CN105039407A - Lentiviral vector with improved transcription readthrough effect and application of lentiviral vector - Google Patents

Lentiviral vector with improved transcription readthrough effect and application of lentiviral vector Download PDF

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CN105039407A
CN105039407A CN201510353122.3A CN201510353122A CN105039407A CN 105039407 A CN105039407 A CN 105039407A CN 201510353122 A CN201510353122 A CN 201510353122A CN 105039407 A CN105039407 A CN 105039407A
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ltr
fragment
lentiviral vector
lentiviral vectors
read
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曾凡一
张敬之
高越
何佳平
方彧聃
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Shanghai City Children Hospital
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Shanghai City Children Hospital
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Abstract

The invention discloses a lentiviral vector with an improved transcription readthrough effect and application of the lentiviral vector. Lentiviral vector delta U3 and foamy virus R-U5 zones are prepared in LTR (long terminal repeat) regions of an HIV-1 (human immunodeficiency virus-1) lentiviral vector to form a chimera LTR (FH LTR), or two copied chicken beta-Globin HS4 (heparan sulfate 4) insulator complete sequences (2.4C<->, 2*1.2-kb) are reversely inserted onto a framework of a lentiviral vector on the basis of an HIV-1, so that the lentiviral vector can be obtained. The lentiviral vector and the application have the advantages that diversified methods for reducing a transcription readthrough rate of the lentiviral vector are screened; the transcription readthrough rate of the lentiviral vector is reduced and reaches 15% approximately after the chimera FH LTR is used; the transcription readthrough rate is greatly reduced, and accordingly the safety of the third-generation lentiviral vector can be improved to a great extent during gene therapy.

Description

A kind of lentiviral vectors and application thereof improving read-through
Technical field
The invention belongs to biological technical field, be specifically related to a kind of lentiviral vectors and the application thereof that improve read-through.
Background technology
Human immune deficiency I C-type virus C is the pathogenic agent of acquired immune deficiency syndrome (AIDS)-be commonly called as acquired immune deficiency syndrome (AIDS).It is I type virus of AIDS that HIV-1 is also commonly called as.Speak of virus of AIDS, first reaction of people is frightened and uneasy.Scientists is but transformed into gene transfer vehicle this virus, is referred to as the lentiviral vectors based on HIV-1.
The effective application of lentiviral vectors in clinical gene therapy substantially increases the requirement of researcher to its security.Lentiviral vectors in a case where may induced tumor: lentiviral vectors random integration enters in chromatin to cause the property inserted sudden change, integration site is positioned at inside or the upstream of proto-oncogene or cancer associated gene seat just, and the characteristic due to lentiviral vectors with read-through causes downstream proto-oncogene transcriptional activation or transcript to increase.Because on chromatin, encoding sequence only accounts for 5%, therefore, the possibility simultaneously possessing above-mentioned condition is very little.But these insecurity factors exist, and current lentiviral vectors is often used to the seriously disease that some cannot carry out conventional treatment in clinical gene therapy.The people such as 1999-2002, Hacein-Bey-Abina utilize γ-RVs to carry out gene therapy to X chain severe combined immunodeficient disease (X-SCID) patient.Although cured the 9 routine patients connect in subject 10 examples, but among after treatment 31-68 month, 4 routine patients are had in succession to obtain leukemia.Follow-up research finds, before γ-RVs carrier prefers and is incorporated into proto-oncogene (lmo2, bmi1 or ccnd2), and activates the expression of these genes.The reason of γ-RVs bearer activation downstream gene expression has 2 points: 1, γ-RVs5 ' holds the enhancer element in long terminal repeat (LTR) can activate the expression of gene near integration site; 2, γ-RVs3 ' holds LTR to have very high read-through activity, thus causes integration site downstream gene transcriptional expression.There is read-through phenomenon in lentiviral vectors, especially SIN-LVs equally.SIN-LVs deletes promotor in LTRU3 and enhancer element on the basis of earlier generations LVs.The prioritization scheme of SIN-LVs reduces the probability producing regroup live virus (RCLs).But meanwhile SIN-LVs read-through rate but adds greatly.This is because its U3 district is partly deleted, containing transcription terminator element USE in U3 district.Research finds, the read-through rate of SIN-LVs may add about 10 times than the rate of reading over before transformation.So high read-through rate makes SIN-LVs while security is promoted, and turn increases new security risk.
In order to improve the read-through of lentiviral vectors, just need the transcription terminator element LTR optimizing lentiviral vectors, to strengthen its Transcription Termination ability.But the complicacy of LTR structure and function significantly increases the difficulty improved from deactivation lentiviral vectors read-through rate.LTR is not only containing overlapping transcription initiation/transcription terminator element, also in viral wrapping process, participate in transcribing of virus genome RNA, in host cell, participate in the process that viral RNA reverse transcription becomes cDNA, and participate in process in viral integrase to cyto-chromatin.Therefore, the impact on all above-mentioned viral physiological processs must be considered on the optimization design of LTR simultaneously.This improvement research of reading over rate to lentiviral vectors adds difficulty.
Scientists has attempted the method for many reduction SIN-LVs read-throughs.Yang etc. once attempted in SIN-LVsLTR, add I type leukemia virus polyadenylation signal element respectively, and the growth hormone gene 3 ' of people holds little intron element and sudden change tRNA motif element to improve the read-through of SIN-LVs.Found that these all optimization methods not only could not increase the Transcription Termination ability of SIN-LVs, add the read-through of SIN-LVs on the contrary.The core parts (0.25kb) that the people such as Hanawa choose chicken β-Globin5 ' HS4 separaant are inserted in lentiviral vectors, find that the read-through of lentiviral vectors is improved.And oppositely inserted by this element, the improvement of read-through is more obvious.β-Globin5 ' HS4 separaant the core sequence of the T cell separaant (α/δ BEAD-1) of people and chicken merges and obtains a new element by the people such as Ramezani, is called FB element (77bp).They study discovery, and FB element has the same effective enhanser block function of 5 ' HS4 separaant complete with 1.2kb.Be inserted in SIN-LVs by FB element, the conversion risk of carrier significantly reduces.In SIN-LVsLTR, supplement USEs element also contribute to improving read-through.The people such as Schambach have selected seven kinds of upstream Polyadenylations and strengthen element USEs (from viral or cytogene), are inserted into the △ U3 region of SIN-LVsLTR, to improve read-through.Insert the USE element (2 × SV40USE, long 80bp) from SV40 virus in the △ U3 of research discovery 3 ' LTR, the read-through rate of virus vector significantly reduces, and its titre increases by 2.8 times, and genetic expression also has 1.6-2.2 increase doubly.But aforesaid method also could not eliminate read-through phenomenon, read-through level is not made to be reduced to safety range yet.
Summary of the invention
The object of the present invention is to provide a kind of lentiviral vectors and the application thereof that improve read-through.
Improve a lentiviral vectors for read-through, described carrier the LTR region of HIV-1 lentiviral vectors is transformed into its △ U3 and foamy virus (foamyvirus) R-U5 district forms mosaic LTR (FHLTR); Or the skeleton of the lentiviral vectors based on HIV-1 oppositely inserts the complete sequence (2.4C of the chicken β-GlobinHS4 separaant of two copies -, 2 × 1.2-kb);
The sequence of described HIV-1 lentiviral vectors is as shown in sequence table SEQ IDNO.1;
The sequence of described mosaic LTR is as shown in sequence table SEQ IDNO.2.
Described 2.4C -sequence as shown in sequence table SEQ IDNO.3.
Described mosaic FHLTR refers to lentiviral vectors △ U3 and foamy virus (foamyvirus) R-U5 district and merges and form brand-new mosaic LTR.
A kind of application of lentiviral vectors in expressing gene engineering product improving read-through.
Beneficial effect of the present invention: the read-through rate of lentiviral vectors is quantized by the method based on RT-qPCR detection lentiviral vectors read-through of designed, designed by the present invention.Successfully determine the third generation from deactivation lentiviral vectors (FUGW virus packaging system) the read-through rate in 293T cell, be about 68%.And the rate of reading over of wild-type HIV-1 is about 24%.The present invention has filtered out the method reducing lentiviral vectors read-through rate.Wherein, in 293T cell, the rate of reading over is reduced to about 15% after using mosaic FHLTR in lentiviral vectors.The significantly reduction of read-through rate, improves the security of third generation lentiviral vectors in gene therapy to a great extent.But the titre delta data of virus also needs to be supplemented by subsequent experimental after transformation.
Accompanying drawing explanation
Fig. 1 is experimental road line chart of the present invention.
Fig. 2 is FUGW, FLV carrier schematic diagram.
Fig. 3 is the read-through rate that RT-qPCR detects the transfer vector of each optimization.
Fig. 4 is that FACS detects EGFP positive rate.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be further described.
Simulated virus vector integration is entered the situation in chromatin by the present invention, detects the change carrying the lentiviral vectors provirus read-through rate of mosaic FHLTR.In addition, the present invention have also been devised other several new prioritization schemes to improve lentiviral vectors read-through level.The present invention devises 10 kinds of prioritization schemes (see table 1) to being optimized the read-through rate of SIN-LVs, and the validity (experiment route as shown in Figure 1) of inspection optimization scheme in two different aspects respectively.
First, pEGFP-N1 report carrier systems axiol-ogy is utilized to insert the Transcription Termination ability of the later LTR of these optimization elements.On this basis, utilize provirus carrier system analog carrier to be incorporated into situation on chromatin, detect and verify that prioritization scheme improves the validity of lentiviral vectors read-through.
Table 1 improves 10 kinds of schemes of lentiviral vectors read-through rate
One, optimization design and the structure thereof of lentiviral vectors read-through is improved
The structure of 1.1FLV carrier
FUGW carrier is the transfer vector of the third generation in deactivation lentiviral vectors packaging system be widely used at present.The carrier is carrier of the present invention using FUGW carrier as research lentiviral vectors read-through rate.By the method for chemosynthesis (Invitrogen), the R-U5 fragment of synthesis Human foamy spumavirus (HumanFoamyVirus), by fusion DNA vaccine, is fused into mosaic LTR (FHLTR) with the △ U3 on lentiviral vectors.Be cloned into the two ends of FUGW subsequently, replace the original LTR in its two ends, formation simulation integration enters the provirus carrier on chromatin.。。
R (repeatsequence) is one section of sequence in HIV-1 long terminal repeat LTR, is positioned at the both sides of HIV-1 genome sequence.Virus transcribe with process of reverse-transcription in all to get up very important effect.Because R element contains the core sequence polyAsignal of Transcription Termination, improve the read-through of lentiviral vectors by increasing a R element.After detecting and increasing R element, whether virus vector can initiation transcription smoothly, needs to build these two carriers (Fig. 2) of FGW and FGW-2R.
Containing an AflII restriction enzyme site in the sequence of R, in AflII site, R element is opened, insert R in the sequence (fusion sequence of AflII downstream sequence and AflII downstream sequence is called for short 2R-insertsequence) of the sequence of AflII sites downstream and AflII site upstream thus the tumor-necrosis factor glycoproteins (2R element) obtained containing two R elements.
FUGW carrier respectively has a R element at 5 ' end and 3 ' end, and for the ease of cutting operation to the enzyme of R element, contriver first removes the partial sequence in FUGW carrier, makes it only containing 3 ' the R element held.Cut FUGW carrier with ApaI and PacI enzyme, remove the sequence between ApaI-PacI.After skeleton fragment fills, connect with ligase enzyme, carrier is from successivelyying win to obtain intermediate carrier FUGW-A/P.The basis of intermediate carrier FUGW-A/P operates R element.
Synthesize with primer AflII-2R-F and AflII-2R-R (sequence is in table 2) of number of base complementation, carry out extensions acquisition 2R-insertsequence with High fidelity PCR enzyme Pyrobest, then carry out enzyme with AflII and cut and obtain 2R-AflII fragment.In addition, with AflII, enzyme is carried out to FUGW-A/P plasmid DNA and cut acquisition FUGW-A/P-AflII fragment.2R-AflII fragment and FUGW-A/P-AflII fragment are connected acquisition intermediate carrier FUGW-A/P-(5 '-2R) under the effect of ligase enzyme.Next the sequence (sequence between ApaI-PacI) of excising in FUGW-A/P (-5 '-2R) carrier to be refilled.FUGW carrier first carries out complete degestion with PmeI, then carries out with PstI the fragment (FUGW-PmeI/PstI-3523 fragment) that partially digested (37 DEG C, 5min) obtain 3523bp.Intermediate carrier FUGW-A/P-2R PmeI and PstI carries out complete degestion and obtains FUGW-A/P (-5 '-2R)-PmeI/PstI fragment.FUGW-PmeI/PstI-3523 fragment and FUGW-A/P-2R-PmeI/PstI fragment carry out being connected acquisition intermediate carrier FUGW-5 '-2R with ligase enzyme.FUGW and FUGW-5 '-2R all carries out double digestion with PacI and BamHI, cuts the sequence containing Ubiqutin promotor between PacI-BamHI, fills from successivelyying win to obtain final carrier FGW and FGW-5 '-2R (being called for short FGW-2R).
1.2pEGFP-N1 serial carrier builds
PEGFP-N1 is a business-like eukaryotic expression vector (Invitrogen), carries the reporter gene EGFP in CMV promoter and downstream thereof.There is a multiple clone site MSC between CMV and EGFP, be convenient to exogenous array or gene clone in carrier.Various optimised LTR element is cloned in MSC to detect the Transcription Termination effect of these elements by contriver.
Construct and carry FHLTR, Δ LTR, Δ LTR-C -, Δ LTR-C +, Δ LTR-U +with Δ LTR-C -u +pEGFP-N1 carrier (N1-Δ LTR, N1-C -, N1-Δ LTR-C +, N1-U +and N1-C -u +), and it has best Transcription Termination effect to have proved FHLTR.Here, also need to build the pEGFP-N1 control vector of carrying wLTR.
1.2.1N1-wLTR vector construction
Wild-type HIV-1 viral integrase enters after in the genome of people, and both sides can form a long terminal repeat LTR.The third generation in order to reduce the probability producing recombinant virus, carries out part deletion to the LTR sequence of HIV-1 from deactivation type lentiviral vectors (as FUGW).LTR after deactivation is deleted, contriver is referred to as Δ LTR; And the LTR that wild-type HIV-1 virus is carried is referred to as wLTR.Contriver utilizes and carries the upstream primer LTR-K-U3 of KpnI and carry the downstream primer LTR-B-U5 of BamHI, amplifies wLTR sequence (about 750bp) by the method for PCR.Carry out double digestion with BamHI and KpnI again and obtain wLTR-BamHI/KpnI fragment.In addition, same BamHI and KpnI enzyme is cut pEGFP-N1 and is obtained skeleton fragment pEGFP-N1-BamHI/KpnI.Ligase enzyme connects wLTR-BamHI/KpnI fragment and pEGFP-N1-BamHI/KpnI fragment.
1.2.2N1-2R vector construction
Δ LTR-2R fragment is obtained by the method amplification of fusion DNA vaccine.With LTR-X-U3 (containing XhoI restriction enzyme site) and AflII-2R-R for primer, take FUGW as template (containing Δ LTR) amplified fragments Δ U3-R-Δ R fragment.With LTR-P-U5 (containing PstI restriction enzyme site) and AflII-2R-F for primer, FUGW is template amplification fragment Δ R-R-U5 fragment (primer sequence is in table 3).Δ U3-R-Δ R fragment and Δ R-R-U5 fragment are mixed in sex change in boiling water, then make its naturally cooling anneal, add PCRbuffer, Pyrobest and dNTPs, in PCR instrument, 72 DEG C extend 10min.In system, add primer LTR-XhoI-U3 and LTR-PstI-U3, carry out normal PCR circulation, amplify Δ LTR-2R fragment (about 680bp).PCR primer is through sequence verification, and sequence is entirely true.With XhoI and PstI, double digestion is carried out to Δ LTR-2R fragment, obtain Δ LTR-2R-XhoI/PstI fragment.In addition, same XhoI and PstI enzyme is cut pEGFP-N1 and is obtained skeleton fragment pEGFP-N1-XhoI/PstI.Ligase enzyme connects wLTR-XhoI/PstI fragment and pEGFP-N1-XhoI/PstI fragment obtains carrier pEGFP-N1-Δ LTR-2R (being called for short N1-2R).
1.2.3N1-2RC -u +vector construction
With LTR-X-U5 and LTR-d-R for primer, N1-C -u +for template, pcr amplification U3C -u +fragment.U3C is cut with XhoI enzyme -u +fragment obtains U3C -u +-XhoI fragment.N1-2R XhoI and EcoRV enzyme are cut and are obtained skeleton fragment N1-2R-XhoI/EcoRV.Connect U3C -u +-XhoI fragment and N1-2R-XhoI/EcoRV fragment obtain N1-2RC -u +.
1.2.4N1-FHLTR vector construction
FHLTR is the product R+U5-10+10 sequence (being called for short hFVp sequence) in U3 and the hFVLTR of lentiviral vectors merged.First synthesize hFVp sequence in Invitrogen company, then obtain FHLTR fragment by fusion DNA vaccine.With LTR-X-U3 and U3-R for primer, take FUGW as template amplification fragment 1.With U3-F and FHU5-R (containing ApaI restriction enzyme site) for primer, with hFVp sequence for template amplification fragment 2.Fragment 1 and fragment 2 are carried out fusion amplification and obtain FHLTR.With XhoI and ApaI, double digestion is carried out to FHLTR fragment, obtain FHLTR-XhoI/ApaI fragment.In addition, same XhoI and ApaI enzyme is cut pEGFP-N1 and is obtained skeleton fragment pEGFP-N1-XhoI/ApaI.Ligase enzyme connects FHLTR-XhoI/ApaI fragment and pEGFP-N1-XhoI/ApaI fragment obtains carrier pEGFP-N1-FHLTR (being called for short N1-FHLTR).
The structure of 1.3Fw transfer vector series
1.3.1Fw-LTR, Fw-C -u +the structure of carrier
Fw-LTR, Fw-C -u +carrier is built by contriver before being.FUGW XbaI carries out the EGFP gene that enzyme goes between XbaI two restriction enzyme sites earnestly, certainly successivelys win and to obtain intermediate carrier FUGW-XbaI/XbaI (claiming FUW).Intermediate carrier FUW NdeI and XhoI carries out enzyme and cuts the short-movie section FUW-NdeI/XhoI obtained containing virus sequence.N1-LTR and N1-C in addition -u +all carry out enzyme with NdeI and XhoI and cut acquisition skeleton fragment N1-LTR-NdeI/XhoI and N1-C -u +-NdeI/XhoI.Each 2 skeleton fragments are connected with FUW-NdeI/XhoI fragment respectively and obtain Fw-LTR and Fw-C -u +.
1.3.2Fw-wLTR the structure of carrier
Fw-LTR BamHI and KpnI carries out double digestion and obtains Fw-LTR-BamHI/KpnI skeleton fragment.N1-wLTR also carries out double digestion acquisition wLTR-BamHI/KpnI fragment with BamHI and KpnI in addition.Fw-LTR-BamHI/KpnI skeleton fragment is connected acquisition Fw-wLTR carrier with wLTR-BamHI/KpnI fragment.
1.3.3Fw-2R the structure of carrier
BamHI and KpnI enzyme is cut Fw-LTR and is obtained skeleton fragment Fw-LTR-BamHI/KpnI.In addition, N1-2R and N1-2RC -u +all carry out double digestion with BamHI and KpnI and obtain 2R-BamHI/KpnI fragment and 2RC -u +-BamHI/KpnI fragment.Fragment 2R-BamHI/KpnI and 2RC -u +-BamHI/KpnI is connected with skeleton fragment Fw-LTR-BamHI/KpnI respectively and obtains Fw-2R and Fw-2RC -u +carrier.
The structure of 1.4 provirus analog carrier (proFUGW-X) series
Three step clones build proFUGW-X serial carrier.The first step is introduced a NheI restriction enzyme site and is convenient to add complete LTR at 5 ' end after the 5 ' U5 of FUGW.Second step introduces LTR at 3 ' end.3rd step introduces LTR 5 '.
1.4.1 the structure of intermediate carrier FUGW+NheI
Three fragment connection methods introduce NheI restriction enzyme site in FUGW.With NheI-F and PacI-R for primer, FUGW is that template amplification obtains PCR primer 1, cuts PCR primer 1 obtain fragment 1 with NheI and PacI enzyme.With CMV-MluI-F and U5-NheI-R for primer, FUGW is that template amplification obtains PCR primer 2, cuts PCR primer 2 obtain fragment 2 with MluI and NheI enzyme.Cut FUGW with MluI and PacI enzyme and obtain skeleton fragment 3.The FUGW carrier of band NheI is obtained, called after FUGW+NheI by T4ligase junction fragment 1, fragment 2 and fragment 3.
1.4.2 the structure of intermediate carrier FUGW+NheI-3 '-LTR (FN-3 '-LTR)
FUGW+NheI XhoI and ApaI enzyme are cut and are obtained skeleton fragment FUGW+NheI-XhoI/ApaI.With LTR-X-U3 and U5-ApaI-R for primer, respectively with N1-wLTR, N1-2R, N1-C -u +, N1-2RC -u +, N1-C -, N1-U +for template amplification carries wLTR, LTR-2R, LTR-C of XhoI and ApaI restriction enzyme site -u +, LTR-2RC -u +, LTR-C -and LTR-U +sequence.XhoI and ApaI enzyme is cut these 6 PCR primer and is obtained wLTR-XhoI/ApaI, LTR-2R-XhoI/ApaI, LTR-C -u +-XhoI/ApaI, LTR-2RC -u +-XhoI/ApaI, LTR-C --XhoI/ApaI and LTR-U +-XhoI/ApaI fragment.These 6 fragments are carried out being connected with FUGW+NheI-XhoI/ApaI skeleton fragment respectively and are obtained intermediate carrier FUGW+NheI-3 '-wLTR (FN-3 '-wLTR), FUGW+NheI-3 '-LTR-2R (FN-3 '-2R), FUGW+NheI-3 '-LTR-C -u +(FN-3 '-C -u +), FUGW+NheI-3 '-LTR-2RC -u +(FN-3 '-2RC -u +), FUGW+NheI-3 '-LTR-C -(FN-3 '-C -) and FUGW+NheI-3 '-LTR-U +(FN-3 '-U +).
Fusion DNA vaccine amplification obtains LTR-2.5R.With LTR-XhoI-U3 and R1b+3+R1a for primer, N1-LTR is template, amplification fragment upstream.With U5-ApaI-R and R2b+3+R2a for primer, N1-LTR is template, amplification segments downstream.With LTR-XhoI-U3 and U5-ApaI-R for primer, with the mixture of fragment upstream and segments downstream for template, amplify LTR-2.5R sequence.Cut LTR-2.5R with XhoI and ApaI enzyme and obtain fragment LTR-2.5R-XhoI/ApaI.Fragment LTR-2.5R-XhoI/ApaI is connected with skeleton fragment FUGW+NheI-XhoI/ApaI and obtains intermediate carrier FUGW+NheI-3 '-LTR-2.5R (FN-3 '-2.5R).
1.4.3 the structure of carrier proFUGW-LTR (pro-LTR)
FUGW+NheI, FN-3 '-wLTR, FN-3 '-2R, FN-3 '-2.5R, FN-3 '-C -u +, FN-3 '-2RC -u +, FN-3 '-C -and FN-3 '-U +acquisition 8 skeleton fragment FUGW+NheI-MluI/NheI, FN-3 '-wLTR-MluI/NheI, FN-3 '-2R-MluI/NheI, FN-3 '-2.5R-MluI/NheI, FN-3 '-C is cut with MluI and NheI enzyme -u +-MluI/NheI, FN-3 '-2RC -u +-MluI/NheI, FN-3 '-C --MluI/NheI and FN-3 '-U +-MluI/NheI.With U3-MluI-F, U5-NheI-R for primer, respectively with FUGW+NheI, FN-3 '-wLTR, FN-3 '-2R, FN-3 '-2.5R, FN-3 '-C -u +, FN-3 '-2RC -u +, FN-3 '-C -and FN-3 '-U +plasmid DNA is fragment LTR, wLTR, LTR-2R, LTR-2.5R, LTR-C that template amplification carries MluI and NheI restriction enzyme site -u +, LTR-2RC -u +, LTR-C -and LTR-U +.These 8 fragments are all carried out enzyme with MluI and NheI cut, obtain the fragment of band sticky end: LTR-MluI/NheI, wLTR-MluI/NheI, LTR-2R-MluI/NheI, LTR-2.5R-MluI/NheI, LTR-C -u +-MluI/NheI, LTR-2RC -u +-MluI/NheI, LTR-C -and LTR-U +-MluI/NheI.
Skeleton fragment FUGW+NheI-MluI/NheI is connected the whole carrier proFUGW-LTR of acquisition (being called for short pro-LTR) with fragment LTR-MluI/NheI.Skeleton fragment FN-3 '-wLTR-MluI/NheI is connected the whole carrier proFUGW-wLTR of acquisition (being called for short pro-wLTR) with fragment wLTR-MluI/NheI.Skeleton fragment FN-3 '-2R-MluI/NheI is connected the whole carrier proFUGW-LTR-2R of acquisition (being called for short pro-2R) with fragment LTR-2R-MluI/NheI.Skeleton fragment FN-3 '-2.5R-MluI/NheI is connected the whole carrier proFUGW-LTR-2.5R of acquisition (being called for short pro-2.5R) with fragment LTR-2.5R-MluI/NheI.Skeleton fragment FN-3 '-C -u +-MluI/NheI and fragment LTR-C -u +-MluI/NheI connects the whole carrier proFUGW-LTR-C of acquisition -u +(be called for short pro-C -u +).Skeleton fragment FN-3 '-2RC -u +-MluI/NheI and fragment LTR-2RC -u +-MluI/NheI connects the whole carrier proFUGW-LTR-2RC of acquisition -u +(be called for short pro-2RC -u +).Skeleton fragment FN-3 '-C --MluI/NheI and fragment LTR-C --MluI/NheI connects the whole carrier proFUGW-LTR-C of acquisition -(be called for short pro-C -).Skeleton fragment FN-3 '-U +-MluI/NheI and fragment LTR-U +-MluI/NheI connects the whole carrier proFUGW-LTR-U of acquisition +(be called for short pro-U +).SalI and BamHI enzyme is cut pBC1 carrier (Invitrogen) and is obtained 2 × Chicken β-globininsulator (2 × 1.2kb, about 2.4kb are called for short 2.4C) element.T4Polylimase fill enzyme cut after 2.4C-SalI/BamHI-T4Poly fragment.Pro-LTR carrier XhoI enzyme is cut, and after CIAP process, T4Polymerase fills and obtains fragment pro-LTR-XhoI-CIAP-T4Poly.2.4C-SalI/BamHI-T4Poly fragment Opposite direction connection pro-LTR-XhoI-CIAP-T4Poly skeleton fragment obtains whole carrier proFUGW-LTR-2.4C -(be called for short pro-2.4C -).Pro-U +carrier XhoI enzyme is cut, and after CIAP process, T4Polymerase fills and obtains fragment pro-U +-XhoI-CIAP-T4Poly.2.4C-SalI/BamHI-T4Poly fragment Opposite direction connection pro-XhoI-CIAP-T4Poly-U.Skeleton fragment obtains whole carrier proFUGW-LTR-U-2.4C and (is called for short pro-2.4C -u +) Poly fragment forward connection pro-U +-Xho.2.4C-SalI/BamHI-T4I-CIAP-T4, Poly skeleton fragment obtains whole carrier LTR-U +-2.4C +(be called for short proFUGW-pro-2.4C +u +).
With the N1-FHLTR carrier built for template, use primer U3-M-F (containing Mlu I restriction enzyme site), FH-NheI-R (containing Nhe I restriction enzyme site) amplify fragment laggard performing PCR product glue and reclaim, and use Mlu I, Nhe I double digestion to obtain FHLTR-Mlu I/Nhe I fragment.Use primer LTR-X-U3 (containing Xho I restriction enzyme site), FH-U5-R (containing Apa I restriction enzyme site) amplify fragment laggard performing PCR product glue and reclaim, and use Xho I, Apa I double digestion to obtain FHLTR-Xho I/Apa I fragment.
Use that FHLTR-Mlu I/Nhe I fragment substitutes Mlu I, Nhe I enzyme cuts pro-Δ LTR gained fragment respectively, and FHLTR-Xho I/Apa I fragment substitutes Xho I, Apa I enzyme cuts pro-Δ LTR gained fragment.Carry out ligation by T4Ligase, final acquisition simulates provirus carrier proFUGW-FHLTR (being called for short pro-FHLTR).
Two, lentiviral vectors Characteristics Detection after optimizing
2.1RT-qPCR detects read-through rate
(1) lentiviral vectors transient transfection 293T cell, after 24-48h, collecting cell extracts total serum IgE.The mRNA of lentiviral vectors normal transcription and the mRNA of read-through is comprised in total serum IgE.
(2) residual DNA is removed with DNaseI process RNA.
(3) get 1 μ gRNA as template, carry out reverse transcription using LTR3 as Auele Specific Primer, obtain cDNA-L.The template of cDNA-L be lentiviral vectors transcribe out read over mRNA.Separately get the identical RNA sample of 1 μ g as template, carry out reverse transcription using RP-2 as Auele Specific Primer and obtain cDNA-R.The template of cDNA-R comprises mRNA and the read-through mRNA of lentiviral vectors normal transcription.Therefore the composition of whole cDNA-L is comprised in cDNA-R.
(4) using two kinds of cDNA products (cDNA-L and cDNA-R) as template, JP-2 and RP-2 is as quantitative primer, qLTR carries out quantitative PCR reaction as fluorescent probe, detects the molecular amounts (quantitative primer and probe sequence are in table 2) of two kinds of cDNA.
Table 2 is primer and probe sequence quantitatively
(5) elementary cell using table 3 as preparation quantitative PCR reaction system.The reaction member quantity preparation PCRmix (reaction member quantity=sample size × parallel laboratory test quantity+standard substance sample size+blank quantity of calculation in quantity PCR is carried out according to sample size and parallel stoichiometric number.Generally do 2-3 parallel laboratory test reaction tubes; Molecule number generally got by standard substance is 10 4, 10 510 9the plasmid dilute sample of individual/μ L is 6 reaction members; Blank gets ddH 2o, as template, is 1 reaction member).
Table 3 quantitative PCR reaction system
(6) above-mentioned PCRmix is dispensed in quantitative PCR pipe, often pipe 21 μ L.
(7) in each quantitative PCR pipe, 4 μ LcDNA sample and plasmid standards are added respectively.
(8) pipe cap is covered tightly, centrifugal, be loaded on ABI7500 quantitative PCR apparatus.
(9) software parameter is set by the response procedures of table 4, starts quantitative instrument.
Table 4 quantitative PCR response procedures
(10) according to quantitative result analytical data, calculate cDNA-L and cDNA-R that RNA sample obtains) molecular amounts.According to following formulae discovery read-through rate.
The detection of lentiviral vectors read-through after 2.2 optimizations
2.2.1 detect in FUGW transfer vector system and carry each element to the effect reducing read-through
The read-through of lentiviral vectors occurs in two aspects.First be in the preparation or wrapping process occurring in lentiviral vectors, namely occur in the transcription of transfer vector.Another is then occur in lentiviral vectors to be integrated in the expression process of foreign gene after chromatin forms provirus.Contriver is by the read-through rate of lentiviral vectors after these two aspect inspection optimizations.Before, contriver's experimental result shows, in pEGFP-N1 carrier system, to insert C first in LTR -u +transcription Termination effect after element is better than inserting C separately -or U +transcription Termination effect during element, is better than the Transcription Termination effect of Δ LTR more.Here contriver adopts increases R element (2R), increases C -u +element (C -u +) and superposition use R and C -u +element (2RC -u +) three kinds of prioritization schemes transform the transfer vector in lentiviral vectors packaging system, obtain 3 kinds of transfer vector (Fw-2R, Fw-C optimized -u +and Fw-2RC -u +).3 kinds of transfer vectors optimized and two kinds of control vector (Fw-Δ LTR and Fw-wLTR) difference transient transfections, to 293T cell, are detected by the read-through rate of method to each carrier of RT-qPCR.4 times independently revision test result as shown in table 5 and Fig. 3.Carry out statistical study to data, result shows compared with Fw-Δ LTR, Fw-2R, Fw-C -u +and Fw-2RC -u +read-through rate all have remarkable reduction (p=0.0447, p=0.0027 and p=0.0048).Wherein Fw-C -u +read-through rate reduce the most obvious, be reduced to about 30%.Next is Fw-2RC -u +and Fw-2R, the rate of reading over is respectively 34.3% and about 41.5%.But, Fw-2RC -u +significant difference (p=0.4177) is not had with the result of Fw-2R.
The read-through rate (%) of transfer vector respectively optimized by table 5
Note: compare with the N1-LTR group significance of difference, * P<0.05, * * P<0.01, * * * P<0.001.]
Read-through rate under 2.3 simulation provirus states
The security risk that lentiviral vectors read-through phenomenon is brought mainly occurs in after vector integration enters genome.It is that it carries out gene therapy important safety hidden danger that read-through causes the expression of downstream gene to activate.After lentiviral vectors is integrated into chromatin formation provirus, its transcription properties is subject to the impact of chromatin environment.Consider the complicacy of chromatin environment near the random integration of lentiviral vectors and integration site.Reduce chromatin environment to the impact of virus vector characteristic, contriver simulates the provirus structure of lentiviral vectors by plasmid vector, and in analog carrier, detects the read-through rate of each optimization carrier by transient transfection.
In simulation provirus carrier system, contriver adds prioritization scheme.Except the increase R element (pro-2R) before employing, increase C -u +element (pro-C -u +) and superposition use R and C -u +element (pro-2RC -u +) beyond 3 kinds of prioritization schemes, contriver also increases other 6 kinds of prioritization schemes: increase 1.5R element (pro-2.5R), increase C -element (pro-C -), increase U +element (pro-U +), increase 2.4C -element (pro-2.4C -), increase 2.4C -u +element (pro-2.4C -u +), increase FHLTR element (pro-FHLTR).
Optimize carrier and 2 control vector (pro-LTR and pro-wLTR) transient transfection 293T cell respectively, use the method based on RT-qPCR to detect read-through rate for 9.The result of 6 independent repeated trials is as shown in table 6.
The read-through rate (%) of provirus carrier simulated by table 6
Data statistic analysis result display (table 7), the read-through rate of pro-2R and pro-LTR do not have significant difference, and pro-C -u +, pro-2RC -u +, pro-2.5R, pro-C -, pro-U +, pro-2.4C -and pro-2.4C -u +all there were significant differences compared with pro-LTR group for the read-through rate of test group, and its read-through rate is all lower than pro-LTR.Wherein, pro-2.4C -read-through rate minimum, be reduced to about 19.5%, lower than the level of pro-wLTR (about 24.0%), but pro-2.4C -and there is no significant difference (p=0.1190) between pro-wLTR two groups of data.In addition, pro-C -u +read-through rate (29.2%) equally also close to the level (not having significant difference p=0.3438 between two groups) of pro-wLTR.Pro-2.4C +u +the transcript that experimental group almost can't detect normal transcript and reads over, this illustrates 2.4C +u +element can stop transcribing (titre almost reduces to zero) of virus vector, and therefore this prioritization scheme is infeasible.The read-through rate of pro-FHLTR is minimum (15.7%), lower than the level of pro-wLTR.
The each experimental group of table 7 and pro-LTR group carry out one-way analysis of variance
The present invention relates to 10 kinds of schemes optimized lentiviral vectors and read over to reduce transfection, and (pEGFP-N1 carrier system and proFUGW simulate provirus carrier system) have detected respective improvement effect (as shown in table 8) in two aspects.Wherein, simulation provirus carrier system is integrated into the state in chromatin closest to lentiviral vectors.Certainly, the detection data in this system truly can reflect the read-through characteristic of lentiviral vectors.PEGFP carrier system have detected the Transcription Termination ability of each optimization LTR, for judging whether prioritization scheme effectively provides important reference data.
The validity that each prioritization scheme of table 8 improves read-through in three cover detection systems
Note: "-" represents to be tested; "×" indicates without positive effect; " √ " represents to have unusual effect, P<0.05; " √ √ " represents P<0.01; " √ √ √ " represents P<0.001; " √ √ √ √ " represents P<0.0001.
Increase R element (2R) and increase 2.4C in above-mentioned 10 cover prioritization schemes +u +element (2.4C +u +) this two covers prioritization scheme reads in rate and do not have effect improving lentiviral transcription.Other 7 kinds of prioritization schemes all effectively can improve the read-through of lentiviral vectors, wherein FHLTR, C -u +element and 2.4C -the effect that element improves rate of reading over is the most obvious.Wherein increase C -u +element will be readed over rate and be reduced to about 29% from about 68%, and increase 2.4C -the rate of reading over is reduced to about 20% by element.And the rate of reading over is reduced to about 15% by FHLTR.

Claims (3)

1. improve a lentiviral vectors for read-through, it is characterized in that, described carrier is that the LTR region of lentiviral vectors based on HIV-1 is transformed into its △ U3 and foamy virus R-U5 district forms mosaic LTR; Or the skeleton of the lentiviral vectors based on HIV-1 oppositely inserts the chicken β-GlobinHS4 separaant complete sequence of two copies, insertion sequence called after 2.4C -;
The sequence of the lentiviral vectors based on described HIV-1 is as shown in sequence table SEQ IDNO.1;
The sequence of described mosaic LTR is as shown in sequence table SEQ IDNO.2;
Described 2.4C -sequence as shown in sequence table SEQ IDNO.3.
2. a kind of lentiviral vectors improving read-through according to claim 1, is characterized in that, described mosaic LTR refers between △ U3 district on lentiviral vectors and foamy virus R-U5 district and merges.
3. one kind is improved the application of lentiviral vectors in producer gene engineering product of read-through.
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CN110760543A (en) * 2019-08-21 2020-02-07 常州市第二人民医院 Lentiviral multi-promoter stable expression vector constructed by insulator combination and construction method
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