medicament for treating phenylketonuria.
According to one aspect of the present invention, there is provided a genetically modified cell drug expressing PAH, comprising a cell modified with a human normal phenylalanine hydroxylase (PAH) gene, or a nucleic acid molecule having a nucleotide sequence having at least 75% homology with the nucleotide sequence of the human normal PAH gene, which can continuously and stably express PAH in vivo/in vitro, and which can be used as a "pharmaceutical factory" for producing PAH in vivo for a long period of time when used as a cell therapeutic drug for treating phenylketonuria, thereby effectively solving the problems of short half-life of PAH protein and frequent drug administration for lifetime in the existing phenylketonuria treatment method.
The nucleotide sequence of the human normal PAH is shown as SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO: 2. The nucleic acid molecule having a nucleotide sequence having at least 75% homology to the nucleotide sequence of the human normal PAH gene may be a nucleic acid molecule having at least 75% homology to the nucleotide sequence of the human normal PAH gene, which normally encodes human normal PAH, obtained by at least one of codon optimization based on the nucleotide sequence of the human normal PAH gene, reduction of CpG dinucleotide content, removal of any additional ORFs in sense and antisense directions, and the like.
In some embodiments, cells useful for performing the PAH gene modification (hereinafter referred to as "host cells") may be selected from at least one of stem cells, peripheral blood mononuclear cells.
In some embodiments, the host cell may be a stem cell; among them, the types of stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPS), and the like.
In some embodiments, the mesenchymal stem cells may be selected from at least one of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells.
In some embodiments, the host cell may be an umbilical mesenchymal stem cell. The umbilical cord mesenchymal stem cells have the advantages of convenient material taking, no wound, wide sources, simple in vitro separation and culture, no matter what the physics is in dispute, and the like, have lower immunogenicity and no tumorigenicity, can be used as host cells of cell therapeutic drugs, can not generate allogeneic immune rejection reaction, and have high safety.
In some embodiments, the gene delivery vector may be selected from at least one of viral vectors, transposons, gene knockins, liposomes, nanogold particles, exosomes.
In some embodiments, the gene delivery vector may be a viral vector; among them, types of viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, herpesviral vectors, and the like.
In some embodiments, the gene delivery vector may be a lentiviral vector; among them, packaging systems for lentiviral vectors include, but are not limited to, three plasmid packaging systems and four plasmid packaging systems.
According to another aspect of the present invention, there is provided a method for preparing the above-described genetically modified cell drug expressing PAH, comprising the steps of:
constructing a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:1 or a nucleotide sequence having a sequence corresponding to SEQ ID NO:1, a nucleotide sequence having at least 75% homology to the nucleotide sequence set forth in seq id no; and then introducing the gene delivery vector into cells.
In some embodiments, the gene delivery vector may be a lentiviral vector and the cells may be umbilical cord mesenchymal stem cells.
In some embodiments, the lentiviral vector is made by packaging a recombinant lentivirus, essentially by packaging a recombinant lentivirus expression plasmid, a lentivirus packaging plasmid, and a lentivirus envelope plasmid; the recombinant lentiviral expression plasmid contains EF1 alpha promoter and target gene sequence, and the target gene sequence is shown in SEQ ID NO:1 or a polypeptide having a sequence similar to SEQ ID NO:1, and a nucleotide sequence having at least 75% homology to the nucleotide sequence shown in (1).
In some embodiments, the recombinant lentiviral expression plasmid is made primarily by ligating an EF1 a promoter and a gene sequence of interest into a pRRLSIN plasmid; the preparation method specifically comprises the following steps:
(1) The DNA sequence of PAH gene was artificially synthesized and cloned into pRRLSIN plasmid, and the constructed vector was named: pRRLSIN-PAH;
(2) Amplifying the Nhe I-EF1 alpha-Mlu I fragment by PCR;
(3) Double digestion is carried out on the Nhe I-EF1 alpha-Mlu I fragment and the vector pRRLSIN-PAH by utilizing Nhe I enzyme and MIu I enzyme respectively, and digestion products are recovered;
(4) Connecting the two recovered enzyme digestion products, converting the connection products into escherichia coli Trans5α chemically competent cells, and streaking and plating for culture;
(5) And (3) selecting a monoclonal colony, identifying a recombinant vector through colony PCR, extracting plasmids after positive clone shaking, further verifying the plasmids through sequencing, and naming the plasmids with correct sequences as recombinant lentivirus expression plasmids.
In some embodiments, the lentiviral vector is prepared using a four plasmid packaging system; wherein the lentiviral packaging plasmid may be pMDLg/pRRE (Kan + ) Plasmid and pRSV-REV (Kan + ) Plasmid, lentiviral envelope plasmid may be pMD2.G (Kan + ) A plasmid.
In some embodiments, the preparation of the lentiviral vector comprises the steps of:
recombinant lentiviral expression plasmid pRRLSIN-EF1 alpha-PAH and lentiviral packaging plasmid pMDLg/pRRE (Kan) + ) And pRSV-REV (Kan) + ) Lentiviral envelope plasmid pMD2.G (Kan + ) And co-transfecting the lentivirus into a lentivirus packaging cell by a transfection reagent, packaging the lentivirus, and purifying the lentivirus to obtain the lentivirus vector.
In some embodiments, the transfection reagent may be Polyetherimide (PEI).
In some embodiments, the lentiviral packaging cell may be a HEK293T cell.
In some embodiments, the method of introducing a lentiviral vector into umbilical cord mesenchymal stem cells may specifically comprise the steps of:
treating umbilical cord mesenchymal stem cells with lentiviral vector and polybrene, changing liquid with a culture medium after 18-24 hours, and harvesting cells after culturing for 48-54 hours; wherein, the MOI of the lentiviral vector can be 1-10, and the final concentration of the polybrene can be 1-10 mug/mL.
The invention provides an in vitro gene therapy method aiming at phenylketonuria, firstly provides a 'pharmaceutical factory' for producing PAH by utilizing human umbilical cord mesenchymal stem cells, and provides the umbilical cord mesenchymal stem cells which express the genetic modification of PAH, which are prepared by modifying umbilical cord mesenchymal stem cells in vitro, have good safety, can continuously express high-level PAH in vivo for a long time, are safe and effective, and can be applied to preparing medicines for treating phenylketonuria.
Drawings
FIG. 1 shows the PCR amplification products of NheI-EF1 alpha-MluI fragments detected by agarose gel electrophoresis;
FIG. 2 shows the detection of pRRLSIN-PAH cleavage products by agarose gel electrophoresis;
FIG. 3 shows the identification of recombinant lentiviral expression plasmid pRRLSIN-EF 1. Alpha. -PAH by colony PCR;
FIG. 4 is a diagram of recombinant lentiviral expression plasmid pRRLSIN-EF1 alpha-PAH;
FIG. 5 is a flow cytometry detection of MSC cell phenotype;
FIG. 6 shows the expression of PAH by MSC after lentiviral infection by western blotting;
FIG. 7 shows the expression of PAH by MSC at various time points after detection of lentiviral infection by western blotting.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments. The examples are for illustration only and are not intended to limit the invention in any way. The raw materials and reagents used in the examples were conventional products which were obtained commercially, unless otherwise specified; the experimental methods for which specific conditions are not specified in the examples are generally in accordance with the conditions conventional in the art or in accordance with the manufacturer's recommendations.
EXAMPLE 1 preparation of PAH-expressing genetically modified umbilical mesenchymal Stem cells
1. Construction of recombinant lentiviral expression plasmid pRRLSIN-EF1 alpha-PAH
(1) According to the human phenylalanine hydroxylase gene sequence (NM-000277.3) provided by NCBI, the nucleotide sequence is shown in SEQ ID NO:1, the DNA sequence of the PAH gene was artificially synthesized by general biosystems (Anhui) and cloned between Mlu I and Sal I cleavage sites of pRRLSIN plasmid, and the constructed vector was named pRRLSIN-PAH.
pRRLSIN plasmid used in the invention is provided by Guangzhou Anjie biomedical technology Co., ltd, and is a lentiviral expression plasmid obtained by modifying pRRLSIN.CPPT.PGK-GFP.WPRE lentiviral expression plasmid, and the map of pRRLSIN plasmid after EF1 alpha promoter and PAH are connected is shown in figure 4.
(2) An EF1 alpha promoter is inserted in front of the PAH gene to construct a recombinant lentiviral expression plasmid pRRLSIN-EF1 alpha-PAH, and the specific process is as follows:
s1: the NheI-EF1 alpha-MluI fragment was amplified using pLVX-EF1 alpha-IRES-puro (supplied by Anjie biomedical technology Co., ltd.) as a template and NheI-EF1 alpha-F/EF 1 alpha-MluI as a primer, wherein the primer sequence was as follows:
Nhe I-EF1α-F：5’-CTAGCTAGCGCTCCGGTGCCCGTCAG-3’(SEQ ID NO：3)
EF1α-Mlu I：5’-CGACGCGTTCACGACACCTGAAATGG-3’(SEQ ID NO：4)
PCR reaction parameters: pre-denaturation: 95 ℃ for 5min; denaturation: 94 ℃ for 30s; annealing: 55 ℃ for 30s; extension: 72 ℃,75s; cycling for 32 times; extending at 72 ℃ for 10min;4℃for forever.
PCR amplification products of Nhe I-EF1 alpha-Mlu I fragments were identified and isolated by 1% agarose gel electrophoresis, and the identification results are shown in FIG. 1, wherein Lane1 and Lane 2 are Nhe I-EF1 alpha-Mlu I fragments.
The Nhe I-EF1 alpha-Mlu I amplified fragment was recovered and purified using a DNA gel recovery kit.
S2: double digestion is carried out on the Nhe I-EF1 alpha-Mlu I amplified fragment and the carrier pRRLSIN-PAH by utilizing the Nhe I enzyme and the MIu I enzyme respectively, wherein the digestion products of the Nhe I-EF1 alpha-Mlu I amplified fragment are directly recovered, the digestion products of the carrier pRRLSIN-PAH are identified by agarose gel electrophoresis and then are recovered by gel cutting, and the identification result is shown in figure 2, wherein Lane1 is the digestion fragment of the carrier pRRLSIN-PAH.
S3: the Nhe I-EF1 alpha-Mlu I amplified fragment and the cleavage product of the vector pRRLSIN-PAH were digested with T 4 The DNA ligase was ligated, and the ligation product was transformed into E.coli Trans5α chemically competent cells (supplied by Beijing full gold Biotechnology Co., ltd.) and streaked on plates.
S4: 10 single colonies were picked and identified by colony PCR using Nhe I-EF 1. Alpha. -F/EF 1. Alpha. -Mlu I as primers, and the results are shown in FIG. 3, where the lane 8 and lane 9 clones amplified the desired bands and the bands were apparent, indicating that positive clones were possible.
S5: the plasmid was extracted with a UE plasmid miniprep kit (Yoyland Biotechnology Co., ltd.) after shaking the positive clone, and the plasmid was sequenced, and the sequencing result showed that the sequences were all correct, indicating that the recombinant lentivirus expression plasmid was successfully constructed, designated pRRLSIN-EF1 alpha-PAH, and the plasmid map was shown in FIG. 4.
S6: plasmids were extracted using endotoxin-free plasmid large extraction kit (Endo-free Plasmid Maxi Kit, omega) and stored in a-20℃refrigerator.
2. Lentivirus packaging and purification
(1) The frozen HEK293T cells were recovered and subcultured with DMEM complete medium (DMEM medium+10% FBS). HEK293T cells are inoculated to a 10-layer cell factory, DMEM complete culture medium is added, and the cells can reach 80-90% fusion degree by overnight culture, and plasmid transfection is carried out.
(2) Recombinant lentiviral expression plasmid pRRLSIN-EF1 alpha-PAH and lentiviral packaging plasmid pMDLg/pRRE (Kan) + ) And pRSV-REV (Kan) + ) Lentiviral envelope plasmid pMD2.G (Kan + ) Adding into serum-free DMEM, mixing, standing for 5min to obtain plasmid mixed solution;
adding PEI into serum-free DMEM, uniformly mixing, and standing for 5min to obtain a transfection reagent;
adding the transfection reagent into the plasmid mixed solution, fully and uniformly mixing, and standing for 20min to form a DNA-PEI complex;
(3) Adding the DNA-PEI complex into 1L of DMEM medium containing 5% FBS, mixing thoroughly, replacing the culture solution in the 10-layer cell factory in step (1), placing at 37deg.C and 5% CO 2 Culturing in an incubator;
(4) Collecting culture supernatant after 48h and 72h of transfection, and storing in a refrigerator at the temperature of 2-8 ℃;
(5) Mixing the collected culture supernatants, and removing cells and cell debris using a sac-type filter (Sartorius); then the clarified and filtered lentivirus supernatant is passed through Shibijingzhi tangential flow filtration systemKR 2I) is concentrated for 10-15 times, and the concentrated solution is subjected to chromatographic purification after being filtered by a 0.45 mu m filter membrane; the purified lentivirus was sterilized and filtered through a 0.22 μm filter (Sartorius), sub-packaged and stored in a refrigerator at-80 ℃. The resulting lentivirus was designated pRRLSIN-EF1 alpha-PAH lentivirus.
3. Isolated culture of umbilical cord Mesenchymal Stem Cells (MSCs)
The umbilical cord of a term healthy fetus is collected, immersed in PBS containing 1% penicillin and streptomycin, and transported to a laboratory at low temperature (2-8 ℃). The umbilical cord was removed to a 10cm dish and cut into several small pieces, and washed 3 times with physiological saline and physiological saline containing penicillin and streptomycin, respectively. Placing the washed umbilical cord into a new 10cm culture dish, removing artery and vein, separating gum, and cutting into 1mm pieces 3 The tissue mass was washed twice by centrifugation (500 g,5 min) in physiological saline. Adding the sheared Huatong gel into a culture flask containing serum-free culture medium, slightly oscillating to uniformly disperse tissue blocks, and placing at 37deg.C and 5% CO 2 Culturing in an incubator. Half-changing the culture solution is carried out for 5-7 days, and the culture is continued in the incubator until long spindle-shaped cells grow out from the edge of the tissue block. When the cells covered the bottle bottom by about 50%, the culture medium and tissue blocks in the culture bottle were discarded, and the culture bottle was washed 2 times with physiological saline. Pancreatin was added for digestion and subculture, labeled P1. Pancreatin digestion was added when 90% of the bottom of the bottle was covered with adherent cells, passaging was performed at a ratio of 1:5, and phenotypes were detected by flow cytometry, and the results were positive for CD105, CD73, CD90, and negative for CD34, as shown in fig. 5, indicating successful isolation of MSC cells. Freezing the cells with the freezing solution, establishing an umbilical cord mesenchymal stem cell bank, and marking the corresponding generations.
4. Preparation of PAH Gene-modified umbilical mesenchymal Stem cells
(1) And taking MSC cells in the cell bank, resuscitating, and adding serum-free culture medium for subculture.
(2) Inoculating MSC with good growth state into culture flask, placing at 37deg.C, and 5% CO 2 After overnight incubation in an incubator, pRRLSIN-EF 1. Alpha. -PAH lentivirus (MOI=5) and polybrene (final concentration 6. Mu.g/mL) were added and centrifuged (700 g,1.5 h) and then placed at 37℃with 5% CO 2 Culturing in an incubator.
(3) After 24h of slow virus infection, the liquid is changed, serum-free culture medium is added, and the mixture is placed at 37 ℃ and 5 percent CO 2 The incubator continues the amplification culture.
(4) Cells and culture supernatants were harvested 72h after lentiviral infection, respectively. Wherein the harvested cells are umbilical cord mesenchymal stem cells modified by PAH genes capable of expressing PAH.
As shown in FIG. 6, lane1-3 was MSC supernatant, empty lentivirus-infected MSC supernatant, pRRLSIN-EF 1. Alpha. -PAH lentivirus-infected MSC supernatant, lane4-6 was MSC lysate, empty lentivirus-infected MSC lysate, pRRLSIN-EF 1. Alpha. -PAH lentivirus-infected MSC lysate, and only after Lane3 and Lane6 detected a single band and the size was as expected, indicating that pRRLSIN-EF 1. Alpha. -PAH lentivirus-infected MSC was successfully expressed and secreted outside cells, indicating that the gene modified cells expressing phenylalanine hydroxylase were successfully prepared.
Test example 1
(1) And taking MSC cells in the cell bank, resuscitating, and adding serum-free culture medium for subculture.
(2) MSCs with good growth status were inoculated into 6-well plates, placed in a 37 ℃ 5% co2 incubator for overnight culture, pRRLSIN-EF1 alpha-PAH lentivirus (MOI=5) and polybrene (final concentration 6. Mu.g/mL) were added, and placed in a 37 ℃ 5% co2 incubator for culture after centrifugal infection (700 g,1.5 h).
(3) Collecting supernatant 24 hr after lentivirus infection, changing liquid, adding serum-free culture medium, standing at 37deg.C and 5% CO 2 The incubator continues to culture, and culture supernatants of 48h, 72h, 96h, 120h and 144h after lentivirus infection are respectively collected.
(4) The expression of phenylalanine hydroxylase (PAH) was detected by western blotting at various times after lentiviral infection.
As shown in FIG. 7, lane1-6 was the expression of PAH in culture supernatants of 24h, 48h, 72h, 96h, 120h, 144h after infection of MSC cells with pRRLSIN-EF1 alpha-PAH lentivirus, respectively, which showed that a single band was detected at all time points and the size was expected, and that the protein expression level increased with time, peaked at 96h and then decreased, but remained at a higher level at 144h, indicating that MSC was able to continue to express human normal PAH and to continue to secrete PAH outside the cells after infection of MSC with pRRLSIN-EF1 alpha-PAH lentivirus.
Therefore, the phenylalanine hydroxylase gene modified cell provided by the invention can be used as a therapeutic drug for phenylketonuria, and can be used as a 'drug factory' for producing PAH in vivo after being transplanted in vivo, and the phenylalanine hydroxylase (PAH) is continuously produced, so that phenylalanine (Phe) accumulation caused by PAH gene mutation is compensated, and the defects of short half-life period and frequent drug administration for lifetime of PAH protein in the existing therapeutic method are overcome.
What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.
Claims (6)
1. The preparation method of the gene modified cell medicine for expressing PAH is characterized by comprising the following steps:
constructing a lentiviral vector containing a target gene sequence; then the slow virus vector is led into umbilical cord mesenchymal stem cells to obtain; wherein, the target gene sequence is the coded amino acid sequence shown in SEQ ID NO:2 and has the amino acid sequence as set forth in SEQ ID NO:1, a nucleotide sequence having at least 75% homology to the nucleotide sequence set forth in (1);
the lentiviral vector is mainly prepared by packaging recombinant lentivirus expression plasmid, lentivirus packaging plasmid and lentivirus envelope plasmid with recombinant lentivirus;
the recombinant lentiviral expression plasmid is mainly prepared by connecting EF1 alpha promoter and target gene sequence into pRRLSIN plasmid.
2. The method of claim 1, wherein the target gene sequence is as set forth in SEQ ID NO: 1.
3. The method of claim 1 or 2, wherein the lentiviral packaging plasmid is pMDLg/pRRE Kan + Plasmid and pRSV-REV Kan + Plasmid, the lentivirus envelope plasmid is pMD2.G Kan + A plasmid.
4. The method of preparing according to claim 1 or 2, wherein the method of introducing the lentiviral vector into umbilical cord mesenchymal stem cells comprises the steps of:
treating umbilical cord mesenchymal stem cells with lentiviral vector and polybrene, changing liquid with a culture medium after 18-24 hours, and harvesting cells after culturing for 48-54 hours; wherein the MOI of the lentiviral vector is 1-10, and the final concentration of the polybrene is 1-10 mug/mL.
5. A genetically modified cell drug expressing PAH prepared by the method according to any one of claims 1 to 4.
6. The use of a genetically modified cell drug expressing PAH according to claim 5 for the manufacture of a medicament for the treatment of phenylketonuria.
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| CN111433368A (en) * | 2017-10-02 | 2020-07-17 | 美国基因技术国际有限公司 | Vector with promoter and enhancer combination for treating phenylketonuria |
| CN113874508A (en) * | 2019-03-13 | 2021-12-31 | 世代生物公司 | Non-viral DNA vectors and their use for expressing phenylalanine hydroxylase (PAH) therapeutics |
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| CN101481741B (en) * | 2009-02-12 | 2012-01-25 | 上海交通大学医学院附属新华医院 | Novel mutated PAH gene |
| US11820999B2 (en) * | 2017-04-03 | 2023-11-21 | American Gene Technologies International Inc. | Compositions and methods for treating phenylketonuria |
| EP3692141A1 (en) * | 2017-10-03 | 2020-08-12 | Children's Hospital and Clinics of Minnesota | Vectors and methods of use |
| JP2021522811A (en) * | 2018-05-09 | 2021-09-02 | ビオマリン プハルマセウトイカル インコーポレイテッド | How to treat phenylketonuria |
| US20210348135A1 (en) * | 2018-10-12 | 2021-11-11 | Genzyme Corporation | Generation of improved human pah for treatment of severe pku by liver-directed gene replacement therapy |
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| CN111433368A (en) * | 2017-10-02 | 2020-07-17 | 美国基因技术国际有限公司 | Vector with promoter and enhancer combination for treating phenylketonuria |
| CN113874508A (en) * | 2019-03-13 | 2021-12-31 | 世代生物公司 | Non-viral DNA vectors and their use for expressing phenylalanine hydroxylase (PAH) therapeutics |
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