Translocation of target genes with surface-modified lentiviral vectors

Summary

Two types of surface modifications have been made to lentiviral vectors in order to achieve target gene translocation: (i) taking advantage of the targeting properties of other viruses by incorporating heterologous viral glycoproteins (termed pseudotyping); and (ii) fusion of heterologous peptides with viral glycoproteins to enable lentiviral particles to be re-targeted to the cells of interest. This chapter provides a novel approach to enhance tissue targeting of lentiviral vectors and an example protocol for targeting transduced hematopoietic stem cells ( H S C ). Author: T. Friedman et al, Translated by Wei-Qing et al. This experiment is from "Gene Transfer".

Operation method

Raking of hematopoietic stem cells with surface-engineered lentiviral vectors

Move

Raking of hematopoietic stem cells with surface-engineered lentiviral vectors Materials

reagents

293T cells

Anti-CD34 albumin antibody (BDPharmingen)

C a l p h o s Mammalian transfection kit for calcium phosphate-mediated transfection (Clontedi)
Cellgro medium (serum-free medium; Cellgenix)

DMEM medium with 0.l l g/L sodium pyridoxine and vitamin _B6

DMEM was supplemented with 10% fetal bovine serum, 100 Wow/m l streptomycin< ! >, lOOU/ml penicillin, stored at 4°C.

Fetal Cow Serum (F C S , sterile)

FicoU-PaquePlus (sterile; A m e r s h a m )

H e L a cell

Hematopoietic stem cells (H S C ; purified from newborn cord blood as follows)

Many studies have shown that HSC are present in cell populations that express CD34+ antigen. Approximately ⅔ of the mononuclear cells in cord blood are CD34+.

N O D / S C I D mice

RNA

Lentiviral vector D N A encoding a H I V - I-derived self-inactivating vector with an intrinsic EFl-a promoter

driving the green fluorescent protein (GFP) reporter gene.

Shell glycoprotein expression plasmid

Fusion glycoprotein V S V - G

Glycoproteins that activate and target hematopoietic stem cells: ① TPOHA (T P O fusion H A shell glycoprotein)
white); ② S C F S U x (S C F fused M L V shell glycoprotein).

Viral structural protein (G a g -Pol) Expression Plasmid (p C M V 8. 91)

Neuroacidases Expression Plasmid P C M V - N A

Phosphate buffer (P B S ) , calcium and magnesium free, helium sodium carbonate free, sterile

Digestive reagents'

Trypsin / E D T A

I X H a n k balanced salt solution (H B S S ), calcium and magnesium free, sterile

Instrumentation

C D 34+ cell isolation kit from Miltenyi containing anti-human C D 34 microbeads and blocking reagents
Membrane (0.45um) 45um)

Flow-through system capable of cell sorting and trichromatic analysis

Flow Tubes

MACS Cell Separation Columns (Miltenyi Biotec)

MACS Separator (MiltenyiBiotec)

Standard tissue culture reagents, including 100m m, 6-well and 48-well tissue culture plates

Generation of lentiviral vectors capable of displaying active peptides

1.0 day: one day before transfection, inoculate 2. ^5X106 293T cells in IOOmm tissue culture dish, IOmlDMEM culture.

Day 2.1: Co-transfect 8. 6ug of H IV packaging plasmid and 8. 6ug of lentiviral gene transfer vector and two glycoproteins: V S V - G (1,5 khoo) and TPO H A (1.5 ug) with calcium phosphate transfection reagent. Co-transfection of TPO H A and the plasmid encoding neuraminidase allowed efficient release of the virus from the producing cells. Otherwise, the vector particles would bind to the production cells through the mutual reaction of the H A shell and the salivary acid receptor.

3.2 Days: 15 h after transfection, the medium was replaced with 6 m l of fresh Cellgr◦ medium.
This medium is suitable for culturing CD34+ cells and allows cytokines to be displayed on the surface of the vector to maintain function.

4.3 Days: 36 h after transfection, collect the vector, filter through a 0.45f x m membrane, and store at 80°C. The vector can be stored for 2--3 months. Vectors can be stored for 2--3 months.

Immunoselection of Human CD34+ Cells

5- Dilute the squeeze band blood (C B ) with P B S l : 1 , and gently place 35 m l of diluted blood over I5 m I FicollPaque Plus in a 50 m l tube.

6. Centrifuge cells at 850 g for 30 m i n at 20°C without braking. Collect the layer containing mononuclear cells.
Mononuclear cells are the white bands located at the top of the Ficdl layer.

7 . Wash the collected monocytes with ?83 and 2% ? The collected monocytes were washed with ?83 and 2% ?3 and centrifuged at 850 g for 10 min at 20 °C.

8. Resuspend the cells with PBS and 2% FC S at a concentration of IX ^lO8 ~ ^2X108 cells. Add anti-human CD34+ micromagnetic beads to magnetize the cells according to the manufacturer's instructions. Incubate at 4°C and shake for 30 min.

9-Wash cells to remove unbound antibody and resuspend cells with PBS/2% FCS.

10-Wash M A C S separator column with 200ul P B S / 2 % F C S. Add labeled cells to column and place in M A C S separator. Allow unlabeled cells to pass through the column. Wash once with P B S / 2 % F C S. Remove the column from the separator to elute CD34+ cells. Repeat the procedure once.

The purity of CD34+ cells is typically 9 0 % to 9 5 % .

Titer determination

11-The day before: inoculate H eL a cells in a 6-well plate at 2 X ^IO5 cells/well with a final volume of 2 ml DMEM/well. Incubate overnight.

12.0 day: add a series of diluted vector preparations to H e L a cells and incubate overnight.

Day 13.1: Replace cell culture medium with 2 ml fresh DMEM and incubate for 72 hours.

14.3 days: Digested cells transferred to flow tubes. Determine the percentage of GFP positive cells by flow-through.

Infection titers were expressed as transduction units (TU)/ml, calculated by the following formula:
Titer = % G F P positive cells (number of cells at the time of infection/100) X dilution factor
Multiple infectivity (moi) is the ratio of infectious particles to the number of target cells.

Human CD34+ cell transduction

15. Inoculate C D 34+ C B cells in 48-well plates at 5X ^IO4/well in C e U G r o medium. Transduce fresh lentiviral vector supernatant at a multiple infection level of 20 or 4. Incubate for 24 h.

16. Wash and resuspend the transduced cells with Clariglo medium. Incubate for 48 h. Immunolabel cells with anti-CD 34-PE antibody and analyze GFP expression by flow cytometry to determine transduction efficiency.

NOD/SCID retesting

17- 24 h after transduction of T P O H A demonstrated, S C F S U x demonstrated lentiviral vectors , for intravenous injection of nonlethal radioactive (3.5 G y ) C D 3 4 + C B cells into N O D /S C I D mice without in vivo cytokine administration.

18. Bone marrow is collected from the femur 6 to 8 weeks after transplantation.

19- G F P + human cells of different lineages were detected in N O D / S C I D bone marrow with anti-h C D 45-C y C h r o m e , anti-h C D 19-P E, anti-h C D 14-P E, anti-h C D 13-P E, and anti-h C D 34-P E antibodies by applying a three-color flow-through.
In all cases, P E-coupled mouse IgG controls should be used to assess specificity markers.

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