CN101116747B - Cytosine arabinoside and gland correlated virus composite preparations and uses thereof - Google Patents

Abstract

The invention belongs to the biologic medicine filed and relates to a novel gene therapy compound preparation and the use and application method thereof. When adeno-associated virus is used as gene delivery carrier, a certain dose of cytarabine is combined in use, and obviously increases transfer efficiency of normal cells and tumor cells by the adeno-associated virus, advances expression time of foreign gene mediated by adeno-associated virus, obviously increases positive rate and expression level, thereby improving the gene therapy effect employing adeno-associated virus as carrier, particularly the application in gene therapy for diseases, such as ocular fundus disease, genetic disease and tumor. In tumor gene therapy, the invention has functions of inhibiting tumor cell growth, inducing tumor cell apoptosis, and obviously increases the transfer efficiency and expression level of the gene mediated by adeno-associated virus.

Description

Compound formulation of cytosine arabinoside and adeno-associated virus and uses thereof

Technical field

The invention belongs to biomedicine field, relate to a kind of new gene therapy compound formulation and uses thereof and using method.

Background technology

Adeno-associated virus is called for short AAV, belongs to Parvoviridae, is a kind of linear ssdna virus, outsourcing icosahedral capsid albumen, and diameter 20nm is a kind of of minimum in the animal virus.Can divide multiple different serotype, wherein AAV2 is the most frequently used a kind of in the gene therapy.AAV virus has following characteristic: the first, and AAV does not have obviously pathogenic.It is reported,, do not see that itself and any disease have obvious dependency although 80% individuality presents the AAV seropositivity among the crowd.The second, AAV is a kind of defective virus, can not be independently duplicated.When non-auxiliary virus (as adenovirus, herpesvirus etc.) infected, AAV was " hiding " state.The 3rd, site-specific integration.AAV uniquely a kind ofly is incorporated into eucaryon virus on the human chromosomal in site-specific mode, and now clear and definite integration site is that No. 19 chromosomes are long-armed.Mechanism it be unclear that, may be relevant with rep albumen with the ITRs of virus.The 4th, host range is wide.AAV can infect polytype tissue and cell, not only can infect the division stage cell, and is also responsive to non-division stage cell (as neuron, myocyte, hematopoietic stem cell etc.).The 5th, the AAV carrier that is used for gene therapy is reorganization AAV (claiming rAAV), because the genome capacity of AAV is less, and about 5kb, wherein ITR is that the AAV packing is necessary.Gene therapy has only kept ITR with the rAAV carrier, has removed all encoding genes of adeno-associated virus, to the capacity of the about 4.7kb of exogenous gene.So behind the adeno-associated virus infection cell of reorganization, entrained exogenous gene expression is only arranged, do not follow any viral gene expression, immunogenicity is very weak, causes immunological rejection hardly.The 6th, can mediate therapeutic gene long period stably express.Therefore, be considered to hereditary gene delivery vector preferably.There are 38 schemes to use rAAV at present in the world as carrier.

But, when separately using rAAV, its infect and transduction efficiency not high, show rAAV infect after therapeutic gene time of beginning to express later, expression is lower, so therapeutic effect is not ideal enough, so remain the less a kind of viral vector of application in the gene therapy clinical trial at present.Because rAAV has the advantage that other viral vector do not possess, if can improve infection and the transduction efficiency of rAAV, will obviously improve the therapeutic effect of rAAV, and expand the range of application of rAAV.

So, how to improve infection and the transduction efficiency of rAAV? it is to be the rapid process of multistep of the first step attached to cell surface with virus that AAV successfully infects.Virus is betransported in born of the same parents then by cellular uptake subsequently, in the transporte to cells nuclear, hide in nuclear or under the effect in some cofactor in the new viral genome of cell endoreduplication generation, the process of similar most of viral infection host cells.With the most frequently used II type recombinant adeno-associated virus (rAAV2) is example, rAAV2 at first with Heparan sulfate (heparinsulfate proteoglycans, HSPG) combination, because all adherent cell surfaces all express glycosaminoglycan, this also provides simple an explanation for rAAV2 tropism widely or close preferendum.In addition, also have two kinds of receptors to be found relevant with the rAAV2 infection, they are that α v β 5 integrates plain and desmocyte growth factor-21s (FGFR1).Can not viral interference and the combining but suppressed the endocytosis of cell of cell and integrate plain antibody to virus at α v β 5, seemingly AAV2 is necessary by cell endocytic so infer α v β 5 integration elements, but concrete endocytosis process and following step are still not clear at present.

The activity of oxidoreduction modulin Rac1 can strengthen the endocytosis of AAV2 in the proof cells such as Sanlioglu, they studies show that, cough up alkane dithiocarbonate (pyrrolidinedithiocarbonate through adjoining, PDTC) and after having genotoxic factor (UV or H2O2) associated treatment, cell obviously increases for endocytosis and the absorption of rAAV, but each reagent individual processing cell does not have obvious lifting to the endocytosis of rAAV.

RAAV is a kind of single-stranded DNA viruses (ssDNA viruses), and its genes carried will be expressed the process that needs to convert to through strand two strands (duble-stranded DNA dsDNA).Have only double-stranded DNA just to have transcriptional capability.Have result of study to show, after rAAV entered target cell, can only there be very short time in the rAAV genome of free strand state, and wherein most of DNA that is known for damaged by the enzyme of target cell is degraded.In some cases, AAV undresses, and its single stranded DNA can be transformed into double-stranded DNA behind the shell.Single stranded DNA is transformed into double-stranded DNA and has following two approach.Article one, be to be synthetic another chain of masterplate, have E4ORF6 gene, UV irradiation and the genotoxicity chemical drugs etc. of research report adenovirus can strengthen this process with strand AAV DNA; Another is to form double-stranded from two AAV virions, the annealing of opposite polarity strand.Because AAV is single-stranded DNA viruses, terminal ITR is folded to form hairpin like structure, duplicate be from the folding 3 ' end of AAV genome with AAV genome self as the masterplate synthetic dsdna.Double-stranded DNA unwinds again, and positive and negative chain is packaged into new virion with equal efficient.So positive and negative chain can form double-stranded by annealing behind the infection cell.RAAV has caused the delay and the occurrent invalid transduction of its transduction in intracellular these characteristics.There are some researches show that recently cell interior is called the albumen of FKBP52 and the transduction efficiency that heat shock protein 90 (HSP90) can change AAV.The former phosphorylation form and virus terminal oppositely repeat the D-sequence and interact, and suppress the synthetic of viral second chain, limit genetically modified expression.The latter shows as the part of FKBP52-AAV D-sequence complex in the AAV transduction, but itself does not combine with the D-sequence.So thereby KBP52 and HSP90 all can be by the synthetic effects that reaches change AAV transduction efficiency of second chain that changes AAV.

Cytosine arabinoside is a kind of antitumor drug, claim cytarabin again, cytosine arabinoside, (CAR be abbreviated as in foreign language to arabinosylcytosine, CA, Ara-C), be that a class acts on the cell S pyrimidines antimetabolic medicine of propagation phase, chemical name: 1-β-D-arabinofuranosyl base-4-amino-2 (1H)-pyrimidone hydrochlorate.Molecular formula: C9H13N3O5HCl.Molecular weight: 279.68.Cytosine arabinoside is not present in nature, is on the position of its 2 ' glycan molecule the OH base is arranged with the difference of physiological analog Cyd (Cytidine).Ara-C earlier through deoxycytidine enzyme catalysis phosphorylation, changes activated cytosine arabinoside acid (Ara-CMP) in cell, be converted into diphosphonic acid cytosine arabinoside (Ara-CDP) and ara-CTP (Ara-CTP) again and work.The latter and dCTP competition, competitive inhibition DNA polymerase, and be inserted in the DNA chain, stop the prolongation of DNA chain, and cause chain interruption, thereby it is synthetic to suppress DNA.The former can suppress ribonucleotide reductase, stop nucleotide to change Deoxydization nucleotide into, suppress cell DNA polymerization and synthetic, but RNA and protein synthesis are not had remarkable effect, Ara-CTP can also change the metabolism of endocellular phosphorus fat and glycoprotein, and cytotoxicity caused effect.Ara is a cell cycle specific agents, and is the most responsive to the effect that is in S propagation phase cell, a little less than the effect that suppresses RNA and protein synthesis.Be mainly used in the treatment malignant tumor clinically.But Ara had not been found aspect gene transfer efficient that improves the AAV mediation and the expression and was reporting.Use Ara to improve AAV mediated by protein transduction efficient, gene expression dose and persistent period when also nobody attempts when gene therapy particularly to degeneration and hereditary gene therapy.

Summary of the invention

The purpose of this invention is to provide a kind of new compound formulation that is used for gene therapy, be specifically related to compound formulation of cytosine arabinoside and adeno-associated virus and uses thereof.Comprise the compound formulation that certain density cytosine arabinoside and adeno-associated virus are formed, and the substrate of compound formulation; And cytosine arabinoside and adeno-associated virus are used in associating when oculopathy, heredopathia and malignant tumor gene therapy (while or priority), comprise that system gives cytosine arabinoside and adeno-associated virus or system and gives cytosine arabinoside local adeno-associated virus or local cytosine arabinoside and the adeno-associated virus of using of using, with method and the purposes of transduction efficiency, expression and persistent period of strengthening gland relative virus mediated gene; And cytosine arabinoside is improving gland relative virus mediated gene transfer efficient, expression and persistent period, is improving the new pharmaceutical usage of aav gene therapeutic effect.

Experiment confirm cytosine arabinoside of the present invention and adeno-associated virus form compound formulation, or when giving adeno-associated virus, unite and use cytosine arabinoside can significantly improve the therapeutic effect of gland relevant viral vector.

Compound formulation of the present invention, it contains following material as active component:

(1) antitumor drug cytosine arabinoside;

(2) adeno-associated virus, AAV1, AAV2, AAV5 or other serotype such as AAV3, AAV4, AAV6, AAV7 or AAV8.

The dosage of above-mentioned active component such as following:

(1) dosage of antitumor drug cytosine arabinoside is 0.001-10 μ g;

(2) dosage of adeno-associated virus is 1 * 10e9-1 * 10e12 virion in compound formulation.

The substrate of described compound formulation can be normal saline, water for injection, phosphate buffer or cell culture medium.

Described compound formulation can be lyophilized form or liquid form.

Compound formulation of the present invention can adopt local injection or intravenous injection mode.Wherein the local injection mode comprises subretinal injection, vitreous body intracavitary administration or injected into anterior chambers; Intramuscular injection; Lumbar injection; Joint cavity injection; Injection system in perfusion or the tumor in the conduit liver, in the kidney, in the heart.Also comprise the topical administration adeno-associated virus, while or the antitumor drug of intravenous injection successively cytosine arabinoside mode etc.

Above-mentioned compound formulation provided by the invention and wherein antitumor drug cytosine arabinoside and the adeno-associated virus method of uniting use can significantly improve adeno-associated virus is treated disease as gene delivery vector curative effect.Unite the antitumor drug cytosine arabinoside that uses doses, can obviously increase the transduction efficiency of adeno-associated virus to normal and tumor cell, the gland relative virus mediated expression of exogenous gene time is obviously shifted to an earlier date, positive rate obviously increases, expression obviously improves, simultaneously do not have obvious toxic and side effects again, help to improve with the gene therapy effect of adeno-associated virus as carrier.The present invention especially is useful in the application in the disease gene treatments such as oculopathy, heredopathia and tumor.The pharmaceutical usage that this of cytosine arabinoside is new has crucial meaning for the effect that promotes gland relative virus mediated gene therapy, has broad application prospects.

Purpose of the present invention realizes by following step:

(1) cell culture experiments in vitro confirms that cytosine arabinoside can obviously improve transduction efficiency and the gene expression dose of adeno-associated virus to retina cell and tumor cell.

The A cytosine arabinoside significantly improves transduction efficiency and the gene expression dose of adeno-associated virus to the retina cell of In vitro culture

RPE cell (APRE-19 or the former foster RPE that is commissioned to train) is cultivated and is adopted 6 porocyte culture plates, every hole inoculation 2 * 10 ⁵The RPE cell, or 2 * 10 ⁶Adult's retinal neuronal cell.

In 6 porocyte culture plates, unite and add cytosine arabinoside and rAAV2-GFP.The rAAV2-GFP consumption is each cell 1 * 10 ⁴Virion.The cytosine arabinoside concentration range is 0.0028-4.2 μ g/ml (final concentration).

For the cell that infects rAAV2-GFP, reflect transduction and the expression efficiency of rAAV2 by the expression of GFP.According to the time of GFP fluorescence appearance, the cell quantity of expression GFP fluorescence and transduction efficiency and the entrained expression of gene level that the GFP fluorescence intensity is judged the rAAV2 carrier.The detection of GFP positive cell quantity and GFP fluorescence intensity is finished by fluorescence microscope and flow cytometry analysis.

Use 1 * 10 separately ⁴RAAV ₂-GFP infected person RPE cell observe small amounts of cells expression GFP fluorescence to infecting back the 5th day (120 hours), and fluorescence is very weak.With the prolongation of the time of infection, the GFP positive cell quantity increases but is slower, and fluorescence intensity strengthens not obvious.The result shows, uses rAAV separately ₂The GFP positive cell quantity is few during-GFP, a little less than the fluorescence.

If using rAAV ₂During-GFP infected person RPE cell, add cytosine arabinoside simultaneously, the expression of GFP obviously shifts to an earlier date, expression also obviously increases.When the cytosine arabinoside final concentration is 0.0028 μ g/ml (0.0028 μ g), 0.014 μ g/ml (0.014 μ g), 0.028 μ g/ml (0.028 μ g), 0.14 μ g/ml (0.14 μ g), 0.28 μ g/ml (0.28 μ g), 0.56 μ g/ml (0.56 μ g), 1.4 μ g/ml (1.4 μ g), 2.8 μ g/ml (2.8 μ g), 4.2 μ g/ml (4.2 μ g), GFP was shifted to an earlier date in the expression time of RPE cell, and expression increase.Cytosine arabinoside concentration is high more, and the GFP positive cell quantity is many more, and fluorescence is bright more.With infecting time lengthening, express the cell quantity showed increased of GFP, fluorescence intensity significantly strengthens.Microscopically observation of cell form is good, does not see cytopathy, and Tunel detects apoptosis and contrast no significant difference.

Western blot respectively organizes the GFP expressing quantity more than analyzing, and when using rAAV2-GFP infected person RPE cell separately, the GFP positive signal is very weak.The interior GFP protein content of cell obviously increases behind the use in conjunction cytosine arabinoside, and the degree that increases is relevant with the dosage of cytosine arabinoside.When the cytosine arabinoside concentration of use in conjunction is 1.4 μ g/ml, 2.8 μ g/ml and 4.2 μ g/ml, GFP protein content height in the cell, the band difference of described three concentration is not obvious.What wherein band was the most weak is when cytosine arabinoside concentration is 0.0028 μ g/ml (0.0028 μ g), 0.014 μ g/ml (0.014 μ g), even if but at this moment also significantly better than independent application rAAV ₂Result when infecting the RPE cell.

The above results shows, the cytosine arabinoside of 9 variable concentrations that adopted all can be in various degree raising and accelerate transduction efficiency and the gene expression dose of rAAV to the RPE cell, wherein effective to the concentration of 4.2 μ g/ml with 0.028 μ g/ml, after infection, can make the GFP positive rate rise to 98.5% on the 7th day, mean flow rate reaches 137.7, efficient when being higher than independent application rAAV far away, cytosine arabinoside demonstrate powerful quickening and improve rAAV ₂The effect of genes carried expression efficiency.Simultaneously, behind the rAAV infection cell the 7th day, not obvious to the effect difference that improves the rAAV transduction efficiency between each concentration, cell also all showed good upgrowth situation and form, did not see that cell quantity is less, became phenomenons such as circle and apoptosis.Proved that its safe and effective dosage range was wider when the employing cytosine arabinoside improved rAAV to RPE cell transduction efficient, these characteristics will become a kind of advantage when clinical practice is used.

Except above-mentioned RPE extracellular the present invention who builds strain uses rAAV separately ₂Adult RPE cell, iris pigment epithelial cells and human retina neurocyte that-GFP or associating cytosine arabinoside infector are commissioned to train foster have all obtained and the identical result of RPE cell who builds strain.

The B cytosine arabinoside significantly improves transduction efficiency and the gene expression dose of adeno-associated virus to the tumor cell of cell in vitro cultivation.

Also show identical effect for the kinds of tumor cells cytosine arabinoside, with rAAV ₂-GFP separately or the associating final concentration be 0.56 μ g/ml (0.56 μ g), 1.4 μ g/ml (1.4 μ g), 2.8 μ g/ml (2.8 μ g) cytosine arabinoside infected person non-small cell lung cancer cell NCIH446, human small cell lung carcinoma cell strain NCIH460, human hepatoma cell strain SMMC7721, human stomach cancer cell line SGC7901 and human lung adenocarcinoma cell line A549 respectively.Fluorescence microscope is expressed the cell quantity and the fluorescent brightness of GFP fluorescence, and Taking Pictures recording.The result shows, when cytosine arabinoside and 5 kinds of tumor cells of rAAV2-GFP co-infected, raising rAAV2-GFP that can be in various degree is to the transduction efficiency and the gene expression dose of each tumor cell, wherein obvious with SMMC7721, SGC7901 and NCIH446 effect, this may be relevant to the close preferendum height of described 3 kinds of tumor cells with rAAV2-GFP itself, and cytosine arabinoside has further promoted exogenous gene on this basis at intracellular expression speed and expression.Simultaneously, cytosine arabinoside has also produced the obvious apoptosis effect to tumor cell.Results suggest, according to these characteristics of cytosine arabinoside, can be in tumor therapeutic procedure, chemotherapy is combined with gene therapy, bring into play the antitumor action of cytosine arabinoside simultaneously, make full use of the characteristic that it improves the gene therapy vector transduction efficiency again, thereby improve the therapeutic effect of malignant tumor.

Experiment confirm of the present invention: the antitumor drug cytosine arabinoside can significantly accelerate and improve exogenous gene that rAAV mediates in retina cell and the intracellular expression of RPE when doses concentration, pair cell does not have obvious toxicity, side effect.For the effect of improving the retinopathy gene therapy obvious effect is arranged.In addition, cytosine arabinoside can also obviously improve rAAV when promoting apoptosis of tumor cells ₂The expression of the exogenous gene that is mediated in tumor cell.For the therapeutic effect that improves malignant tumor obvious effect is arranged.

(2) the experiment confirm cytosine arabinoside can obviously improve transduction efficiency and the gene expression dose of adeno-associated virus to live body retina cell, tumor cell and multiple tissue in the body.

The A cytosine arabinoside significantly improves transduction efficiency and the gene expression dose of adeno-associated virus to the live body retina cell, improves therapeutic effect

Choose 8 the week age Balb/c mice, after the anesthesia respectively at retina cavity of resorption injection of AAV ₂-Luc and AAV ₂-Luc+Ara-c.In injection back after 24,48,72 hours and 6 days, 9 days, 21 days, 28 days with mouse anesthesia through toy living imaging visual report gene Luc in the intraretinal expression of mice.The result shows independent application AAV ₂When-Luc carries out subretinal injection, the expression of Luc in the visible retina of the 6th talent; And use in conjunction chemotherapeutics Ara-c, just can judge existing Luc expression of gene in the retina by Luc biotin fluorescence in injection after 24 hours, prolongation along with infection time, reporter gene Luc strengthens in intraretinal expression, and the ophthalmic fluorescence of use in conjunction chemotherapeutics Ara-c is all the time than independent application AAV ₂The ophthalmic fluorescence of-GFP is strong, scope is big.HE sections observation retina inner cell is not seen obvious necrosis, apoptosis and inflammatory reaction.

Get 3 the week age RCS rat be divided into 3 groups at random, respectively at subretinal injection normal saline, AAV ₂-CNTF, AAV ₂-CNTF+Ara-c.The injection back is got eyeball 5 weeks and is made HE section back OLYMPAS microscopically observation counting RCS rat outer nuclear layer cell quantity.Only surplus one deck of subretinal injection normal saline group outer nuclear layer cell even disappearance, simple injection of AAV ₂The nearly 3-4 layer of-CNTF group outer nuclear layer cell number of plies, use in conjunction AAV ₂The outer nuclear layer cell number of plies is at the 5-6 layer when-CNTF and Ara-c.

The B cytosine arabinoside significantly improves adeno-associated virus to transduction efficiency and gene expression dose in intravital multiple tissue and the tumor.

With cytosine arabinoside and adeno-associated virus joint injection in mouse peritoneal,, adeno-associated virus is AAV ₂-Luc, consumption are 1-3 * 10 ¹⁰Individual virion, the cytosine arabinoside consumption is respectively 1ug and 10ug.Living imaging shows that cytosine arabinoside can obviously increase adeno-associated virus in Intraabdominal expression efficiency and persistent period.

Tumor model is selected people's pulmonary carcinoma NCIH460 nude mouse model for use, and is earlier that people's pulmonary carcinoma NCIH460 cell inoculation is subcutaneous to Balb/c nude mouse hind leg, and tumor growth is to the 8-10mm diameter after 4 weeks.Respectively with AAV ₂-Luc 1 * 10 ¹⁰Or 3 * 10 ¹⁰Individual virion is adjusted volume to 50ul with after 0.01 μ g, 0.1 μ g, 1 μ g, 2 μ g, 5 μ g and 10 μ g cytosine arabinosides mix with PBS, is injected directly in the tumor then, or respectively with AAV ₂-Luc is expelled in the tumor, and 1 μ g, 2 μ g, 5 μ g, 10 μ g, 50 μ g and 100 μ g cytosine arabinosides are expelled to intraperitoneal.Detect fluorescence intensity in the tumor in injection living body fluorescent imaging in back 48 hours, 96 hours.Testing result shows that cytosine arabinoside can obviously increase the expression efficiency of adeno-associated virus in tumor.

The virus involved in the present invention and the source of medicine are as follows:

2 type adeno-associated virus AAV of 1 band GFP reporter gene ₂2 type adeno-associated virus AAV of-GFP and band Luc reporter gene ₂-Luc is provided by AGTC Gene Technology Co., Ltd..

2 antitumor drug cytosine arabinosides are the cytosar HC1 vial that Harbin Bolai Pharmaceutical Co., Ltd. produces, and product batch number is 06010702.

The source and the cultural method of various cells involved in the present invention are as follows:

Related various cells have: adult's RPE cell (APRE-19) is the ATCC product.

Former adult RPE cell of being commissioned to train foster, the former concrete cultural method of adult's retinal neuronal cell of being commissioned to train foster are as follows: discarded eyeball 75% alcohol disinfecting after the corneal transplantation, gentamycin and PBS wash repeatedly.Carefully prune away then except that the fatty tissue around the eyeball, eyeball is cut into two parts from the ambitus.Discard the part eyeball that contains cornea; A part contains retina in addition, carefully wipes out the vitreous body in the vitreous chamber; Under anatomical lens careful taking-up of retina put into the graduated centrifuge tube that fills PBS, PBS washes 3 times, adds 37 ℃ of digestion of the long-pending 0.25%Try-EDTA of monoploid 60 minutes.Adult RPE cell directly digested 60 minutes with 0.25%Try-EDTA in eyecup.Add DMEM/F12 culture medium (Invitrogen Inc) the piping and druming cell that contains 15%FBS then respectively behind the sucking-off 0.25%Try-EDTA and become cell suspension.The microscopically counting.With every hole 2 * 10 ⁵Cell quantity divide to 6 orifice plates.At 37 ℃, CO ₂Volume fraction is 5% interior cultivation of incubator.Observe once every day.Neurocyte changed liquid once in 7 days, and the RPE cell changed liquid once in 3 days.

Also relate to various tumor cell strains, comprise people's non-small cell lung cancer cell strain NCIH446, human small cell lung carcinoma cell strain NCIH460, human lung adenocarcinoma cell line A549, human hepatoma cell strain SMMC7721 and human stomach cancer cell line SGC7901.

Description of drawings

Fig. 1-3: fluorescence microscope is taken a picture, respectively by AAV ₂-GFP or AAV ₂GFP expression in the RPE cell that the cytosine arabinoside of-GFP associating variable concentrations infects,

Wherein, Fig. 1 uses AAV separately ₂-GFP 1 * 10e9 infected behind the RPE cell the 5th day, and cell well-grown, fluorescence microscope be visible down, and to express the cell quantity of GFP few, is dispersed in distribution, a little less than the GFP fluorescence intensity.A and B are the fluorescence photo and the black-and-white photograph of RPE cell under the same visual field, * 50; C and D are the fluorescence photo and the black-and-white photograph of RPE cell under the same visual field, * 100.

Fig. 2 AAV2-GFP 1 * 10e9 and 0.0028 μ g/mi cytosine arabinoside infected behind the RPE cell the 5th day simultaneously, and the cell growthform is good, parts of fine cellular expression GFP fluorescence.A and B are the fluorescence photo and the black-and-white photograph of RPE cell under the same visual field, * 50; C and D are the fluorescence photo and the black-and-white photograph of RPE cell under the same visual field, * 100.

Fig. 3 AAV2-GFP 1 * 10e9 and 1.4 μ g/ml cytosine arabinosides infected behind the RPE cell the 5th day simultaneously, most cellular expression GFP, and GFP fluorescence is strong.Cellular morphology is good, does not see that apoptotic cell exists.A and B are the fluorescence photo and the black-and-white photograph of RPE cell under the same visual field, * 50; C and D are the fluorescence photo and the black-and-white photograph of RPE cell under the same visual field, * 100.

Fig. 4: fluorescence microscope is taken a picture, respectively by AAV ₂-GFP or AAV ₂-GFP unites GFP expression among the tumor cell SGC7901 that 0.56 μ g/ml cytosine arabinoside infects,

Wherein, AAV2-GFP 1 * 10e9 and 0.56 μ g/ml cytosine arabinoside infected behind 7901 cells 48 hours simultaneously, most cellular expression GFP, and GFP fluorescence is strong.But part cell rounding apoptosis is floating.A and B are the fluorescence photo and the black-and-white photograph of 7901 cells under the same visual field, * 50; C and D are the fluorescence photo and the black-and-white photograph of RPE cell under the same visual field, * 400.

Fig. 5: flow cytometry analysis, AAV ₂Independent or the AAV of-GFP ₂The RPE cell that-GFP associating variable concentrations cytosine arabinoside infects GFP expression after 7 days.

Fig. 6: flow cytometry analysis, AAV ₂Independent or the AAV of-GFP ₂-GFP unites 0.54 μ g/ml cytosine arabinoside infected tumor cell GFP expression after 72 hours.

Fig. 7: Western blot detects, by AAV ₂Independent or the AAV of-GFP ₂GFP expression behind the-GFP associating variable concentrations cytosine arabinoside infection RPE cell,

Wherein, the cytosine arabinoside of AAV2-GFP 1 * 10e9 associating variable concentrations infected behind the RPE cell the 7th day simultaneously, and Westernblot detects GFP expression in the cell.Along with the increase of cytosine arabinoside concentration, GFP content is also increasing gradually in the cell.When cytosine arabinoside concentration is 1.4 μ g/ml, 2.8 μ g/ml and 4.2 μ g/ml, GFP protein content height in the cell, the stripe size difference is not obvious between each concentration.Be cytosine arabinoside concentration when being 0.56 μ g/ml then, when being respectively 0.28 μ g/ml, 0.14 μ g/ml and 0.028 μ g/ml for cytosine arabinoside concentration once more, the band difference of these three concentration is also not obvious.What band was the most weak is when cytosine arabinoside concentration is 0.0028 μ g/ml (0.0028 μ g), 0.014 μ g/ml (0.014 μ g).Faint when using AAV2-GFP separately because of band, to such an extent as to can't observe.

Fig. 8 toy living imaging is observed AAV2-Luc (right eye) or AAV2-Luc and is united behind 0.1 μ g/ml cytosine arabinoside (left eye) subretinal injection infection conditions to retina cell, wherein, the use in conjunction cytosine arabinoside significantly increases the efficiency of infection of AAV2-Luc to retina cell.

Fig. 9 uses AAV2-CNTF 1 * 10e-10 or use in conjunction AAV2-CNTF 1 * 10e10 and cytosine arabinoside 1 μ g separately, be injected under the 3 week age RCS rat retinas the 5th week behind the gap through Nei Lufa, the cell number that outer nuclear layer retains in the retina when HE sections observation use in conjunction AAV2-CNTF 1 * 10e10 and cytosine arabinoside is many when using AAV2-CNTF 1 * 10e10 more separately

Wherein, apoptosis almost all takes place in RCS rat retina outer nuclear layer cell during the A untreated, forfeiture; * 400,

Apoptosis almost all takes place in RCS rat retina outer nuclear layer cell during B subretinal injection normal saline, forfeiture; * 400

When using AAV2-CNTF 1 * 10e10 separately, C as seen have 3-4 layer outer nuclear layer cell to retain in the RCS rat retina; * 400

As seen there is 5-6 layer outer nuclear layer cell to retain in the RCS rat retina when D use in conjunction AAV2-CNTF 1 * 10e10 and cytosine arabinoside 1 μ g; * 400

The specific embodiment

Transduction efficiency and the gene expression dose of rAAV-GFP to retina cell accelerated and improved to embodiment 1 cytosine arabinoside

By every hole 2 * 10 ⁵With RPE (APRE-19, ATCC) cell inoculation is in 6 porocyte culture plates, cell attachment after 24 hours is respectively with 1 * 10 ⁹RAAV ₂-GFP separately or the cytosine arabinoside of uniting 0.0028 μ g/ml (0.0028 μ g), 0.014 μ g/ml (0.014 μ g), 0.028 μ g/ml (0.028 μ g), 0.14 μ g/ml (0.14 μ g), 0.28 μ g/ml (0.28 μ g), 0.56 μ g/ml (0.56 μ g), 1.4 μ g/ml (1.4 μ g), 2.8 μ g/ml (2.8 μ g), 4.2 μ g/ml (4.2 μ g) concentration join in the cultured cells.Every day is with brightness, cellular morphology and the pathological changes state of fluorescence microscope and film recording infected cells GFP fluorescence then.Trypsinization after 7 days, flow cytometer detection GFP positive cell number and GFP fluorescence intensity, Westernblot detect the GFP expression.

Above-mentioned testing result is as follows, if use rAAV separately ₂-GFP infection cell, the 5th day (120 hours) rise just can be observed GFP and expresses, and a little less than few, the brightness of GFP positive expression cell quantity, cell counting in the time of 7 days, cell quantity are 2.25 * 10 generally speaking ⁵, it is 13.5% that flow cytometer detects the GFP positive rate, mean flow rate 2.75 is seen Fig. 1.If add the cytosine arabinoside of 0.028 μ g/ml (0.028 μ g), 0.14 μ g/ml (0.14 μ g), 0.28 μ g/ml (0.28 μ g), 0.56 μ g/ml (0.56 μ g), 1.4 μ g/ml (1.4 μ g), 2.8 μ g/ml (2.8 μ g), 4.2 μ g/ml (4.2 μ g) concentration simultaneously, GFP expression time obviously shifts to an earlier date, and expression significantly increases.Infect just can be observed more RPE cellular expression GFP fluorescence in back 18 hours, and express the cell quantity of GFP fluorescence and fluorescence intensity this moment apparently higher than independent application rAAV ₂The 7th day situation during-GFP infection cell.With the prolongation of the time of infection, the GFP positive cell number continues to increase in the infected cell, and the mean flow rate of GFP also increases thereupon, and the cell growthform is good simultaneously, does not have the circle of change and apoptotic cells.Infect back 7 days counting cells, cell quantity is respectively 2.17 * 10 by the concentration order of above-mentioned cytosine arabinoside ⁵, 2.21 * 10 ⁵, 2.05 * 10 ⁵, 2.09 * 10 ⁵, 2.12 * 10 ⁵, 2.18 * 10 ⁵With 2.03 * 10 ⁵Flow cytometry analysis showed GFP positive rate is respectively 97.1%, 97.3%, 97.3%, 98.0%, 97.9%, 98.2%, 98.5%.The mean flow rate of GFP is respectively 94.8,93.6,98.6,104.3,110.5,126.6,137.2.When the cytosine arabinoside concentration of uniting use is 0.0028 μ g/ml (0.0028 μ g), 0.014 μ g/ml (0.014 μ g), rAAV ₂Behind-GFP the infection cell 24 hours, can be by fluorescence microscope to a small amount of GFP fluorescence.Counting cells in the time of 7 days, cell quantity is respectively 2.21 * 10 ⁵With 2.20 * 10 ⁵The flow cytometer testing result: the GFP positive rate is respectively 70.3% and 74.5%.The mean flow rate of GFP is respectively 44.1 and 49.7.At this moment, cellular morphology is good, does not have the circle of change and apoptotic cells.See Fig. 2,3,5,7.

Embodiment 2

Transduction efficiency and the gene expression dose of rAAV-GFP to kinds of tumor cells accelerated and improved to cytosine arabinoside

By every hole 1 * 10 ⁵Tumor cell (7721,7901, NCIH446, NCIH460, A549) is seeded in the 24 porocyte culture plates, and cell attachment after 24 hours is respectively with 1 * 10 ⁹RAAV ₂-GFP separately or with final concentration be that the cytosine arabinoside of 0.56 μ g/ml (0.56 μ g), 1.4 μ g/ml (1.4 μ g), 2.8 μ g/ml (2.8 μ g) joins in the cultured cells simultaneously.Infect back 24 hours fluorescence microscopes, infected back 24 hours, use rAAV separately ₂There is no obvious fluorescence in 5 kinds of tumor cells that-GFP infects; In the tumor cell of use in conjunction variable concentrations cytosine arabinoside, GFP fluorescence appears in the part cell in SMMC7721, SGC7901 and the NCIH446, and cellular morphology is good, does not see cell rounding.After 48 hours, use rAAV separately ₂As seen the cell of a small amount of expression GFP fluorescence is arranged in SGC7901, NCIH446 that-GFP infects and the SMMC7721 cell, do not see obvious fluorescence yet in all the other 2 kinds of tumor cells, 5 kinds of tumor cell forms are good, do not see cell rounding.And at rAAV ₂In the cell of-GFP and cytosine arabinoside co-infected, the GFP positive cell further increases among SGC7901 and the NCIH446, and fluorescence is brighter, and GFP positive cell quantity and fluorescence intensity are along with using cytosine arabinoside concentration to increase and increase.GFP fluorescence appears in also visible small amounts of cells in other 2 kinds of tumor cell NCIH460 and the A549.In addition, the tumor cell of every adding cytosine arabinoside was observed in infection in back 48 hours, had all seen the part cell rounding, and the concentration of cytosine arabinoside is high more, and the quantity that becomes the circle cell is many more.Wherein obvious to unite the SMMC7721 cell that adds 2.8 μ g/ml (2.8 μ g) cytosine arabinoside.Infected back 72 hours, with independent use rAAV ₂The tumor cell that-GFP infects is compared, and the cell quantity of expressing GFP fluorescence in the tumor cell of 5 kinds of use in conjunction 0.56 μ g/ml cytosine arabinoside infection is obviously many and bright.But the tumor cell that adds cytosine arabinoside all has in various degree apoptosis and floating, and uses rAAV separately ₂Then growth conditions is good for the tumor cell that-GFP infects, no apoptosis and hydro-planing.The fluidic cell testing result is as follows: use rAAV2-GFP separately and infect NCIH446, NCIH460, SGC7901, SMMC7721 and A549 cell after 72 hours, the GFP positive rate is respectively 30.1%, 26.1%, 31.8%, 34.4% and 23.6%, and mean flow rate is respectively 21.4,12.9,28.1,27.2 and 16.3; The GFP positive rate is respectively 72.3%, 52.5%, 80.5%, 85.1% and 47.4% behind the use in conjunction 0.56 μ g/ml cytosine arabinoside, and mean flow rate is: 57.4,48.1,62.5,60.5 and 31.5.When the concentration of cytosine arabinoside is 1.4 μ g/ml (1.4 μ g), 2.8 μ g/ml (2.8 μ g),, can't record the streaming result because of cell rounding, floating serious.Reflect rAAV according to GFP positive rate and fluorescent brightness ₂Transduction efficiency, then use rAAV separately ₂During-GFP infected tumor cell, rAAV ₂Slightly higher in SGC7901 endocellular transduction efficient, in general, all on the low side than other 4 kinds of tumor cells.Behind rAAV2-GFP and 0.56 μ g/ml cytosine arabinoside co-infected cell, GFP positive cell quantity and brightness obviously improve in 5 kinds of tumor cells, be apparent that SMMC7721 cell and SGC7901 cell the most, the GFP positive rate is brought up to 85.1%, 31.8% by 34.4% respectively and is brought up to 80.5%, and mean flow rate rises to 60.5,28.1 by 27.2 respectively and rises to 62.5.Be NCIH446, NCIH460 and A549 cell then successively.See Fig. 4,6.

Above-mentioned experimental result shows that it is lower to retina and the isocellular transduction efficiency of tumor as gene delivery vector to use rAAV2 separately, and gene expression dose is also lower, and gene to begin to express the needed time longer.Transduction efficiency and the gene expression dose of rAAV2 in retina cell can be accelerated and improve to the antitumor drug cytosine arabinoside safely and effectively in range of doses.When cytosine arabinoside concentration is 0.028 μ g/ml (0.028 μ g), 0.14 μ g/ml (0.14 μ g), 0.28 μ g/ml (0.28 μ g), 0.56 μ g/ml (0.56 μ g), 1.4 μ g/ml (1.4 μ g), 2.8 μ g/ml (2.8 μ g), 4.2 μ g/ml (4.2 μ g), both can significantly accelerate and improve the expression of GFP, do not produce the growth of obvious suppression cell again, side effect such as cell death inducing.Therefore, for adopting cytosine arabinoside to improve rAAV2 this new method of transduction efficiency in retina cell, the safe and effective dosage range of cytosine arabinoside is more extensive.In addition, cytosine arabinoside also can significantly improve transduction efficiency and the gene expression of rAAV2 in tumor cell when making apoptosis of tumor cells.

Embodiment 3

The experiment confirm cytosine arabinoside can obviously improve transduction efficiency and the gene expression dose of adeno-associated virus to the live body retina cell in the body, improves therapeutic effect

The A cytosine arabinoside improves the expression of gene in the live body retina cell of AAV mediation

Choose 8 the week age Balb/c mice, after the anesthesia respectively at retina cavity of resorption injection of AAV ₂-Luc and AAV ₂Each 2ul of-Luc+Ara-c.Concrete dosage: left eye AAV ₂-GFP 2 * 10 ⁹(2ul) individual virion, right eye AAV ₂-GFP2 * 10 ⁹Individual virion associating Ara-c 0.001 μ g.Respectively at subretinal injection after 24,48,72 hours and 6 days, 9 days, 21 days, 28 days with mouse anesthesia through toy living imaging visual report gene Luc in the intraretinal expression of mice.The result shows independent application AAV ₂When-Luc carries out subretinal injection, the expression of Luc in the visible retina of the 6th talent; And use in conjunction chemotherapeutics Ara-c, just can judge existing Luc expression of gene in the retina by Luc biotin fluorescence in injection after 24 hours, prolongation along with infection time, reporter gene Luc strengthens in intraretinal expression, and the ophthalmic fluorescence of use in conjunction chemotherapeutics Ara-c is all the time than independent application AAV ₂The ophthalmic fluorescence of-GFP is strong, scope is big.Get eyeball after 1 week of subretinal injection and the back paraformaldehyde perfusion of 2 weeks, fix 24 hours behind 5% glacial acetic acid, serial section after the paraffin embedding, the about 6 μ m of slice thickness, the retina inner cell is observed in HE dyeing back, does not see obvious necrosis, apoptosis and inflammatory reaction.See Fig. 8.

B. adenovirus improves the therapeutic effect of the gene transfer of AAV mediation

Get 3 the week age RCS rat, male and female are not limit, and are divided into 3 groups at random, respectively at subretinal injection normal saline (2ul), AAV ₂-CNTF 1 * 10 ¹⁰(2ul) individual virion, AAV ₂-CNTF 1 * 10 ¹⁰Individual virion+Ara-c 0.001 μ g (2ul altogether).Get eyeball after the 5 all paraformaldehyde perfusions of injection back, fixing behind 5% glacial acetic acid, paraffin embedding, section is observed in HE dyeing back.The OLYMPAS microscopically is observed counting RCS rat outer nuclear layer cell quantity.Only surplus one deck of subretinal injection normal saline group outer nuclear layer cell even disappearance, simple injection of AAV ₂The nearly 3-4 layer of-CNTF group outer nuclear layer cell number of plies, use in conjunction AAV ₂The outer nuclear layer cell number of plies is at the 5-6 layer when-CNTF and Ara-c 1 μ g, and the p value is all less than 0.05 between three groups, and difference has statistical significance.See Fig. 9.

Claims (17)

1. the compound formulation of cytosine arabinoside and adeno-associated virus comprises active component and substrate, it is characterized in that described active component is following material:

(1) antitumor drug cytosine arabinoside, (2) adeno-associated virus,

The consumption of described adeno-associated virus is 1 * 10 ⁹～1 * 10 ¹³AAV virion, the consumption of described cytosine arabinoside are 0.001～10 μ g.

2. compound formulation according to claim 1 is characterized in that described adeno-associated virus is AAV1, AAV2, AAV5, AAV3, AAV4, AAV6, AAV7 or AAV8.

3. compound formulation according to claim 1 is characterized in that the substrate in the described compound formulation is water for injection, normal saline, phosphate buffer or cell culture medium.

4. compound formulation according to claim 1 is characterized in that described preparation is lyophilized form or liquid form.

5. compound formulation according to claim 1 is characterized in that its administering mode adopts local injection or intravenous injection mode.

6. compound formulation according to claim 5 is characterized in that described administering mode is that cytosine arabinoside mixes the back with the form of mixtures administration with adeno-associated virus, or gives cytosine arabinoside and adeno-associated virus respectively.

7. compound formulation according to claim 6, it is characterized in that the described administering mode that gives cytosine arabinoside and adeno-associated virus respectively is: inject cytosine arabinoside and adeno-associated virus respectively in the part, or while intravenous injection cytosine arabinoside and adeno-associated virus, or local injection adeno-associated virus or intravenous injection cytosine arabinoside.

8. compound formulation according to claim 6, it is characterized in that in the described administering mode, time of administration is to inject cytosine arabinoside and then injection adeno-associated virus in advance in proper order, or injects cytosine arabinoside and adeno-associated virus simultaneously, or injects cytosine arabinoside again behind the injection adeno-associated virus earlier.

9. the described compound formulation of claim 1 improves gland relative virus mediated gene transfer efficient, expression and the persistent period and/or improves purposes in the aav gene therapeutic effect medicine in preparation.

10. by the purposes of claim 9, it is characterized in that described purposes is the gene therapy of preparation oculopathy, heredopathia gene therapy, malignant tumor gene therapy, treats various types of arthritis, treats various types of heart diseases, treats the various metabolic diseases of liver and/or the purposes in treatment muscle or moving back property of the nervous system change medicine.

11. purposes according to claim 10 is characterized in that wherein oculopathy gene therapy, its dosage is 0.001-1 μ g cytosine arabinoside; 1 * 10 ⁹-1 * 10 ¹²The AAV virion; Adopt subretinal injection, vitreous body intracavitary administration or injected into anterior chambers mode.

12. purposes according to claim 10 is characterized in that wherein heredopathia gene therapy, its dosage is 0.01-10 μ g cytosine arabinoside; 1 * 10 ¹⁰-1 * 10 ¹³The AAV virion; Adopt intramuscular injection; Lumbar injection or in the conduit liver, in the kidney reperfusion mode.

13. purposes according to claim 10 is characterized in that wherein malignant tumor gene therapy, its dosage is 0.01-10 μ g cytosine arabinoside; 1 * 10 ¹⁰-1 * 10 ¹³The AAV virion; Adopt injection or lumbar injection mode in the tumor.

14. purposes according to claim 10 is characterized in that wherein treating various types of arthritis, its dosage is 0.001-1 μ g cytosine arabinoside; 1 * 10 ¹⁰-1 * 10 ¹²The AAV virion; Adopt in the articular cavity or its surrounding injection mode.

15. purposes according to claim 10 is characterized in that wherein treating various types of heart diseases, its dosage is 0.01-10 μ g cytosine arabinoside; 1 * 10 ¹⁰-1 * 10 ¹³The AAV virion; Employing is reperfusion mode in the conduit heart.

16. purposes according to claim 10 is characterized in that wherein treating the various metabolic diseases of liver, its dosage is 0.01-10 μ g cytosine arabinoside; 1 * 10 ¹⁰-1 * 10 ¹³The AAV virion; Employing is through conduit liver perfusion mode.

17. purposes according to claim 10 is characterized in that wherein treating muscle or the change of moving back property of nervous system, its dosage is 0.01-10 μ g cytosine arabinoside; 1 * 10 ¹⁰-1 * 10 ¹³The AAV virion; Adopt injection system in the tumor.

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