CN109423525A - A kind of retrovirus clearance detection method for expressing CAR - Google Patents
A kind of retrovirus clearance detection method for expressing CAR Download PDFInfo
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Abstract
It lets pass and detects the present invention provides a kind of retrovirus for expressing CAR, this method comprises: expression CAR virus infection efficiency inspection, the inspection of CAR+ cell quantity, the detection inspection of bacterial fungus endotoxin, detection of mycoplasma, the detection of retrovirus reproducibility (RCR), external exogenous virus factors check.The retrovirus of expression CAR by strict inspections a series of has the characteristics that homogeneity, stability, safety.It is more suitably applied to batch, industrialization production CART cell.
Description
Technical field
The present invention relates to field of pharmaceutical biology, and in particular to a kind of retrovirus clearance detection method for expressing CAR.
Background technique
Gene therapy is one forever in the field of expansion, is generally defined as the importing of therapeutic genes material.From 1989
Transgenic research starts in year first man body, and the clinical trial for being related to transgenosis emerges one after another.Gene therapy is most apparent
Application be exactly genotype genetic disease amendment, such as hemoglobinopathy, acquired immunodeficiency syndrome and dysbolism.?
Past 20 years, researcher find transgenic technology can be a large amount of diseases, the genopathy being not only in traditional concept,
New method is provided.Being continuously increased for the database of laboratory and zooscopy illustrates the potential benefit of gene therapy.Current
Challenge is that the clinical safety for being technical application and effective means develop necessary technical capability.
Candidate stem cell is still attractive target in gene therapy methods.This cell is highly suitable for body
Outer operation, organ transplantation technique is very perfect, and heredity, pernicious and viral disease can be thin by the hematopoiesis of transgenosis
Born of the same parents take aim at fixed.It is anticipated that hematopoietic progenitor cells will be subjected to largely expanding, carrier has stable transfection target cell function, target gene
Daughter cell can be transmitted to.Currently, retroviral vector is in Transfection of Hematopoietic cell processes because have become a kind of selection,
It has stable transfection, and not will cause significant cell death and chromosome breakage or other may have negative shadow to target cell
Loud adverse factor.Developing first viral vectors is the virus for entering clinical test based on retrovirus and first
Carrier.Most of retrovirus be based on murine leukemia virus (oncogenic retrovirus, it is subsequent referred to as inverse in the present invention
Retroviral), and have the advantages that the breeding cycle of unique retrovirus, so that it is allowed the deletion of viral-coding sequences
(gag, pol and env), and use and delete encoding hiv protease sequence.There is no the foreign gene of copy function to the area
Domain is replaced, this is both the requirement of a clinical application, but a technological difficulties: how RNA being packed in virion.This
A challenge is resolved by the package cell line of retrovirus.Initial package cell line is female thin from mouse fiber
Born of the same parents system, the most common NIH3T3 cell.Plasmid expression gene needs the formation of Vims particle structures.These design cell line and
It is not factory of the plasmid as virion protein of building, therefore viral gene is almost impossible is combined into as virion
Son.The transfection of one (such as: viral LTR and interested transgenosis) plasmid for containing vector construction, results in carrier
The release of particle product in the medium.
In mouse body, retrovirus is by integrating susceptible oncogene nearby, and adjusting oncogene mistake
LTR enhancer, Lai Yinqi malignant tumour are usually used in expression.The step is typically considered to need the change of multiple oncogene,
Therefore single carrier is typically considered to be loaded with the pernicious of unusual low-risk.Previously, available statistics indicate that retrovirus turn
The mutated gene being inserted into after dye is also very low, but is not absolutely not.It is comprehensive immune being actively engaged in X-linkage severe
There are two patients to occur in 11 patients of dcc gene Experiment on therapy, a carrier being incorporated near LMO2 gene and white blood
It is sick related.It is very complicated why these children can develop into leukaemia, between the reality of retroviral gene therapy before
Test it is no pernicious, but primary evidence show this research (cytokine receptor of common γ chain) transgenic also show as
A possibility that oncogene.The adverse events of these replication defectives virus strengthen the carrier (RCR) for excluding to have replication capacity and produce
The importance of the vigilant test of product accidental pollution.
In retrovirus product, the most probable source RCR is carrier and viral gene used in virus packaging
Between recombination.RCR was once frequent in the production strain of earlier version (all viral genes are all expressed on the same plasmid)
Be detected.The productivity of RCR is reduced by reducing carrier and producing the sequence homology between strain, and is divided
Opening production strain of the gag-pol and env gene on different plasmids successfully reduces the frequency of RCR generation.However, much containing
The novel RCRs of Endogenous Retrovirus sequence has been generated.Chong etc. reports a RCR in GP+envAM12 cell
5`LTR, gag and most pol gene source are in reverse transcription sequence raw in thermophilic parent's property.Garrett etc. confirmed a RCR
It appears in the GP+envAM12 cell line that the package carrier of thermophilic parent's property transfected.This RCR contains the endogenous sequence of recombination
Column, including producing the endogenous sequence that strain is transferred to from the thermophilic parent's property of GPE+E86.These are statistics indicate that when RCR is generated, it is possible to more
Increase after a recombination, and endogenous sequence may be contained.When producing virus using human cell line, this incidence can be more
It is few, although must take into account a possibility that recombinating with the endogenic reversion rate virus sequence of the mankind.
So far, the clinical use for detecting RCR in carrier relies on the combination of bioassay and Molecular Detection.In the U.S.,
The bioassay of recombination is the derivative and label marker rescue assay of the S+/L- test of the virus expanded in the cell line of license.S+/
L- test in specified cell line (such as cat cell line PG-4) for expanding the assessment of virus.PG-4 is referred to as a kind of S+/L-
Cell line, the sarcoma positive (S+) and leukaemia are negative (L-).PG-4 cell contains murine sarcoma virus genome, but only in same table
Phenotypic change can be induced in up to the cell of murine leukemia virus.If test material contains RCR, the cell of transfection can be
It is detected on PG-4 cell.It can be quantified by the viral level to the limited dilution of test sample, test sample.Phase
Anti-, label marker rescue assay is arrived using the cell line containing the carrier moved in the presence of RCR, label transgenosis
The movement of one designated cell proves RCR.
The retrovirus for expressing CAR will detect, especially to it before doing infection T cell by stringent clearance
The detection of safety such as RCR and exogenous virus detection, are the precondition for guaranteeing safety in production CART cell, emphasis solution of the present invention
The certainly safety issue of retrovirus.
Summary of the invention
For the above reason, the present invention provides the sides that standard set detects the clearance of the retrovirus of expression CAR
Method, particular content see the table below.
The retrovirus clearance detection project and method of the expression of table 1 CAR
The invention has the following advantages:
1) operation of the present invention is simple, and the retrovirus safety of the expression CAR of preparation is good, and uniformity is high.
2) instrument used in detection method of the invention is all laboratory Conventional compact instrument, does not need large-scale instrument.
3) detection method of the invention is easier to promote, and lays the foundation for the industrialization production of CART.
Detailed description of the invention
Fig. 1 flow cytometer detection expresses the efficiency of the retroviral infection PBMC of CAR
Fig. 2 PG4 cell adds the cytopathy figure after positive-virus
Specific embodiment
Embodiment
The present invention is described in further detail by reference to following EXPERIMENTAL EXAMPLE.These embodiments are merely for explanation
Property purpose provide, be not intended to it is restrictive, unless otherwise prescribed.Therefore, the present invention should not be interpreted in any way as being limited to
Lower embodiment, but should be interpreted as including since introduction provided herein will become apparent from any and whole variation.
Present invention specifies the real-time method of several special detections, method that conventional detection can refer to Chinese Pharmacopoeia.
1. retroviral infection PBMC Efficiency testing of embodiment
The expression of PBMC lymphocytic cell surface CAR albumen is as follows after flow cytomery infection:
72 hours after infecting CART cells are collected by centrifugation respectively, PBS abandons supernatant after washing 1 time, pats test tube and mixes carefully
Born of the same parents are added PBS after corresponding antibody is protected from light 30min and wash, are resuspended, add suitable Viability dyestuff
(invitrogen), it is protected from light incubation 15 minutes.Last flow cytomery CAR
The result of the present embodiment is as shown in Figure 1, the CART cell of the CD3 positive is greater than the CAR expression rate of 95%, CART cell
Greater than 50%.
The detection of 2. retrovirus reproducibility of embodiment
Sample and 293 cells co-culture and PG-4S+L- is tested
1 293 plating cells of Day
(1) disinfection by ultraviolet light 30min opens Biohazard Safety Equipment headlamp and blower into laboratory.
(2) culture medium is taken out from 4 DEG C of refrigerators, is restored to room temperature.
(3) 293 cells after taking out cultured digestion process in advance, sampling count, and are resuspended and are adjusted with culture medium after centrifugation
Whole cell concentration is 5 × 105/ml。
(4) adjustment concentration is 5 × 10 by the 6 orifice plates for taking TC-treated (corning)5293 cell inoculations of/ml are in 6
In orifice plate, the cell suspension of 1ml is added in every hole.Negative control group, experimental group and positive controls are inoculated with 6 orifice plates respectively.
(5) 1ml culture medium is added in every hole in 6 orifice plates.37 DEG C are placed in, 5%CO2Overnight incubation in incubator.
Sample and 293 cells co-culture
(1) experimental group:
Negative group: 293 cell itself, with experimental group operation repetitive；
Experimental group: sample to be tested；
Positive group: wild type GALV virus liquid.
(2) it discards 293 culture supernatant of negative control group first, 1ml D10 complete medium and 8ug/ml is added
polybrene；Next discards 293 cells and supernatant of experimental group, is added experimental group sample 1ml's and 8ug/ml to be measured
polybrene；293 cells and supernatants of positive group are finally discarded, positive group viral supernatants 1ml is added, and (GALV virus stock solution used is dilute
Release 10 times) and 8ug/ml polybrene；37 DEG C are placed in, 5%CO22h is cultivated in incubator.Positive group uses individually biology
The reagents such as safety cabinet and culture medium, avoid pollution sample sets.
(3) fresh complete medium, the hole 2ml/ are changed to after 2h.Operation order are as follows: negative group-experimental group-positive group.
18 293 cell continuous passage of Day 3-Day
(1) Day3-Day4 observes the cell confluency degree in 6 orifice plates to 70-80% or so, and digestion is transferred to 10cm culture dish
In continue to cultivate.Operation order are as follows: negative group-experimental group-positive group.Positive group uses individual Biohazard Safety Equipment and culture
The reagents such as base, avoid pollution sample sets.
(2) 18 group of cells of Day 5-Day passes at same time point, is passaged twice a week, and passage 5 times or more.Operation
Sequentially are as follows: negative group-experimental group-positive group.Positive group avoids dirt using reagents such as individual Biohazard Safety Equipment and culture mediums
Contaminate sample sets.
Day 19-Day 21 collects 293 cells and supernatant of each group
(1) pass in last the 3rd day Day 21, group of cells convergence degree reaches 100%, is changed to fresh culture.
Operation order are as follows: negative group-experimental group-positive group.
(2) Day 22 collects each 2ml of each group culture supernatant, marks, and is saved after the filtering of 0.45um filter to -80 degree ice
Case.At this time note that sample must not exceed 2h at room temperature.Operation order are as follows: negative group-experimental group-positive group.
Collect the detection of each group supernatant: Q-PCR detects GALV copy number；PG-4S+L- method detects cytopathy.
It is same that Q-PCR detects the detection of GALV copy number.
PG-4S+L- method detects cytopathy
(1) 1 Day, PG-4 plating cells.Cultured PG-4 cell in advance is taken out, sampling counts, with complete after centrifugation
It is 1 × 10 that adjustment cell concentration, which is resuspended, in culture medium5/ml.Adjustment concentration is 1 by the 6 orifice plates for taking TC-treated (corning)
×105The PG-4 cell inoculation of/ml adds the cell suspension of 1ml in 6 orifice plates in every hole, and adds 1ml complete medium.
37 DEG C are placed in, 5%CO2Overnight incubation in incubator.Negative control group, experimental group and positive controls are inoculated with 6 orifice plates respectively.Its
In, the positive sample in experimental group also needs individually to be inoculated in 6 orifice plates, operates parallel with positive controls.
(2) PG-4 cell is infected in 2 Day.
A) experimental group:
Negative control group: PG-4 cell itself, with experimental group operation repetitive；
Experimental group: the supernatant being collected into；
Positive group: the virus liquid of 10 gradients of wild type GALV virus liquid stoste and 10 times of dilutions.
B) it discards negative control group PG-4 culture supernatant, 1ml D10 complete medium and 8ug/ml is added
polybrene；Next discards experimental group PG-4 cells and supernatant, is added experimental group sample 1ml's and 8ug/ml to be measured
Polybrene, positive sample and positive controls operation repetitive in experimental group；Finally discard the training of positive controls PG-4 cell
Supernatant is supported, the polybrene of positive group GALV viral supernatants 1ml and 8ug/ml is added；37 DEG C are placed in, 5%CO2It is trained in incubator
Support 2h.
C) fresh D10 complete medium, the hole 4ml/ are changed to after 2h.Operation order are as follows: negative group-experimental group-positive group/
Experimental group positive sample.Positive group avoids pollution sample sets using reagents such as individual Biohazard Safety Equipment and culture mediums.
(3) Day3-Day7, PG-4 cell change liquid and observe the plastidogenetic lesion of PG-4.According to PG-4 cell state, often
Primary fresh D10 complete medium is replaced every two days, until making when observing that negative control group PG-4 cell confluency degree is 100%
Observed with inverted microscope and photograph to record cytopathy as a result, observation multiplying power be 4 × object lens.All samples are same
It is observed.
Testing result determines: PG-4S+L- detection cytopathy should be negative, and experimental group PG-4 cell is without lesion, and positive group
Visible cell lesion.
The positive findings and negative findings of the present embodiment are shown in Fig. 2.
The external exogenous virus factors check of embodiment 3.
Viral based article is related to the use of the animal derived material such as FBS, pancreatin, cell line in production, exists outside causing
Therefore the possibility of source factor contamination in order to guarantee the quality of virus liquid, needs to carry out the detection of the exogenous virus factor.According to medicine
Allusion quotation biological products general rule biological products produce the calibrating preparation of zooblast matrix and vertification regulation.
Living cells (or using considerable amount of cell lysate when suitable) is prepared with cell culture supernatant to be checked, inoculation is dynamic
Exogenous virus factors check is carried out in object.Cell to be checked should at least be inoculated with suckling mouse, (two groups not on the same day for adult mice and chicken embryo
Age) it is 4 groups total, for example newly-built cell also needs inoculation cavy.Primary MK cells also need thin with rabbit Inoculation method or rabbit kidney
Born of the same parents' cultivation checks monkey bleb B virus.Animal dead is more than in 24 hours after inoculation
2 0%, test is invalid.
One, in suckling rat brain and intraperitoneal inoculation cell suspension
1,1x10 is prepared7The cell PBS suspension of/ml；
2, suckling mouse in taking be born for 24 hours, quantity 20；
3,0.25ml glass syringe, 4.5# syringe needle, boiling sterilization；
4, the region of suckling mouse cranium, the midpoint of-two ear line of eye-ear, trigonum are the positions for being commonly used to injection；Left hand
Fixed suckling mouse head (attention is unable to weight) exposes above mentioned position, and the right hand keeps steady syringe and fix needle core should not be random
It is dynamic, syringe needle is penetrated into above mentioned trigonum centre, depth about 2mm injects emitter to injection volume 0.01ml with index finger
(head for having to fixed stably suckling mouse) is taken advantage of a situation with the index finger of fixing head or thumb by the needle on scalp while extracting syringe needle
Eye is rubbed with the hands, to prevent the exposing of injected material.
5, the intraperitoneal inoculation of suckling mouse, when injection, by syringe needle first it is intradermal walk a distance then through subcutaneous, give
Medicine slowly presses intradermal position of threading a needle with cotton swab after extraction syringe needle, can prevent medical fluid from flowing out, and 0.1 milliliter of injection volume.
6, observation index: the death rate, dead animal should carry out gross anatomy observation and histological examination in the observation period, with
Determine the cause of death.
Two, adult rats intracerebral and intraperitoneal inoculation cell suspension
1,1x10 is prepared7The cell PBS suspension of/ml；
2, by mouse forehead iodine tincture disinfection, then with alcohol iodine is taken off；
3, left hand thumb and index finger catch two ears and skin of head fixes mouse, the right hand be cased with plastic tube, needle point reveals
2 millimeters of long No. 5 syringe needles are directly hit exactly by forehead and are pierced into intracerebral, i.e. injectable out.Can also by mouse ether light anesthesia,
Make syringe and skull at about 45 degree of angles, is pierced at 2 millimeters on the outside of the middle line, the position bone is thin, injection shot 0.03
Milliliter/only；
4, it is raised 21 days after putting back to mouse cage；
5, small white mouse makees intraperitoneal inoculation, slightly raises rear quarters when preferably using Baoding of lying on the back, inoculation, its internal organ is made to be inclined to ante-chamber,
It in abdomen trailing flank insertion needle, first pierces under fell, answers non-resistance when entering abdominal cavity, injection afterwards, skin also swells；
6, observation index: the death rate, dead animal should carry out gross anatomy observation and histological examination in the observation period, with
Determine the cause of death.
Three, chick embryo allantoic cavity inoculating cell suspension
10 pieces of allantoic cavity > 5X10 of chicken embryo 9-11 age in days60.2mL 3-4 days
1, the selection and hatching of chicken embryo should select fresh fertile egg of the health without diseased chicken group or SPF chicken group.For convenient for according to egg sight
Examine, since navigate egg or other white shell eggs preferably.With case hatching of hatching, to pay special attention to temperature, humidity and egg-turning.Incubation condition
Be typically chosen relative humidity be 60%, 36 DEG C of minimum temperature, general 37.5 DEG C.Daily egg-turning minimum 3 times, starting can be by chicken embryo
Traverse, 2d is stood up before inoculation, and major part is upward, pays attention to chicken embryo position, as embryo tends on one side easily death.
Hatch 3~4d, can be observed with candler in darkroom.When chick embryo development is normal, it is seen that the clearly chicken of blood vessel and work
Embryo, blood vessel and its major branches are obvious, are in cerise, and chicken embryo can activity.Unfertilized and dead germ idiosome is fixed at one end not
It is dynamic, it can't see blood vessel or blood vessel dissipate, should reject.Depending on route of inoculation and inoculation material, yolk bag connects the age in days of chicken embryo
Kind, with the chicken embryo of 6~8 ages in days；Chorioallantoic cavity inoculation, with instar chicken embryo on the 9th~10；Allantocherion vaccination, with 9~13 ages in days
Chicken embryo；Intravascular injection, with instar chicken embryo on the 12nd~13；Amniotic cavity and intracerebral injection, with instar chicken embryo on the 10th.
2, the preparation before being inoculated with
Fluent material, added with antibiotic (100~500 μ g of 100~500IU of penicillin and streptomysin) set 1h or refrigerator in room temperature
12~for 24 hours high speed centrifugation take supernatant, or through filter filtration sterilization.The position for marking gas chamber, embryo with pencil according to egg, marks phase
The position answered.Punching is sterilized on the eggshell at inoculation with the tincture of iodine, and is punched at this.
3, the inoculation of chicken embryo
Egg inoculation is generally injected with tuberculin syringe, seals inoculation hole using the paraffin of fusing after having injected
It closes.
1) injection takes instar chicken embryo on the 9th~10 in chorioallantoic cavity, with awl after the disinfection of alcolhol burner flame, in gas chamber
Top and gas chamber lower edge are inserted under gas chamber along aperture without an aperture, syringe needle is respectively bored at blood vessel, deep 1.5cm has had passed through
Periostracum and there is half span from injection volume about 0.1~0.2mL away from embryo.With paraffin closed pores after injection, set straight in incubator
It is vertical to be incubated for.
2) yolk intracapsular injection takes instar chicken embryo on the 6th~8, (syringe needle is inserted into 3~3.5cm) can be inoculated with from gas chamber top side, because of embryo
Tire and yolk bag position have been determined, and can also insert the needle into yolk sac inoculation from flank hole.Side inoculation is not easy to injure chicken embryo,
But syringe needle extracts rear portion and divides inoculation liquid excessive sometimes, need to be wiped with cotton ball soaked in alcohol.Remaining is the same as allantois intracavitary administration.
4, it is checked after being inoculated with
After inoculation for 24 hours in dead chicken embryo, chicken embryo is impaired when being due to inoculation or other reasons and death, it should discard,
After for 24 hours, daily shine egg 2 times, such as find chicken embryo death be immediately placed in refrigerator, in cannot be lethal in certain time chicken embryo be also put into
Refrigerator is freezed to death.Dead chicken embryo, which sets 1~2h in refrigerator, can be taken off collecting material and checks chicken embryo lesion.
Four, chicken embryo lecithocoel inoculating cell suspension
10 pieces of yolk bag > 2X10 of chicken embryo 5-7 age in days60.55 day
Ibid
Five, cavy intraperitoneal inoculation cell suspension
Cavy 350-500g 5, in intraperitoneal inoculation cell density > 4X105/ ml, 5ml.Observation at least 42 days, observation end
Phase dissects all animals.
Make intraperitoneal inoculation in rabbit, cavy, small white mouse, slightly raises rear quarters when preferably using Baoding of lying on the back, inoculation, make its internal organ
It is inclined to ante-chamber, in abdomen trailing flank insertion needle, is first pierced under fell, answers non-resistance when entering abdominal cavity, injection afterwards, skin also swells.
Six, rabbit is subcutaneous and intradermal vaccination cell suspension
Rabbit 1.5-2.5kg 5, in subcutaneous/intradermal vaccination cell density > 2X105At/ml, 9ml/0.1mlX10, see
It examines 21 days.
Rabbit inoculates: rabbit being lain prostrate or lain on the back Baoding by assistant, is dredged in its back side or veutro subcutaneous connective tissue
Loose part cropping disinfection, the right hand syringes of patient, with left hand thumb, index finger and middle finger have pinched skin and have made into a triangle wrinkle
Pleat, or with tweezers pick up skin, in its bottom inserting needle, feeling that syringe needle can be stirred arbitrarily indicates that insertion is subcutaneous.When push-in is injected
Feel when object it is fluent it is unimpeded be also illustrated in subcutaneous, by pin hole and slightly massaged when transfering to injection needle with sterilized cotton ball.
Rabbit intradermal vaccination: when writer's rabbit intradermal vaccination, it is both needed to assistant Baoding animal, Baoding method is the same as subcutaneous vaccination.
Patient picks up skin with left hand thumb and index finger when inoculation, and right hand syringes are inserted between thumb and index finger with fine needle
Syringe needle insertion is unsuitable too deep in skin, while being inserted into angle and wanting small, and when injection feels have on skin after having resistance and injecting
Small hard blister is as injected intradermal.Intradermal vaccination is slow, disseminates infection to prevent making skin spalling or flowing out injected material from pin hole.
Seven, evaluation of result
In observation period, the analysis of causes should be carried out by being such as vaccinated animal and exception or disease occur, dead animal in the observation period
Gross anatomy observation and histological examination should be carried out, to determine the cause of death.It, then should be using training if animal shows virus infection
The method of supporting or molecular biology method identify virus (to be more than 20% animal appearance death, and can define in such as observation period
Caused by being judged to baiting because of animal), it is invalid that test is determined as, should retry.When observing the end of term, meets following condition and be judged to qualification.
1. suckling mouse and adult mice inoculation group should at least have 80% inoculation animal is strong to deposit, and mouse does not show propagability
The factor or other virus infections.
2. the chicken embryo of egg inoculation group yolk sac inoculation should at least have 80% survival, and not show virus infection；Allantois
The chicken embryo of chamber inoculation should at least have 80% survival, and allantoic fluid hemagglutination test (HA test) is feminine gender.
3. cavy inoculation group should at least have 80% inoculation animal is strong to deposit, and animal do not show the propagability factor or other
Virus infection.
4. rabbit inoculation group should at least have.
Claims (8)
1. a kind of antiretroviral agents, which is characterized in that the retrovirus of expression Chimeric antigen receptor CAR.
2. the retrovirus of expression CAR according to claim 1 will be examined by stringent clearance.
3. the clearance inspection of the retrovirus of expression CAR according to claim 2, which is characterized in that expression CAR reverse transcription
The inspection of virus infection PBMC efficiency, the inspection of CAR+ cell quantity, the detection inspection of bacterial fungus endotoxin, detection of mycoplasma, retrovirus
The detection of reproducibility (RCR), external exogenous virus factors check.
4. described in claim 3, expressing CAR virus infection PBMC efficiency >=20%.
5. detection bacterium, fungi, mycoplasma are all negative before according to claim 3, expression CAR retrovirus is let pass.
6. detection endotoxin residual quantity is < 0.5EU/ml before according to claim 3, expression CAR retrovirus is let pass.
7. RCR is detected as feminine gender before according to claim 3, expression CAR retrovirus is let pass.
8. according to claim 3, the expression external exogenous virus factors check of CAR retrovirus is feminine gender.
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