“慢病毒载体”专题
biotcm先发一篇生物通的技术点评，大家了解一下大概的发展情况，呵呵。
www.ebiotrade.com/emgzf/ebiotech/44-01.pdf
为了普及逆转录病毒和慢病毒载体的应用，特提供载体互换（只限研究交换用，恕不赠送，请谅解）：
逆转录病毒载体：
pLNCX2
pQCXIH
pVpack-GP
pVPack-VSV-G
慢病毒载体：
pLVX-DsRed-Monomer-N1
pLP1
pLP2
pLP/VSVG
等
想使用此系统者，请PM。
逆转录病毒、慢病毒生产纯化方法：
安全性考虑，大家了解一下，不用太担心：
telomerase：
我还没有做包装病毒与滴度测定，实验还在计划之中。
我准备要直接买病毒的，直接回来感染细胞。
和公司的人谈了下，他们也不确定悬浮细胞感染的方法。
查了个方法，想麻烦你帮看看，哪些地方需要重点注意和改进的。
Production of Lentiviral Vectors: Packaging of the Vectors
1. Maintain 293T cells in complete culture medium in a 37ºC incubator with 5% CO2. Twenty-four hours before transfection, plate exponentially growing 293T cells in 100-mm tissue culture dishes at 4 x 106 cells/plate.
Cells should be ~80% confluent for transfection.
2. Prepare 1 ml of calcium phosphate-DNA suspension for each 100-mm plate of cells as follows:
i. Set up two sterile tubes for transfection of one plate. Label the tubes 1 and 2.
ii. Add 0.5 ml of 2X HBS-LT to Tube 1.
iii. Add TE/10 to Tube 2.
The volume of TE/10 to add is 440 µl minus the volume of the DNA solution (see Step 2.iv).
iv. Add the DNA solution (15 µg of the transfer vector containing the transgene, 15 µg of pCgp, 5 µg of pCMV-rev, and 5 µg of pCMV-G) to Tube 2 and mix.
v. Add 60 µl of the 2 M CaCl2 solution to Tube 2 and mix gently.
vi. Transfer the contents from Tube 2 to Tube 1 dropwise with gentle mixing.
vii. Allow the suspension to sit for 30 minutes at room temperature.
3. Mix the precipitate well by pipetting or vortexing.
4. Add 1 ml of the suspension to the 100-mm plate containing cells (from Step 1). Add the suspension slowly, dropwise, while gently swirling the medium in the plate. Return the plates to the 37ºC incubator for 4 hours.
5. After 4 hours, replace the old medium with 6 ml of fresh culture medium. Add 60 µl of 0.6 M sodium butyrate. Return the cells to the incubator.
6. After 48 hours of culture, collect the supernatant. Freeze it at -80ºC, or proceed to Step 7
Concentration of the Vectors
7. Centrifuge the supernatant (from Step 6, freshly collected or thawed from the freezer) at 900g for 10 minutes to remove any cell debris.
8. Filter the supernatant through a 0.2-µm syringe filter.
9. Transfer the supernatant to autoclaved polyallomer tubes. Concentrate the supernatant by ultracentrifugation for 1.5 hours at 4ºC in a Beckman SW 28 swinging bucket rotor at 24,500 rpm.
10. Remove the supernatant, and resuspend the pellet in an appropriate amount of culture medium, for example, 300 µl for 30 ml of original supernatant if a 100-fold concentration is desired.
11. Divide the concentrated vector into 10-50-µl aliquots. Store the aliquots at -80ºC until use.
Titration of the Vectors
12. Seed 5 x 104 HT1080 cells/well in a 12-well plate in complete medium. Culture the cells overnight in a 37ºC incubator with 5% CO2.
13. Add serial diluted vector stock (from Step 11) and 4 µl/ml polybrene to the cultured cells. Continue to culture for 48 hours.
14. Trypsinize the cells. Following centrifugation, remove the supernatant, and resuspend the pellet in 300 µl of 3.7% formaldehyde.
15. Determine the percentage of EGFP-positive cells by FACS analysis.
The titer will be represented as transduction units (TUs) per milliliter concentrated vector (TU/ml
Transduction of Lentiviral Vectors to Target Cells
For transducing cultured hematopoietic cells, follow Steps 16-20. For transducing primary hematopoietic stem cells, follow Steps 21-29.
Transducing Cultured Hematopoietic Cells
16. Seed exponentially growing cells at 2 x 105 cells/well in 1 ml of culture medium into a 24-well plate.
17. Add various amounts of concentrated vector stock (from Step 11), depending on cell type.
For the K562 cell line (CML leukemia cell line), a multiplicity of infection (MOI) of 10 can achieve virtually 100% transduction.
18. Add 4 µg/ml polybrene. Return the cells to a 37ºC incubator.
19. After overnight incubation, centrifuge the cells, discard the supernatant, and resuspend the cells with fresh culture medium. Return the cells to the incubator.
20. Determine transduction efficiency by FACS analysis 48 hours after transduction.
biotcm：
你如果买现成的病毒液的话，须保证他们给你的滴度在10的8次方以上。
其次你尚需优化感染时你细胞的密度和MOI值，
我不知你的试验目的是什么，如果你转染的目的蛋白或其他没有毒性，如果感染率还低，你可以进行流式细胞筛选这样就把阳性细胞富集到然后扩大培养就好了。
当然你也可以直接这么做的，呵呵，一般几代之内整合的基因不会丢失。
如果你目的蛋白有毒性建议你采用诱导表达的启动子慢病毒载体再采用后面的方法就可以了，希望有帮助
你要是筛库的话就麻烦了是不是你包装的假病毒含有各种各样的基因啊 那效率低岂不是许多都拿不到克隆？
建议好好优化一下吧 或者能否考虑换细胞系
我们先来看看各种载体的比较：
Properties of the most common gene delivery vectors (Gao et al., 2007; Kootstra and Verma,
2003; Moroziewicz and Kaufman, 2005; Waehler et al., 2007).
Pros Cons
Baculovirus * High titers (1010-1012 pfu/ml) * Inactivation by complement
* Large insertion capacity > 100 kB * Immunogenic
* Non-human pathogen, safety * Large size
Retro-and * Stable gene expression * Risk of insertional mutagenesis
lentiviruses * Insert capacity 8-9 kB * Risk ofreplication competent
* No pre-existing immunity virus formation
6 10
* Moderate titers 10 -10 TU/ml * Inactivation by complement
Adenovirus * High titers (1010-1012 pfu/ml) * Pre-existing immunity:
* Insert capacity 7-8 kB, for gutless vectors neutralizing antibodies
36 kB * Acute inflammatory and
* Broad tropism immunological responses
* High short-term gene expression * Complicated vector genome
* Oncolytic potential
Adeno- * Stable gene expression possible * Small insert capacity, 4.6 kB
associated * Nonpathogenic * Slow onset of gene expression
virus (AAV) * Highly stable virions * Risk of insertional mutagenesis
* Small size (22 nm) * Production requires helper viruses
* No need for viral genes in vectors * Large-scale production difficult
Vaccinia * Well established safety profile * Immunogenicity
* Oncolytic potential
Herpes * Long-term expression inneuronal cells,* Host immune responses,
simplex neurotropism inflammation and toxicity
8 11
virus (HSV) * High titers, 10 -10 pfu/ml * Complicated vector genome
* Transgene capacity 30 kB, for amplicons
152 kB
* Oncolytic potential
Nonviral * Low degree of toxicity, non-infectious * Low transfection effic. in vivo
vectors * Easy and simple production * Only transient expression
* High efficiency in vitro * For some vectors acute immunity,
* No insert size limit toxicity, aggregation in vivo
要利用慢病毒载体，最好首先对HIV的生活周期有个大概的了解： What are lentiviral vectors?
Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell.Lentiviruses can be used to provide highly effective gene therapy as lentiviruses can change the expression of their target cell's gene for up to six months.They can be used for nondividing or terminally differentiated cells such as neurons, macrophages, hematopoietic stem cells, retinal photoreceptors, and muscle and liver cells, cell types for which previous gene therapy methods could not be used.HIV is a very effective lentiviral vector because it has evolved to infect and express its genes in human helper T cells and other macrophages.The only cells lentiviruses cannot gain access to are quiescent cells (in the G0 state) because this blocks the reverse transcription step (Amado and Chen, 1999).To understand how HIV is a good vector for gene therapy, we must understand the structure of HIV and how it functions and infects its host.
Structure of HIV
Why HIV is a good vector for gene therapy?
The preintegration complex of the human immunodeficient virus (HIV), which allows the vector assess inside human cells, dividing or non-diving, is composed of the enzyme integrase, the product of the vpr gene (an accessory gene), and a protein encoded by the gag gene (an essential structural gene) called matrix.This matrix protein contains a localization sequence which is recognized by the import machinery of the nucleus of a cell.The virus is surrounded by a lipid bilayer with protruding membrane proteins.One of these proteins, gp120, is recognized by the host helper T cell CD4 receptor protein. Then HIV binds to a secondary receptor (CCR5 or CXCR4) and triggers a membrane fusion-mechanism with the gp41 transmembrane protein.This allows the virus asses to the cell interior and the virus content is released into the cytoplasm of the cell (Adler, Gifford, and Sumner; Schmidt, The HIV Page).Once inside of the cell in the cytoplasm, the matrix protein of the HIV contains a localization sequence that is recognized by the nuclear import machinery, which docks the complex at a nuclear membrane pore.This enables the preintegration complex of the HIV lentiviral vector to pass into the nucleus (Amado and Chen, 1999).
* Components of HIV Provirus
It is useful to understand the components of HIV and how it affects its host cell.The major protein components of the HIV virus can be seen in Table 1.
Table 1: The major protein components, which are expressed by all retroviruses and are necessary for virus replication. They are encoded by three major transcripts: gag, pol, env. These proteins are synthesized as fusion proteins, which are post-translationally cleaved by the virus-encoded protease. HIV has some additional genes (from Schmidt, The HIV Page).
Name: Protein: Fuction:
MA Matrix Matrix protein (gag gene); lines envelope
CA Capsid Capsid protein (gag gene); protects the core; most
abundant protein in virus particle
NC Nucleocapsid Capsid protein (gag gene); protects the genome;
forms the core
PR Protease Essential for gag protein cleavage during maturation
RT Reverse transcriptase Reverse transcribes the RNA genome; also has
RNAseH activity
IN Integrase Encoded by the pol gene; needed for integration of
the provirus
SU Surface glycoprotein The outer envelope glycoprotein; major virus
antigen
TM Transmembrane protein The inner component of the mature envelope
glycoprotein
How are HIV lentiviral vectors made?
To obtain a lentiviral gene therapy vector, a reporter gene or therapeutic gene is cloned into a vector sequence that is flanked by LTRs and the Psi-sequence of HIV.The LTRs are necessary to integrate the therapeutic gene into the genome of the target cell, just as the LTRs in HIV integrate the dsDNA copy of the virus into its host chromosome.The Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the reporter or therapeutic gene in virions. Viral proteins which make virus shellsare provided in the packaging cell line, but are not in context of the LTRs and Psi-sequences and so are not packaged into virions.Thus, virus particles are produced that are replication deficient, so are designed to be unable to continue to infect their host after they deliver their therapeutic content (Schmidt, HIV as a Vector for Gene Therapy). HIV Vectors Have A Three Plasmid Expression System
Lentiviral vectors are usually created in a transient transfection system in which a cell line is transfected with three separate plasmid expression systems.These include the transfer vector plasmid ( portions of the HIV provirus), the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (ENV) of a different virus (Amado and Chen, 1999).The three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell (Kalapana, 1999).The virus portions of the vector contain insert sequences so that the virus cannot replicate inside the cell system (Adler, Gifford, and Sumner).
Transfer Vector Plasmid
The transfer vector plasmid contains cis-acting genetic sequences necessary for the vector to infect the target cell and for transfer of the therapeutic (or reporter) gene and contains restriction sites for insertion of desired genes.The 3’ and 5’ LTRs, the original envelop proteins, and gag sequence promoter have been removed (Adler, Gifford, and Sumner; Naldini et al., 1996).
Packaging Plasmid
The packaging plasmid is the backbone of the virus system and is also known as pCMVAR9.In this plasmid are found the elements required for vector packaging such as structural proteins, HIV genes (except the gene env which codes for infection of T cells, or the vector would only be able to infect these cells), and the enzymes that generate vector particles (Amado and Chen, 1999).Also contained is the human cytomegalovirus (hCMV) which is responsible for the expression of the virus proteins during translation.The packaging signals and their adjacent signals are removed so the parts responsible for packaging the viral DNA have been separated from the parts that activate them.Thus, the packaging sequences will not be incorporated into the viral genome and the virus will not reproduce after it has infected the host cell (Adler, Gifford, and Sumner; Naldini, 1996).Previous HIV vectors used two plasmids as the packaging plasmid contained the viral envelop gene.However, in the newer, better vectors the packaging plasmid lacks a viral envelop gene because this has been shown to be more desirable in terms of titer (minimum volume needed to cause a particular result in titration), stability, and broad range of target cells (CFAR at UC San Diego).
Envelop Gene of Third Plasmid
The third plasmid’s envelope gene of a different virus specifies what type of cell to target and infect instead of the T cells (Amado and Chen, 1999).Normally HIV can infect only helper T-cells because they use their gp120 protein to bind to the CD4 receptor.However, it is possible to genetically exchange the CD4 receptor-binding protein for another protein that codes for the different cell type on which gene therapy will be performed (Schmidt, HIV as a Vector for Gene Therapy).This gives the HIV lentiviral vector a broad range of possible target cells.There are two types of heterologous envelope proteins.The amphoteric envelop of MLV, another type of vector, is transcribed first followed by the transcription of the G glycoproteins of the vesicular stomatitis virus, known as VSV-G.Both of these help to provide stability to the vector by bringing together the particles that were made by the packaging plasmid pCMVAR9 (Adler, Gifford, and Sumner; Naldini, 1996).
How are HIV lentiviral vectors used?
* Delivery Into Patients' Target Cells
The HIV-based vector can be delivered directly into the body without in vitro manipulations of the patient’s cells (Adler, Gifford, and Sumner).Additionally, lentiviral vectors have been shown to be superior to murine retroviral vectors.Ex vivo manipulations that activate stem cells with growth factors to induce cell division must be carried for the retrovirus to be able to enter the stem cells.However, it has been shown that ex vivo stem cell stimulation is not necessary with lentiviral vectors, so the vectors can be inserted directly into the patient and will find their way to the target cell (Amado and Chen, 1999).
Previous gene therapy using retroviral vectors required that cells be dividing, limiting therapy to proliferating cells in vivo or ex vivo.In the ex vivo method, the target cells are removed from the patient's body, treated to stimulate replication and then transduced with the vector before being returned to the patient.However, with lentiviral vectors there is no need for ex vivo treatment, and the target cells need not be dividing.The HIV-based vector is simply injected into a patient, upon which it seeks out its target cells based on cell membrane receptor proteins.Immune responses to the lentiviruses have not been found (Peel, 1998).
* Uses for HIV Lentiviral Vectors
Scientists have recently been using the HIV lentiviral vector to repair neurons.HIV is also being developed as a delivery system to provide successful gene therapy in many diseases such as metabolic diseases, cancer, viral infection, cystic fibrosis, muscular dystrophy, hemophilia, retinitis pigmentosa, and maybe even Alzheimer’s disease (Adler, Gifford, and Sumner; Naldini et al.; Amado and Chen, 1999; Planelles).
* Concerns With Using HIV Lentiviral Vectors
There is still concern with using lentiviral vectors for safety reasons.One concern involves the possibility that the HIV could self-replicate and could be produced during manufacture of the vector in the packaging cell line or in the target cells by a process of recombination.Thus, the person undergoing gene therapy would also be infected with HIV in addition to the new therapeutic gene.A self-replicating infectious vector could cause cancer by inserting itself into the host genome and activate a neighboring proto-oncogene, thus causing mutagenesis (Amado and Chen, 1999).
Back to Index
Current research involving lentiviral vectors
Because scientists have shown that lentiviruses, such as HIV, are successful and efficient gene delivery vehicles, the field has now turned its attention to producing vectors with built-in safety features to prevent the development of replication competent lentiviruses (RCL).However, even the earliest studies with HIV lentiviral vectors did not generate RCL in vitro or in vivo (Amado and Chen, 1999), but precautions are still very important.
* Safety Modifications
HIV lentiviral vectors are being produced whose packaging plasmid does not contain the necessary HIV genes.This does not interfere with efficient vector production and is a great increase in safety because potential RCL’s cannot use the HIV genes necessary for replication of HIV in humans.The drawback to these vectors is that they cannot transduce macrophages because the accessory gene vpr is needed for HIV infection of this type of cells.Thus, scientists are showing that the type of lentiviral vector necessary is dependent on the type of cell chosen as target, so the HIV vectors will be made with different accessory genes (Amado and Chen, 1999).
Researchers at the Salk Institute are creating HIV lentiviral vectors that are self-inactivating.The scientists are working on packaging a defective HIV genome that contains only the necessary elements for gene transduction into a virion that has a broad host range.HIV normally targets human CD4 (helper T cells) through interactions with membrane-bound target proteins, but to broaden the host cell targets a surrogate targeting molecule (VSV-G) was inserted into the viral membrane.The HIV genome was modified to produce a minimal construct and the cytomegalovirus promoter and green fluorescence protein as a marker were added (Sikorski and Peters, 1998).A deletion in the LTR region at the end of the virus genome is also created.These are unique cis-acting sequences that are essential to the virus life cycle.The deletion inactivates the LTR promoter and eliminates the production of vector RNA.The gene to be transferred by the vector is expressed from an exogenous viral or cellular promoter that is inserted into the lentiviral vector.Inactivation of the promoter activity of the LTR reduces the possibility of insertional mutagenesis as the lentiviral products are integrated into the host genome.Also, as expression of the vector RNA is eliminated, the potential for RCL production in the target cell is further minimized (Amado and Chen, 1999).
Other safety methods include using specific internal promoters that regulate gene expression either temporally or with tissue or cell specificity so as to prohibit gene expression that would cause replication of HIV in the gene therapy target cell (Amado and Chen, 1999).
* Use of Non-Human Lentiviral Vectors
By using non-human lentiviruses, scientists hope to bypass the issue of host infection by the gene therapy vector.Researchers are developing non-human lentiviruses such as the feline immunodeficiency virus (FIV)to be used in gene therapy (Amado and Chen, 1999).FIV infects two to twenty percent of domestic cats worldwide and causes a disease similar to human AIDS.While humans have been exposed to this virus through cat bites, humans have never been shown to be infected by the virus.It has been shown that evolutionarily FIV diverged early on from HIV and other lentiviruses.Researchers at the University of San Diego, though, have found that while nonprimate lentiviruses may provide safer alternatives to HIV they have highly restricted host range of infection.However, promoter substitution of FIV enabled an env-deleted, three plasmid, human cell-FIV lentiviral vector system to express high levels of FIV proteins and FIV vectors in human cells.The researchers replaced the U3 element within the 5’ LTR of FIV with the human cytomegalovirus early gene promoter.Pseudotyped FIV vectors were shown to be able to efficiently transduced dividing, growth-arrested, and postmitotic human targets.The researchers also showed that human cells supported mechanisms of the FIV life cycle needed for efficient lentiviral vector transduction.It is the U3 element in FIV that is the only restriction to the productive phase of FIV replication in human cells.The researchers concluded that lentivirus-specific properties of FIV vectors are retained in human cells, and they speculate that eventually FIV vector will have advantages in human clinical use.Additionally, vectors derived from FIV may represent a safer alternative to HIV vectors, even those with deleted nonstructural proteins, because they cannot induce HIV-reactive antibodies in recipients.Overall, FIV has experimental advantages over HIV (Poeschla, Wong-Staal, and Looney, 1998).
Researchers at the University of North Carolina at Chapel Hill are working with equine infectious anemia virus (EIAV) to be used as a lentiviral vector in humans.EIAV is a lentivirus that normally infects horses, donkeys, and mules.It has been shown to be able to infect mature macrophages, and thus has the potential to infect quiescent cells, and has relatively simple genome organization.The researchers constructed separate plasmids encoding EIAV proteins, a viral envelop, and an EIAV vector.They attempted to broaden the host range of the vector to human cells by using non-EIAV enhancer/promoter elements to drive expression and a non-EIAV envelop glycoprotein.They succeeded in transducing up to about 60 to 70 percent of human CFT1 cells which were placed in a culture dish.This is still quite a bit lower than the transduction level obtained using murine retroviruses, but more work with EIAV will hopefully increase the efficiency of this procedure.In addition, the fact that both EIAV-based and HIV vector can mediate gene transfer and expression to non-dividing human cells suggests that nuclear targeting mechanisms of equine and human lentiviruses are functionally conserved (Olsen, 1998).
* Lentiviral Vectors for Hematopoietic Stem Cells
Many recent studies with lentiviral vectors have focused on modifying the hematopoietic stem cell which has the capacity to self-renew and to differentiate into all of the mature cells of the blood and immune systems.Thus, by introducing therapeutic genes into stem cells many diseases that affect these systems could be treated (Amado and Chen, 1999).
* Gene Therapy for Cystic Fibrosis
Researchers at the Institute for Gene Therapy at the University of Pennsylvania evaluated a replication-deficient vector based on HIV for gene transfer directly into the lung to correct the genetic defects of cystic fibrosis (CF).They expanded the target range of the vector by adding the vesticular stomatitis virus G protein into the HIV vector envelop.LacZ was the reporter gene in the HIV-based vector, so the level of transduction was assessed based on the expression of lacZ.The researchers were successful at transducing nondividing airway epithelial cells in vitro, whereas they were unsuccessful when using murine-based retroviral vectors.Thus, the vector corrected the CF defect in proliferating airway cells. There were complications with differentiated epithelial lung cells as the vectors did not effectively transduce these cells.The blockage appeared to be at the level of entry, the researchers reported.Further experimentation is being conducted to examine the problems of cell entry into differentiated cells (Goldman, et al., 1997).
* Liver-Directed Gene Therapy
Initial research aimed at delivering genes to the liver in vivo with HIV-based lentiviral vectors showed promising results, reported Ganjam Kalpana of Albert Einstein College of Medicine this year.This scientist developed a crippled version of HIV and used it as a vehicle for in vivo gene therapy on low-density lipoprotein receptor-deficient Watanabe heritable hyperlipidemic rabbits.A eukaryotic humanized gene fluorescent protein gene was cloned into the transfer vector to act as the reporter gene for successful cell transduction.The HIV vector was highly superior to previous methods of gene therapy using retroviral vectors which were highly invasive to the patient.There was also no host mediated cellular immune response to the lentiviral vector (Kalpana, 1999).This is another application to HIV-based gene therapy vectors that has been shown to be successful.
* Therapy Against Retinitis Pigmentosa
Retinitis pigmentosa is an inherited genetic disease which causes the retina to degenerate leading to loss of visual field and night blindness.Genetic defects of photoreceptor cells of the visual system are the cause of this disease.A vector for gene therapy of retinitis pigmentosa should only target photoreceptor cells, which are located in the outer nuclear layer of the retina.Miyoshi, Takahashi, Gage, and Verma conducted an experiment using an HIV-based vector with a gfp-gene (green fluorescent protein) as a reporter.The vector was injected into rat retina.It was shown that the HIV-based vector did achieve long-term gene expression in the photoreceptor cells when a rhodopsin-promoter was used in the vector.This is only active in the photoreceptor cells, so the vector only targets these cells and not others in the retina.Thus, the researchers were successful in performing gene therapy on their rat patients (Schmidt, HIV as a Vector For Gene Therapy).
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