CN1252006A - Photodynamic therapy generated oxidative stress for temporal and selective expression of heterologous genes - Google Patents
Photodynamic therapy generated oxidative stress for temporal and selective expression of heterologous genes Download PDFInfo
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Abstract
The present invention describes a method for the selection and temporal expression of heterologous genes. The invention consists of methods to obtain selective and temporal expression of heterologous genes in target tissues. Promoters inducible by photodynamic therapy or heat are used to express genes of interest under conditions of heating or Photodynamic Therapy-(PDT-) induced oxidative stress. Selective and temporal expression of heterologous genes (such as cytokines, toxins, tumor suppressor genes, antisense molecules and anti-angiogenic factors) are of significant therapeutic benefit in the treatment of tumors, vascular proliferation and tissue hypertrophy. Gene therapy targeted by laser induced heating, other heating sources (such as microwave, ultrasound or radiofrequency induced currents), or PDT enhances treatment effectiveness by inducing expression of therapeutic genes in a controlled and localized manner.
Description
Background of invention
The present invention has partly utilized the fund that obtains by NIH's delegation to be accomplished.Therefore, federal government enjoys certain right in the present invention.
Invention field
The present invention generally relates to the clinical treatment of entity tumor.More particularly, the present invention relates to controllable limitation mode inductive treatment expression of gene.
The description of correlative technology field
Photodynamic therapy (PDT) is a kind of clinical treatment method (Fisher, people such as A.M.R., Laser Surgery Medicine 17:2-31 (1995) of solid malignant; Marcus, S.L. and Dugan, M.H., LaserSurgery Medicine, 12:318-24 (1992); And Henderson, B.W. and Dougherty, T.J., Photochem.Photobiol., 55:931-48 (1992)).Location character and the photochemistry generation reactive species of oxygen of photosensitizer in tumor tissues combined with the accurate transfer of the light of laser instrument generation, produced and given the scheme of killing tumor promotion in effective part (Henderson, B.W., Photodermatology 6:200-11 (1989); And Wilson, B.C. and Jeeves, W.P.PHOTOMEDICINE (Ben Hur, E. and I.Rosenthal edit) 2:127-67 (1987)).A kind of hematoporphyrin derivative that is called photofrin (photofrin) is the photosensitizer that is used for most of clinical experiments.The PDT of photofrin mediation has obtained the FDA approval recently and has been used for the treatment of the esophageal carcinoma, and this chemical compound also obtains authorities' approval in Canada, Holland and Japan.PDT has shown goodish prospect in treatment bronchus, bladder, skin, head/neck and cervical cancer and non-malignant tumors disease (as psoriasis and the degeneration of macula relevant with the age).
For the exploitation of the new photosensitizer of the pharmacology that shows improvement, photochemistry and photophysics character and the interest also growing (Gomer, C.J.Yearly Review:Photochem.Photobiol., 54:1093-1107 (1991)) of clinical evaluation.The second filial generation photosensitizer that is standing clinical experiment comprises this C.I. Natural Red 8 of stannum (SnET2), list-1-asparaginyl group chlorin e 6 (NPe6), benzoporphyrin derivative (BPD), meso-four-(hydroxy phenyl) chlorin (mTHPC) and 5-amino-laevulic acid (ALA).These chemical compounds show many think with photofrin quite or than its better character, comprise chemical purity, photonic absorption increase under longer wavelength, the stagnation of improved tumor tissue growth, from normal structure, remove rapidly, the high light quantum output of reactive species of oxygen and minimum dark toxicity (dark toxicity) (Dougherty, T.J., Photochem.Photobiol.58:895-900 (1993); Moan, J. and Berg, K., Photochem.Photobiol.55:93 1-48 (1992); And Kessel, Photochem.Photobiol., 50:169-74 (1989)).ALA is the metabolic precursor thereof of endogenous protoporphyrin IX.
The reactive species of oxygen that photochemistry produces during PDT can damage (Moan, J., Photochem.Photobiol.43:681-90 (1986)) to lipid, albumen and nucleic acid.In fact, in many normal and malignant cells, for the injury of cell membrane, organelle, enzyme and DNA (Prinze, C. etc., Biochim.Biophys.Acta., 1038:152-57 (1990) all on the books; And Hillf, R., etc., Cancer Res., 44:1483-88 (1984)).Oxidation destruction for all these positions is enough to cause cell death.Yet the inductive death of the PDT target of cutter system and effect really waits to illustrate.
PDT induces early stage response gene (c-fos, c-jun, c-myc, egr-1) and the stress protein expression of gene (Luna that belongs to heat shock protein (HSP), glucose regulated protein (GRP) and ferroprotoporphyrin oxygenase family, M.C. etc., Cancer Res., 54:1374-80 (1990); Gomer, C.J., etc., Photochem.Photobiol., 53:275-279 (1991); Gomer, C.J., etc., Cancer Res., 51:6574-79 (1991); And Gomer, C.J., etc., Cancer Res. (publishing) (1997)).Inducing of stress gene is in transcriptional level, but is unknown but be responsible on specific target and/or the signal transduction path inducing the information of stress response gene expression.PDT has also induced apoptosis, and it seems that apoptosis comprised signal transduction pathway (Agarwal, M.L. etc., Cancer Res., the 51:5993-96 (1991) that begins from cell membrane; He, X.Y., etc., Photochem.Photobiol.59:468-73 (1994) and Zaidi, S.I.A., etc., Photochem.Photobiol.58:771-76 (1993)).Observe in photoactivated cell that distinctive dna fragmentationization, chromatin concentrate and a kind of activation of composing type endogenous nucleic acid enzyme.Apoptosis also has been considered to early stage behavior in PDT-handles body in the tumor.
Cell has different stress responses.These reply each maybe stress cause by one or more environmental hazards, this inductive main consequence is to have increased the stress tolerance level.It is to have a kind of of feature most in these stress responses to reply that heat shock is replied.According to 1962, be exposed to and make the new puff (puff) of appearance on fruit bat (Drosophila busckii) salivary gland under non-lethal high-temperature (heat shock) and some chemical substance.These apparent puffs under optical microscope represent that but the heat shock induced gene has high transcriptional activity, and the RNA of the messenger RNA of generation heat shock protein is synthetic.Recently, show, in gene expression, similar significant change occurs to high vertebrate vast biology because of heat shock from antibacterial.
Replying of heat shock not only induced the gene of heat shock protein (HSP) consumingly, but also checked most of genes of expressing before this is induced.The mode that the albumen synthesis mechanism of genetic transcription mode and cell selects mRNA to translate all has significant change.
The variation of transcribing that heat shock causes is owing to there is the heat shock element to exist in the heat shock gene promoter.This element is the required DNA sequence of specificity inducible transcription in heat shock is replied.In colibacillary hot inducible promoters, the consensus sequence of heat shock element is CTGCCACCC, is positioned at nucleotide-44 with respect to transcriptional start site to-36.In eukaryotic hot inducible promoters, it adjoins the repetitive sequence of 5bp sequence NGAAN, arranges the upstream that is positioned at the TATA box element with arbitrary orientation.
For experiment purpose, heat shock is replied to rise with temperature usually and is induced.Inductive optimum temperature depends on (animal) kind, but it is higher 1 or 2 ℃ than the maximum temperature that allows growth usually.Equally, reply normally temporary.
Except heat stress, synthetic other inducer in addition of heat shock protein.They comprise several potential cytotoxicity chemical substance and the physiological status that may produce high mars free radical.These inducers may can both at high temperature cause accumulating in the cell albumen of unusual or partial denaturation.
The molecular weight of glucose regulated protein (being GRP) is 78,000 and 94,000, and they have and the homologous sequence of heat shock protein.The albumen of GRP family by the change of glucose hunger, anoxia, cellular calcium matter, be exposed under inhibitor or the glycosylation and by the oxidative stress co-induction of PDT-mediation (Gomer, C.J. is etc., CancerRes.51:6574-79 (1991); And Li, L-J., etc., J.Cell Physiol.153:575-82 (1992)).Molecular weight is that the sequence of 78,000 GRP is identical with the sequence of immunoglobulin heavy chain binding protein, and GRP78 and GRP94 are arranged in endoplasmic reticulum (ER).GRP78 is with newborn, excretory and transmembrane protein is instantaneous combines, with unusual folding or finished albumen permanent bond among the ER.Process when disturbed when the albumen among the ER, think GRP78 when cellular stress and stress after protecting function is arranged.Lee etc. show, make the grp78 transcriptional activation can be used for directed gene therapy by tumor hypoxia.The grp78 promoter of the truncate that most of base components are removed demonstrates and has promoted the high level expression of reporter gene in the hypoxia mouse tumor (Gazit, G. is etc., Cancer Res.55:1660-63 (1995)) effectively.
The present invention confirmed the oxidative stress of PDT-mediation be stress protein (those albumen that particularly belong to heat shock protein Bai nationality) transcribe transducer by force.Therefore, the present invention relates to a kind of directed gene therapy method, this method uses the recombination to construct thing with HSP-or GRP-inducible promoters to promote the high-level local expression of cytotoxin or immunomodulator, kills function of tumor to strengthen PDT.
Therefore, also there is not to produce the method for high-level local expression cytotoxin or immunomodulator in the prior art.The present invention has satisfied this demand and the hope of this area since the midium or long term.
Summary of the invention
An object of the present invention is to provide the local new method of killing tumor (or angiogenesis inhibitor) character of a kind of raising PDT, the molecular switch that this method is expressed on room and time as gene with these therapeutic modalities, with the direct effect that strengthens PDT or with this effect synergism.The present invention is better than present existing strategy, existing strategy is attempted to send the treatment of localization cytotoxic gene with composing type effect promoter, and promptly the viral system has adopted genetic transcription is limited in selected intracellular tissue specificity receptor or tissue specificity reinforce.Promoter with the composing type effect can not be from time adjusted gene expression, because the cytotoxic gene product has the higher baseline expression in normal cell and the tissue.The present invention's (but induced gene treatment and PDT or heating are combined) has strengthened the control of locality tumor, and does not increase normal tissue toxicity (increasing treatment benefits).The transcriptional activation of PDT or hot inducible promoter be controlled in the given tissue volume and special time in.Therefore, this step had both been utilized PDT or thermoinducible direct cytotoxicity, had utilized the targeting potentiality of PDT or heat to come the adjusting of inducing cytotoxic genetic transcription on room and time again.
PDT or hot target gene therapy adopt the construction that contains PDT or hot inducible promoter to strengthen local cells toxicity, this promoter in coding PDT or thermal treatment zone by the upstream of the cDNA of the heterologous gene of transcriptional activation.People such as Weichselbaum have reported the similar program (Weichselbaum, R.R. wait the people, Cancer Res.54:4266-69 (1994)) that adopts ionizing radiation.
An object of the present invention is to provide the method for target tissue in a kind of temporary Local treatment individuality, this method comprises the following steps: to give described individuality a kind of expression vector, wherein said carrier is the express therapeutic heterologous gene under photodynamic therapy or the control of heating inducible promoter, and described target tissue is exposed under described photodynamic therapy or the heating.The various examples of this specific purpose of the present invention comprise by heat energy laser, microwave, ultrasound wave or rf wave and come heat production.
Another object of the present invention provides the method for target tissue in a kind of temporary Local treatment individuality, and this method comprises the following steps: to give described individual photosensitizer and makes photodynamic therapy; Give described individuality a kind of expression vector, wherein said carrier is the express therapeutic heterologous gene under the control of photodynamic therapy inducible promoter; Described photosensitizer and described expression vector are absorbed by described target tissue; With described target tissue is exposed under the illumination, the combining of wherein said illumination and described photosensitizer can produce reactive species of oxygen and induce and can be caused described therapeutic allogeneic gene expression by the inductive described promoter of photodynamic therapy.A particular instance of this purpose of the present invention comprises: photosensitizer is selected from photofrin, this C.I. Natural Red 8 of stannum, list-1-asparaginyl group chlorin e 6, benzoporphyrin derivative, meso-four-(hydroxy phenyl) chlorin and 5-amino-laevulic acid.
The various examples of two purposes of the inventive method comprise whole body or topical administration carrier.The preferred embodiments of carrier of the present invention comprises that described carrier is examples such as retroviral vector, adeno-associated virus vector or liposome dna vector.
The good especially example of two purposes of the present invention is that described promoter is heat shock protein (hsp) promoter or glucose regulated protein promoter.Better example comprises that wherein said heterologous gene is an immunomodulatory gene, particularly cytokine.In addition, example comprises that wherein said heterologous gene is tumor suppressor gene, a kind of antisense DNA or a kind of angiogenesis inhibitor gene.
In two any purposes of purpose of the present invention, preferable example comprises that wherein said target tissue is the unusual zone of tumor or tissue growth.
Other aspects, features and advantages of the present invention can obviously be found out from the following description of preferred embodiments of the present invention.The purpose that provides these examples is for open.
The accompanying drawing summary
Appended accompanying drawing has been included in herein, so just can know and at length understand above-mentioned feature of the present invention, advantage and purpose.These accompanying drawings have constituted the part of description.Yet it should be noted that accompanying drawing described preferred embodiments of the present invention, should not think that these accompanying drawings have limited scope of the present invention.Fig. 1-5 shown beta galactosidase (selectivity and transient expression in the RIF HB-3 cell of reporter gene under being exposed to heat or PDT of β-gal):
Fig. 1: the betagalactosidase activity (recording as substrate) after heat (45 ℃) is handled 1.5,10,20,30 and 40 minutes in the RIF HB-3 cell of transfection with ONPG (neighbour-Nitrobenzol-galactopyranose).High heat effect was collected sample after 6 hours.Each data point is represented with the mean+/-standard error of three independent experiments at least.
Fig. 2.(dosage is 600-5400 joule/cm through the PDT of NPe6-mediation
2) handle the betagalactosidase activity (recording as substrate) in the RIF HB-3 cell of back with ONPG.Illumination was collected sample after 6 hours.Each data point is represented with the mean+/-standard error of three independent experiments at least.
Fig. 3.The kinetics collection of illustrative plates (recording as substrate) that beta galactosidase after 45 ℃ are handled 20 minutes in the RIF HB-3 cell is expressed with ONPG.Each data point is represented with the mean+/-standard error of three independent experiments at least.Detect enzymatic activity (19 milliunits/milligram albumen) after 3 hours at heat treatment.When 6 hours and 12 hours, β-gal activity reaches peak value (100 and 125 milliunits/milligram albumen) under heat exposes.
Fig. 4.(light dosage is 3000 joules/cm through the NPe6-PDT processing
2) beta galactosidase in the RIF HB-3 cell of the back kinetics collection of illustrative plates (recording as substrate with ONPG) of expressing.Each data point is represented with the mean+/-standard error of three independent experiments at least.After handling 3 hours, detect the minimum level of enzymatic activity.Behind the PDT of NPe6 mediation between 6 hours and 12 hours, reach peak activity.
Fig. 5.The betagalactosidase activity (recording as substrate) of growth and the RIF HB-3 tumor handled with heat or NPe6-PDT in C3H/HeJ mouse with ONPG.Heat treatment is included in 45 ℃ and exposes 20 minutes down.Three kinds of illumination doses of test PDT.Each bar figure represents the different mean+/-standard error of measuring 5 times.Finding behind high heat treatment has maximum to reply (8.25 milliunits/milligram albumen).The inductive β of NPe6-PDT-gal enzymatic activity is between 1.08-3.48 milliunit/milligram albumen.But what control tumor or the tumor of only handling with illumination or NPe6 obtained is the record (result who does PDT processing acquisition with PH and Sn ET2 is suitable) that does not have detection level.
Fig. 6 to 12 shown chloramphenicol acetyltransferase (CAT) reporter gene under being exposed to heat or PDT RIF HC-2 cell and selectivity and transient expression in the RIF RHC-7 cell.
Fig. 6.CAT activity after heat (45 ℃) is handled 1.5,10,20 and 40 minutes in the RIF HC-2 cell of transfection.High heat effect was collected sample after 24 hours.The radioactivity of counting gained TLC flat board is calculated the amount that changes acetylation chloromycetin from chloromycetin into.
Fig. 7.CAT activity after heat (45 ℃) is handled 1.5,10,20 and 40 minutes in the RIF RHC-7 cell of transfection.High heat effect was collected sample after 24 hours.The radioactivity of counting gained TLC flat board is calculated the amount that changes acetylation chloromycetin from chloromycetin into.
Fig. 8.(dosage range is 600-5400 joule/cm through the PDT of NPe6 mediation
2) handle the CAT activity of the RIFHC-2 cell of back transfection.Illumination was collected sample after 24 hours.The radioactivity of counting gained TLC flat board is calculated the amount that chloromycetin changes acetylation chloromycetin into.
Fig. 9.Through the active kinetics collection of illustrative plates of the CAT of 45 ℃ of processing RIF HC-2 cell of transfection after 20 minutes.Collect sample after heat effect 3-48 hour.The radioactivity of counting gained TLC flat board is calculated the amount that chloromycetin changes acetylation chloromycetin into.
Figure 10.Through the active kinetics collection of illustrative plates of the CAT of 45 ℃ of processing RIF RHC-7 cell of transfection after 20 minutes.Collect sample after heat effect 3-48 hour.The radioactivity of counting gained TLC flat board is calculated the amount that changes acetylation chloromycetin from chloromycetin into.
Figure 11.Growth and heat the RIFHC-2 cell of tumor and the CAT activity of RIF HC-2 tumor in C3H/HeJ mouse with heating in water bath cell or induced with laser.Heat treatment comprises that 45 ℃ were handled 20 minutes.Heat collecting cell culture sample after 24 hours, heat and collect tumor sample after 3,6 or 24 hours.The record that control tumor obtains is that the CAT activity level can not detect.
Figure 12.Growth and with induced with laser heating, the RIF HC-2 that NPe6-PDT or PH-PDT handle and the CAT activity of RIF RHC-7 tumor in C3H/HeJ mouse.Heat treatment comprises 45 ℃ of effects 20 minutes.Control tumor, or the tumor of only handling with illumination, NPe6 or PH, but the record of acquisition is not have detection level.
Detailed Description Of The Invention
It will be understood by those skilled in the art that in that do not break away from obviously can be to disclosed herein under the spirit and scope of the invention The present invention does various replacements and modification.
Term used herein " photodynamic therapy " or " PDT " refer to give tumor-localizing light in general After quick dose with visible light (usually by non-heat production laser produce) processing entities tumour (see Fisher, A.M.R., etc., Laser Surgery Medicine 17:2-31 (1995); Marcus, S.L. and Dugan, M.H., Laser Surgery Medicine, 12:318-24 (1992); And Henderson, B.W. and Dougherty, T.J., Photochem. Photobiol., 55:931-48 (1992)). The photochemical reaction that sensitising agent and induced with laser produce has produced reactivity Oxygen kind (such as simple substance oxygen), thus induce the oxidation of subcellular fraction target (film, organelle, enzyme and DNA) broken Bad effect. Clinically, PDT be used for treating various entity tumors (oesophagus, bronchus, bladder, brain, eye, Head/neck, skin and cervix) and the non-malignant tumors disease (such as the macular degeneration relevant with the age and ox-hide Tinea). In PDT, adopted various sensitising agents, comprise photofrin (PH), this purpurine of tin (SnET2), single-1-asparaginyl group chlorin e 6 (NPe6), benzoporphyrin derivative (BPD), meso-four-(hydroxy phenyl) Chlorin (mTHPC) and 5-ALA (ALA).
Term used herein " heat shock gene " refers to the base of transcribing with high level in the temperature that raises is replied Cause.
Term used herein " CAT " or " CAT test " refer to measure in the eukaryotic promoter sequence body The test of effect. The CAT of CAT gene code by one in two hydroxyls that make antibiotic chloramphenicol or Two acetylations come this medicine of deactivation. Because the not synthetic CAT of eukaryotic, so this gene opens with performing an analysis The reporter of mover is especially in mammalian cell. CAT uses according to the method for thin-layer chromatography and surveys Fixed, the method general [14C] chloramphenicol and the acetylation inactivation derivative branch that only when the CAT enzyme exists, just synthesizes Open.
Term used herein " β-gal " or " beta galactosidase test " refer to for measuring the eukaryotic promoter order The test of row vivo effect. Beta galactosidase is that a kind of terminal irreducibility beta galactose residue of hydrolysis that passes through is incited somebody to action Beta galactose glycosides (such as lactose) is hydrolyzed into the enzyme of component sugars. In the research of promoter effect, use Escherichia coli LacZ Gene merges so that lacZ exists in the translation box between gene of interest and LacZ gene as reporter Studied the lower expression of promoter control. Measure beta galactose with neighbour-nitrobenzene-galactopyranose as substrate then The glycosides enzymatic activity just can be measured the activity of promoter.
Term used herein " glucose regulated protein " or " GRP " refer to by glucose hunger, anoxic, The change of cellular calcium matter, be exposed under inhibitor or the glycosylation and by the oxidative stress condition association of PDT-mediation The protein families of isogeneous induction.
Term used herein " tumor suppressor gene " is pointed out to participating in the normal control of Growth of Cells and division not Genoid with the aspect. The common trait of these genes is that their inactivation (usually passing through mode of inheritance) can be led Cause the generation tumour.
Term used herein " immunomodulatory gene " refers to a kind of gene, and the expression metering needle of this gene is to special The immunoreactive process of sexual stimulus or multiple stimulation. Example comprise interleukin-4, interleukin 10, Tumor necrosis factor α etc.
Term " cytokine " used herein " refer to that it can influence and instruct the process to the immunne response of differential stimulus by a kind of little protein of immune system cell generation.
Term used herein " angiogenesis inhibitor gene " refers to the coding minimizing or stops angiopoietic proteinic gene.
Term used herein " antisense DNA " refers to the DNA of encoding antisense RNA.These antisense RNAs have the ability that forms the RNA-RNA duplex with natural " justice is arranged " mRNA transcript of its gene, thereby have stoped the translation of " justice is arranged " RNA.Antisense RNA provides a kind of mode of deactivation expression of specific gene, and it is applicable to simple and complicated eukaryotic cell.
According to the present invention, may adopt conventional molecular biology, microbiology and DNA recombinant technique known to those skilled in the art.These technology have in following document fully to be described, for example referring to Maniatis, and Fritsch﹠amp; Sambrook, " molecular cloning: laboratory manual " (1982); " dna clone: practical approach " I and II volume (D.N.Glover edits, 1985); " oligonucleotide is synthetic " (M.J.Gait edits, 1984); " nucleic acid hybridization " (B.D.Hames ﹠amp; S.J.Higgins edits, (1985)); " transcribe and translate " (B.D.Hames﹠amp; S.J.Higgins edits (1984)); " animal cell culture " (R.I.Freshney edits, (1986)); " immobilized cell and enzyme " (IRL Press, (1986)); B.Perbal, " molecular cloning practical guide " (1984).
Therefore, if occur at this paper, following term should have following definitions.
" carrier " is a kind of replicon, for example plasmid, phage or cosmid, and it can be connected with another DNA sections, connects sections and produces and duplicate thereby make.If mammalian receptors can tolerate this carrier that gives, claim that then this carrier is " receivable on the pharmacology ".If dosage has obvious effect on physiology, claim that then this preparation is to give with " treatment effective dose ".If the existence of reagent causes the mammalian receptors physiology to change, then said preparation has physiologic meaning.For example, in treatment during retroviral infection, the chemical compound that can reduce gradient of infection or reduce the physiological injury that is caused by infection will be considered to be in treatment and go up effectively.
" dna molecular " refers to the polymer form with the deoxyribonucleotide of single stranded form or double-stranded spiral form (adenine, guanine, thymus pyrimidine or cytosine).This term only refers to the elementary and secondary structure of molecule, and is not limited to any three grades of specific forms.Therefore, this term comprises the double-stranded DNA of especially finding in linear DNA molecule (as restriction fragment), virus, plasmid and chromosome.When the structure of this paper is discussed,, only provided along DNA non-transcribed chain (being the homologous chain of sequence and mRNA) 5 ' to the sequence of 3 ' direction according to routine.
" replication origin " refers to participate in synthetic those DNA sequence of DNA.
DNA " coded sequence " is when placing suitable regulating and controlling sequence control can transcribe and translate into the double chain DNA sequence of polypeptide down in vivo.The border of coded sequence is determined by 5 ' (amino) end start codon and 3 ' (carboxyl) end translation stop codon.Coded sequence can include but is not limited to the cDNA of protokaryon sequence, eukaryotic mrna, from the genomic dna sequence of eucaryon (for example mammal) DNA, or even synthetic DNA sequence.Polyadenylation signal and transcription terminator are usually located at 3 of coded sequence ' end.
Transcribing and translate control sequence is the DNA regulating and controlling sequence, for example the promoter that need provide at host cell inner expression for coded sequence, enhancer, polyadenylation signal, terminator etc.
" promoter sequence " is and to cause the DNA regulation and control zone that downstream (3 ' direction) coded sequence is transcribed in conjunction with the intracellular rna polymerase.For the present invention is defined, 3 of promoter sequence ' end links to each other with transcriptional start site, and upstream (5 ' direction) but extend the initiation comprised minimum number and be higher than the background detection level and transcribe required base or element.In promoter sequence, can find a transcriptional start site (s1 nuclease collection of illustrative plates commonly used is determined), and be responsible for protein binding domain (consensus sequence) in conjunction with RNA polymerase.Eukaryotic promoter (but not always) usually contains " TATA " box and " CAT " box.Prokaryotic promoter except have-10 and-35 consensus sequences also contain the Shine-Dalgarno sequence.Available various promoter drives carrier.
" expression control sequenc " is control and regulates the DNA sequence that another DNA sequence is transcribed and translated.When RNA polymerase is transcribed into mRNA with coded sequence, when mRNA was translated into the coded sequence encoded protein then, coded sequence was in " under the control " of transcribing and translating control sequence.
Before coded sequence, can comprise " signal sequence ".This sequential coding the signal peptide that links to each other with polypeptide N end, this signal peptide and host cell are got in touch to instruct polypeptide to transfer to cell surface or polypeptide is secreted in the substrate, before albumen left cell, this signal peptide was downcut by host cell.Can find that signal sequence is associated with protokaryon and eukaryotic various native protein.
The new method of tumor (or angiogenesis inhibitor) performance is killed in the part that the present invention relates to a kind of enhancing PDT (or heating), the molecular switch that this method spatially go up to be expressed with the time as gene with these therapeutic modalities, with the direct effect that strengthens PDT or with this effect synergism.The present invention is better than present existing strategy, and existing strategy is attempted to send the treatment of localization cytotoxic gene with composing type effect promoter, and promptly the viral system has adopted genetic transcription is limited in selected intracellular tissue specificity receptor or tissue specificity reinforce.Promoter with the composing type effect can not be from time adjusted gene expression, because the cytotoxic gene product in normal cell and the tissue has the higher baseline expression.The present invention's (but associating induced gene treatment and PDT or heating) has strengthened the part control to tumor, and does not increase the toxicity (having increased the therapeutic benefit) to normal structure.The transcriptional activation of PDT or hot inducible promoter be controlled in the given tissue volume and special time in.Therefore, this step had both been utilized PDT or thermoinducible direct cytotoxicity, had utilized the targeting potentiality of PDT or heat to come the adjusting of inducing cytotoxic genetic transcription on room and time again.
PDT or hot target gene therapy adopt the construction that contains PDT or hot inducible promoter to strengthen local cells toxicity, this promoter in coding PDT or thermal treatment zone by the upstream of the cDNA of the heterologous gene of transcriptional activation.
An object of the present invention is to provide the method for target tissue in a kind of temporary Local treatment individuality, this method comprises the following steps: to give described individuality a kind of expression vector, wherein said carrier is at photodynamic therapy or add express therapeutic heterologous gene under the control of heat inducible promoter, and described target tissue is exposed under described photodynamic therapy or the heat.
For the application of gene therapy, the those of ordinary skill of molecular biology and oncology need not too much experiment just can determine the suitable dose and the route of administration that adopt in the new method of the present invention.
The invention describes the method for selectivity and transient expression's heterologous gene.The present invention includes two kinds of methods and make the cell of incubation growth and heterologous gene selectivity and the transient expression in the interior cell (as the entity tumor) of growing of body.Under induced with laser heating or the inductive oxidative stress of photodynamic therapy (PDT), express interested gene with HUMAN HEAT SHOCK PROTEINS Hhsp HSP70 (hsp) promoter.The selectivity of heterologous gene (as cytokine, toxin, tumor suppressor gene, antisense molecule and anti-angiogenesis) and transient expression have the obvious treatment effect in treatment tumor, blood vessel hyperplasia and tissue hypertrophy.The gene therapy of induced with laser heating, other heating source (as the electric current of microwave, ultrasonic or radio-frequency induced) targeting is by having strengthened therapeutic effect with in check localization mode inductive treatment expression of gene.
Providing the following example purpose is to describe each example of the present invention, but is not to limit the present invention by any way:
Embodiment 1
Cell line:
With radiation-induced fibrosarcoma cell (RIF) as the parental generation control cells, and as recipient cell with hsp promoter reporter gene construction transfection or transduction.Set up three hsp promoter reporter gene cell lines and test.Two cell line expression plasmid transfections, but a cell line is transduceed with the abduction delivering retroviral vector.Make StressGen Biotech Corp. plasmid p2500-CAT (chloramphenicol acetyltransferase (CAT) is provided under the control of hsp70 promoter but abduction delivering) and p173OR (under the control of hsp70 promoter, provide beta galactosidase (but abduction delivering of β-gal)), and be grown in super competence (supercompetent) escherichia coli from the plasmid pMC1Neo (constitutive expression of neomycin resistance gene is provided under the control of thymidine kinase promoter) of Stratagene, behind alkaline lysis and purification, separate acquisition.With calcium phosphate DNA sedimentation with each StreeGen expression plasmid and pMC1Neo (ratio is 5: 1) cotransfection in the RIF-1 cell.Cell is grown, with clone's ring picking institute DCRP in 600 μ g/ml G418.Make G418 resistance clone expansion (expand), and measure β-gal or CAT activity as positive derivant with heating.Behind PDT, test the activity of positive colony then.β-gal or the active indivedual RIF-1 clones of CAT are expressed in acquisition behind PDT.
Being used for 3 cell lines that proof heating or PDT can be used as the switch of " startup " allos reporter gene is:
1.RIF the HC-2 cell, it expresses CAT under the control of 2.5kbhsp promoter;
2.RIF the HB-3 cell, it expresses beta galactosidase under the control of 2.5kb hsp promoter; With
3.RIF RHC-7 cell, it obtains by the modified GINA retroviral vector (contain the 300bphsp promoter, promoter links to each other from phosphotransferase (neo resistance) gene of 5 ' LTR with chloramphenicol acetyl transferasegene and constitutive expression) of transduction.By in G-418, growing, set up stable transfectant, screen the activity of the chloramphenicol acetyltransferase of heating induction expression then.
All these three cell lines all have the expression vector of stable integration, and the flank that all cells system is injected C3H/HeJ mouse is to produce entity fibrosarcoma lump.
Treatment conditions:
The high heat treatment of cultured cells:
Preceding 24 hours of heat treatment is with cell (2 * 10
6) be inoculated in the T-75 bottle.Make cellular exposure in 45 ℃ temperature controlled water bath, transmit heat.
The photosensitizer of cultured cells is handled (PDT):
Detect three kinds of different photosensitizer: photofrin (PH), list-1-asparaginyl group chlorin e 6 (NPe6), this C.I. Natural Red 8 of stannum (SnET2).With cell (2 * 10
6) be inoculated in the 100mm Petri dish 24 hours, cultivated 16 hours with a kind of photosensitizer subsequently.It is 25 μ g/ml that the photosensitizer of PH and Npe6 is cultivated concentration, and SnET2 is 0.75 μ g/ml.Cell was cultivated 16 hours in the dark place in the culture medium that contains 5% serum with photosensitizer.Then cell was cleaned 30 minutes in the culture medium that contains 15% serum, be exposed to again under the HONGGUANG of classification (graded) dosage.For Npe6 and SnET2, the employing close rate is 2mW/cm
2The 664nm diode laser.For the cell of cultivating together with PH, the close rate that adopts the 30W fluorescent lamp by one group of parallel connection to produce is 0.35mW/cm
2Wide range (570-650nm) HONGGUANG.
Be grown in the heat treated of the intravital tumor of mice:
The hyperpyrexia of tumor (recording diameter is 6-7mm) exposes 20 minutes under handling and being included in 44.5-45 ℃.These temperature are by being 270mW/cm with transmitting power density
2The diode laser irradiation tumor of 810nm laser realize.
Be grown in the PDT treatment of the intravital tumor of mice:
Photodynamic therapy (PDT) is handled tumor (recording diameter is 6-7mm) and is comprised the PH of intravenous injection 5mg/kg or the SnET2 of Npe2 or 1.5mg/kg.After giving medicine 4-5 hour (for Npe6) or 24 hours (for PH and Sn ET2), begin with non-heating laser irradiation tumor.For the PDT of PH mediation, adopt the HONGGUANG of 630nm, for the PDT of Sn ET2 and Npe6 mediation, adopt the light of 664nm.The illumination dose rate is 75mW/cm
2
Cell and organize beta galactosidase test:
Place 4 ℃ of microcentrifugal tubes centrifugal 2 minutes cell lysate ((Promega) obtaining) or tumor lysate (obtaining) by homogenization RIF HB-3 solid tumor in containing the reporter lysis buffer of Polytron by cultivating RIF HB-3 cell and reporter lysis buffer (Reporter LysisBuffer).Get 150 μ l supernatant, add the Assay 2X buffer (Promega) that 150 μ l contain substrate ONPG (o-nitrophenyl-B-D-galactose pyranoside), cultivate reaction test in 3 hours betagalactosidase activity for 37 ℃.Add 500 μ l 1M sodium carbonate cessation reactions, read absorbance under the 420nm with spectrophotometer.Betagalactosidase activity is represented with milliunit/milligram albumen (albumen BIO-RAD albumen test determination).
Embodiment 4
Cell and organize CAT test:
For cell, make 10 μ g cell proteins (obtaining) and 35 μ l 1M TrisCl (pH7.8), 10 μ l 6.0mg/ml S-acetyl-coenzyme-As, 2.5 μ l C-14 chloromycetin (ICN Pharmaceuticals by the cell freeze thawing, Inc., CostaMesa, CA, catalog number (Cat.No.) are 12060) and water to be mixed to final volume be 75 μ l.Reactant mixture was cultivated 30 minutes in 37 ℃ of water-baths.Add 1 milliliter of ethyl acetate, eddy oscillating 1 minute, under maximal rate centrifugal 5 minutes, extracting went out acetylation chloromycetin from reactant mixture.Remove ethyl acetate layer and in speed change vacuum (speed vac) dry 45 minutes.In 30 μ l ethyl acetate, point sample is on silica gel thin-layer chromatography TLC plate with sample dissolution.Make the TLC flat board contain 200 milliliters of chloroforms: developed 45 minutes in the container of methanol (95: 5), air drying, and the exposure X-ray film spends the night.Downcut acetylation and not acetylizad point on the TLC plate, in scintillation counter, count, calculate the percent that CAT changes.
For tissue sample, CAT experimental evidence " molecular cloning " Sambrook, Fritsch, Maniatis, the method among the ColdSpring Harbor Laboratory Press (1989) is improved a little and is carried out.Make 65.5 μ g protein samples cultivate 10 minutes down with the deactivation deacetylase at 65 ℃.In every duplicate samples, add 50 μ l 1MTrisCl (pH7.8), 10 μ l 6.0mg/ml S-acetyl-coenzyme-As, 4.3 μ l C-14 chloromycetin (ICNPharmaceuticals, Inc., Costa Mesa, CA), and to add entry to final volume be 130 μ l.Cultivate sample 6 hours for 37 ℃, in sample, added the 6.0mg/ml S-acetyl-coenzyme-A solution of 5 μ l prepared fresh every 2 hours.Cultivate after 6 hours, in every duplicate samples, add 1 milliliter of ethyl acetate, eddy oscillating 1 minute, centrifugal 5 minutes, remove organic layer, air drying under the speed change vacuum adds 30 μ l ethyl acetate in every duplicate samples, with the sample point sample to the TLC flat board.Make the TLC flat board contain 200 milliliters of chloroforms: developed 45 minutes in the container of methanol (95: 5), the air drying and the x-ray film that exposes spend the night.Downcut acetylation and not acetylizad point on the TLC plate, in scintillation counter, count, calculate the percent that CAT changes.
Embodiment 5
PDT inducible promoter/TNF-alpha expression carrier:
Initial plasmid obtains from StressGen (p2500CAT that contains the hsp promoter) and ATCC (pUC-R10173 that contains complete TNF-α coded sequence).With HindIII and BamH1 digestion, remove the CAT gene of p2500CAT.Commercial adapter with New England Biolands is modified resulting end.With aforementioned annealed adapter 1105 (EcoR1-XmnI) and 1107 (HindIII-XmnI) the HindIII end is transformed into EcoRI.With adapter 1105 (EcoR1-XmnI) and 1 106 (BamH1-XmnI) the BamH1 end is transformed into EcoR1.After inserting connexon, carrier is linked to each other with the EcoR1 fragment of the pUC-R10173 of purification.With these cyclisation plasmid transformed competence colibacillus escherichia coli.Cut the micropreparation thing of selected bacterium colony with Ava1 and BglII, to determine the orientation of insert.Amplification has the bacterium colony of correct insert, with gained plasmid (pHspTNF) and pMC1Neo transfection together in the RIF cancerous cell.Separate the tumor cell line that acquisition contains the hsp promoter/TNF-alpha expression carrier pHspTNF of stable integration.
All patents mentioned in this description or publication have all illustrated one of ordinary skill in the art's of the present invention level.And these patents and publication are all included this paper in as a reference, as specifically describe separately each publication include in this paper for referencial use.
Those skilled in the art are readily understood that the present invention is fit to be used for realize and obtain target mentioned in this article, purpose and advantage very much, and the intrinsic target of this paper, purpose and advantage.Embodiment described herein and method, step, processing, molecule and concrete chemical compound are the representatives of preferred embodiments, and the property enumerated does not limit the scope of the invention.Those skilled in the art include among the determined thinking of the present invention of claim scope its variation of doing and other application.
Claims (18)
1. the method for a reinforcing gene expression in individual body inner target tissue, this method comprises the following steps:
Expression vector is delivered to the target tissue of described individuality, and wherein said carrier is the express therapeutic heterologous gene under the control of a promoter, and this promoter can be by photodynamic therapy or heating induction, and wherein said gene is allogenic for described promoter; With
Described target tissue is exposed under described photodynamic therapy or the heating, and wherein said photodynamic therapy or heat have strengthened described expression of gene.
2. method according to claim 1, wherein general gives described carrier.
3. method according to claim 1, the wherein described carrier of topical administration.
4. method according to claim 1, wherein said carrier are retroviral vector, adeno-associated virus vector or liposome dna vector.
5. method according to claim 1, wherein said promoter are heat shock protein hsp promoter or glucose regulated protein promoter.
6. method according to claim 1, wherein said heterologous gene are immunomodulatory gene, tumor suppressor gene, antisense DNA or angiogenesis inhibitor gene.
7. method according to claim 6, wherein said immunomodulatory gene is a cytokine gene.
8. method according to claim 1, wherein said promoter can be induced by reactive species of oxygen.
9. method according to claim 1, wherein said target tissue are unusual zone of tumor, tissue growth or the unusual zone of angiogenic growth.
10. method according to claim 1, wherein said heating is produced by heat production laser, microwave, ultrasound wave or rf wave.
11. method according to claim 1, wherein said generation heat is between 44.5 to 45 ℃.
12. a method that strengthens the gene expression in the individual body inner target tissue, this method comprises the following steps:
Give described individuality with the photosensitizer of photodynamic therapy;
Expression vector is delivered to the described target tissue of described individuality, and wherein said carrier is the express therapeutic heterologous gene under the control of photodynamic therapy inducible promoter, and wherein said gene is allogenic for described promoter;
Described photosensitizer and described expression vector are absorbed by described target tissue; With
Described target tissue is exposed under the illumination, and wherein said illumination combines meeting generation reactive species of oxygen and induces and can be strengthened described therapeutic expression of heterologous genes by the inductive described promoter of photodynamic therapy with described photosensitizer.
13. method according to claim 12, wherein said photosensitizer are selected from photofrin, this C.I. Natural Red 8 of stannum, list-1-asparaginyl group chlorin e 6, benzoporphyrin derivative, meso-four-(hydroxy phenyl) chlorin and 5-amino-laevulic acid.
14. method according to claim 12, wherein said promoter are heat shock protein hsp promoter or glucose regulated protein promoter.
15. method according to claim 12, wherein said carrier are retroviral vector, adeno-associated virus vector or liposome dna vector.
16. method according to claim 12, wherein said heterologous gene are immunomodulatory gene, tumor suppressor gene, antisense DNA or angiogenesis inhibitor gene.
17. method according to claim 16, wherein said immunomodulatory gene is a cytokine gene.
18. method according to claim 12, wherein said light is produced by laser instrument.
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| US81540297A | 1997-03-10 | 1997-03-10 | |
| US08/815,402 | 1997-03-10 |
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| JP (1) | JP2001514658A (en) |
| KR (1) | KR20000076115A (en) |
| CN (1) | CN1252006A (en) |
| AU (1) | AU731502B2 (en) |
| CA (1) | CA2283108A1 (en) |
| IL (1) | IL131776A0 (en) |
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| US6811788B2 (en) * | 2000-01-19 | 2004-11-02 | Baofa Yu | Combinations and methods for treating neoplasms |
| US7927612B2 (en) | 2000-01-19 | 2011-04-19 | Baofa Yu | Combinations and methods for treating neoplasms |
| ATE552855T1 (en) * | 2000-02-10 | 2012-04-15 | Massachusetts Eye & Ear Infirm | COMBINATIONS AND PREPARATION FOR USE IN PDT FOR THE TREATMENT OF EYE DISORDERS WITH UNDESIRABLE CHORIODAL NEOVASCULARIZATION |
| US20050048109A1 (en) * | 2003-08-26 | 2005-03-03 | Ceramoptec Industries, Inc. | Non-polar photosensitizer formulations for photodynamic therapy |
| WO2006116789A2 (en) * | 2005-05-03 | 2006-11-09 | Veterinärmedizinische Universität Wien | Permeable capsules |
| CN101138634A (en) | 2006-09-07 | 2008-03-12 | 于保法 | Composition for treating tumour |
| WO2016086070A1 (en) | 2014-11-26 | 2016-06-02 | Baofa Yu | Hapten-enhanced chemoimmunotherapy by ultra-minimum incision personalized intratumoral chemoimmunotherapy |
| WO2020231236A1 (en) * | 2019-05-15 | 2020-11-19 | 동성제약주식회사 | Photodynamic therapy method mediated by chlorin e6 photosensitizer composite for treating and preventing obesity |
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| US5571797A (en) * | 1994-05-11 | 1996-11-05 | Arch Development Corporation | Method of inducing gene expression by ionizing radiation |
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| KR20000076115A (en) | 2000-12-26 |
| WO1998040105A1 (en) | 1998-09-17 |
| ZA981948B (en) | 1999-09-09 |
| IL131776A0 (en) | 2001-03-19 |
| EP1005375A1 (en) | 2000-06-07 |
| NZ337693A (en) | 2001-07-27 |
| JP2001514658A (en) | 2001-09-11 |
| AU6453998A (en) | 1998-09-29 |
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