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慢病毒的原理制备与应用-徐学明(GeneCopoeia)


慢病毒的原理, 制备及应用
Principles, Production and Application of Lentivirus

Xueming Xu,

M.D. & Ph.D.

徐学明
GeneCopoeia,?Inc.?USA

广州复能基因有限公司

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1

Lenti?or?others?

HIV Viron

3

Retro, HIV and Lenti
Retrovirus

HIV

Lenti?Virus
5 ‐LTR R/U5
? /RRE/cPPT

CMV

ORF

SV40

GFP

IRES

Puro

3 ‐LTR WPR  U3/R

4

HIV?infection
1. Attachment 2. Entry 3. Reverse transcription 4. Transcription 5. Translation 6. Genome replication 7. Assembly 8. Exit

Lentivirus?Reverse?transcription.

gRNA (‐)?DNA (+)?DNA

Pluta?K?and?Kacprzak?MM.?Acta?Biochim?Pol.?2009;56(4):531‐95.

Lentivirus?packaging
Materials:? ? Lenti?Vectors
1. 2. Tat‐Dependent?(wild?type?5'‐LTR?):?Lv200?(GCI),?pLVX?(Clonteck),?TRIPZ?(Thermo? Scientific). Tat‐Independent?(hybrid?5'‐LTR?(CMV?or?RSV):?GCI?HIV?series,?pLenti4,?6?and?7? (Invitrogen),??pLKO(Sigma),?

? Packaging?mix:?gag‐pol,?VSVG,?Rev,?Tat. ? Cells:?293T ? Tranfection?reagent:?Ca++,?Liposome,?Polymers.?

Lenti‐Vector
CMV
cPPT

ORF

SV40

GFP

IRES

Puro
WPRE

pLvRCMV-GFP Puro
RRE

3’‐LTR ? U3/U5

5’‐LTR ?
U5/RSV(U3)

Ampr

pUC ori.

wt?5’‐LRT:?U3‐R‐U5,?Tat?dependent. Hybrid?5’‐LTR:?RSV(CMV)‐R‐U5,?Tat?independent. SIN 3’‐LTR:?Self?Inactivated.

Mammalian?Constitutive?promoters

Qin?JY,?et?al.?(2010)?PLoS?ONE?5(5):?e10611.

Packaging?mix

+
CMV
cPPT

ORF

SV40

GFP

IRES

Puro
WPRE

pLvRCMV-GFP Puro
RRE

3’‐LTR ? U3/U5

5’‐LTR ?
U5/RSV(U3)

Ampr

pUC ori.

Packaging?system?
Table? 1.?Host? cell?range?of?virus? pseudotyped with? different?envelope?proteins.?

Ecotropic

Amphotropic

Dualtropic

Pantropic

Envelope

GP70

4070A

Transfection?methods
? Ca++ precipitation. ? Lipid?mediated:?LF2000?(Invitrogen)?and?FuGENE?HD?(Roche). ? Polymers:??EndoFectin?(Fulengen?复能基因).

Lentivirus?preparation?
Day?1 Day?2

Plate?293T?cells?for?transfection.
24?‐ 48hours

Prepare?DNA?mix?+?EndoFectin
20‐30min

Add?to?293T?cells
6‐16?h

Change?Medium.
48‐72hours?after?transfection Day?4 Day?4‐7

Harvest?medium?(Viruses). Lentivirus?titration. Lentivirus?purification?and?concentration.?(optional) Store?Lentiviruses?in?single?use?aliquot?at?‐80°C

Packaging?Transfection?48?hours?
1‐100ms‐48h 2‐100ms‐48h 3‐100ms‐48h

Titration?of?the?Lentiviruses
Cells?for?Lentivirus?titration?:??Hela,??HT1080,?or?H1299?cells.? 1. Florescent colony counting or FACS counting: Infection units (IU/ml) 2. Resistant colony counting: Infection units (IU/ml) 3. RT-qPCR: copy number of the virus, not the infection unit

Titration?of?the?Lentiviruses?(cont.)
Flurescent colony counting: Transduction in series dilutions (0.02, 0.1, 1.0 and 5.0 ?l) in 6-well or 24well plates, count the fluorescent colonies under microscope 48-72h later. ? 10 x 10 eye field in microscope: Diameter = 1.9-2.2 mm; Area = 3 (2.8-3.8) mm2 For example: 0.1 ?l supernatant transduce one well (6-well plate or 24-well plate), and yield 20 GFP colonies in one eyefield under 10 x 10 magnification. ?Titer (6-well )= 20 x 320(950/3) x 10,000 ( 1,000/0.1ul) = 6.4 x 107 IU/ml ?Titer (24-well) = 20 x 63 (190/3) x 10,000 ( 1,000/0.1 ul) = 1.2 x 107 IU/ml

Titration?of?the?Lentivirus?in?24‐well?plates??
1‐‐ 2.5?x?107?IU/ml?? 2‐‐ 1.8?x?107?IU/ml? 3‐‐ 3.5?x?107?IU/ml?

0.1ul

1‐‐ 2.5?x?107?IU/ml??

2‐‐ 1.8?x?107?IU/ml?

3‐‐ 3.5?x?107?IU/ml?

1.0?ul

Titration?of?the?Lentiviruses(cont.)
Selection resistant colony counting: 1.Transduce cells in series dilutions, 2.Add selection antibiotics 24-48h later 3.Stain with crystal blue and count the resistant cell colonies after 1-2 weeks.

Titration?of?the?Lentiviruses(cont.)
RT-qPCR:
1.Extract RNA from Virus medium (dual RNA copy in virus) 2.Reveres transcription 3.qPCR (including a standard serial templates)

Primers: locate on the Lentivector backbone for universal use. Specific
primers may also be used.

Standard Curve setup:
lenti-vectors in 1x102, 103,104, 105, 106, 107, 108 copies/?l. The copy number of 1ng of a 9.3 kb vector = 1 (ng) / [9300(bp) x660] x10-9 x 6.022 x1023 (Avogadro’s constant ) = 9.811 x 107 molecules

Purification?and?Concentration?of?Lentiviruses
Pre‐step:??Filtration?(polyethersulfone?(PES)?filters)??or?general?centrifugation.?

1.??Traditional?CsCl?ultracentrifugation. 2.??Ultra?filtration: using?filter?<?0.01??M?(200KD)? 3.??Ion?exchange:?ViraBind? Concentration?&?Purification?Kits?(Cell?
Biolabs)?;?Fast‐Trap?Virus?Purification?and?Concentration?Kits? (Millipore).

4.??Precipitation:?LentiPacTM Lentivirus?Concentrator(复能基因).
? ? ? ? Quick?and?simple?concentration?of?lentiviral?particles(2‐3?hours). Increase?titer?(IU/ml)?by?10X?– 100X.? No?ultracentrifugation?is?required High?recovery?:??>?90%

Purification?and?Concentration?of?Lentiviruses?(Cont.) A. Pre?0.2ul B. Conc.?4h?0.02ul C. Conc.?16h?0.02ul

Figure.?Concentration?of?Lentiviruses:?GFP?containing?lentiviruses?(2x107IU/ml)?were?concentrated?10?times?using? LentiDens?and?then?transduced?the?H1299?cells?in?24‐well?plates.?The?images?were?taken?48?hours?after?the?transduction.? A:?0.2?ul?of?pre‐concetrated?lentiviruse;?B:?0.02ul?of?concentrated?lentivirus?(incubated?4?hours?with?LentiDens?before? centrifuge).?C:?0.02ul?of?concentrated?lentivirus?(incubated?16?hours?with?LentiDens?before?centrifuge).?

Application?of?Lentiviruses ?
1. 2. 3. 4. 5.

In?vitro:
Expression?of?genes?of?interest,?miRNA,?shRNA,?miRNA?inhibitors, etc.?in?cells Efficient?delivery?of?genes?to?some?hard‐to‐transfect?cells:?primary?cells,?stem? cells,?neural?cells… Easily?setup?stable?cell?lines. Delivery?of?reporting?system?for?cell?based?assay:?miRNA?target, signal?pathways,? prompters… Arrays?for?high?throughput?screen.?miRNA?(?Inhibitor),?shRNA,?Gene?expression.

?
1. 2.

In?vivo:
Deliver?genes?of?interest,?trackers?and?reporters?systematically in?animal?studies.? Gene?therapy?for?cancer,?infection?diseases,?genetic?diseases?…

Application?of?Lentiviruses?(in?vitro)
1.????Arrays?for?high?throughput?screen.?miRNA?(?Inhibitor),?shRNA,?Gene?expresson. ? Array?Lenti?library?generation?in?96‐well?or?384‐well?plates ? Plate?cells?into?wells?for?transduction ? Challenge?cells?for?drug?screen?or?gene?function?analysis. 2. Targeting?delivery?to?cell?genomes:? Non‐Integrative?Lentivirus?for?recombinase‐mediated?cassette?exchange?(RMCE).

Torres?R,?et?al?(2011):?PLoS?ONE?6(5):?e19794

Application?of?Lentiviruses?(In?Vivo)

Animal?studies?&?Gene?therapy?for?cancer,?infection?diseases,?genetic?diseases?.

Universal?delivery:?widespread?distribution?to?tissues?and?organs.
1. 2. Normal?lentiviruses:?Cancer?and?Genetic?Diseases. Integration?deficient?lentiviruses:?neuron?cells?and?other?non‐dividing?cells.?

Application?of?Lentiviruses?(targeting?delivery)
1.????Pseudotype:?Change?
the?tropism?(envelope?GPs)? of?the?lentiviruses.?

Cronin?J,?et?al.?Curr.? Gene?Ther.?(2005).?5(4):? 387

Application?of?Lentiviruses?(targeting?delivery,?conti.)
2.????Chimeric?GPs:?GPs?fusion?with?antibody,?ligands,?peptides.? ZZ‐SV?GPs scFV‐VSV‐G ZZ‐VSV‐G RGD‐SV?GPs

Ohno??K.?et?al.?Nat?Biotechnol.(1997)?15:763 Dreja?and??Piechaczyk.?Virology?J.?(2006)?3:69 Kameyama?Y?et?al.?J?Virol?Methods.?(2008)?153:?49 Morizono?K,?et?al.?J?Gene?Med?(2009)?11:?549

Pre‐clinic?Application?and?Clinic?Trial
? Lentiviral?vectors:?their?molecular?design,?safety,?and?use?in? laboratory?and?preclinical?research.?Dropuli? B. Hum?Gene?Ther.?(2011)?Jun?
22(6):649

? Gene?therapy?for?the?peripheral?nervous?system:?repair?the? injured?nerve.?Mason?MR,?et?al.?Curr?Gene?Ther.?(2011)?11:75;?Meunier?and?Pohl.?Gene?
Therapy?(2009)?16,?476.?

? lentiviral?vector‐mediated?gene?therapy?through?neural?stem? cells?&?cancer?stem‐like?glioblastoma.?Huszthy?PC?et?al.?PLoS?One.?(2009)?
4:e6314;?Rath?P?et?al.?Curr?Stem?Cell?Res?Ther.?(2009)?4:44.?

? Hematopoietic?stem?cell?gene?therapy?with?lentiviral?vectors.?
Neschadim?A?et?al.??Biol?Blood?Marrow?Transplant.?(2007)?3:1407;?Scaife?MD?et?al.?Expert?Opin? Biol?Ther.?2009?Jun;9(6):749 – Human?β‐thalassaemia:??Cavazzana‐Calvo?M?et?al.?Nature. (2010)??467:?318 – X‐linked?adrenoleukodystrophy?(ALD):?Cartier?N?et?al.?Science. (2009)?326:818;?

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