Non-viral gene transfer experiments with Trojan horse liposomes that can cross the blood-brain barrier

Summary

Non-viral vector plasmid DNA can be delivered to the human brain via the vascular route across the blood-brain barrier by intravenous injection of DNA encapsulated in Trojan horse liposome (T H L , also known as P E G y-Iates immunoliposome, P I L ). The IOOnm surface of the liposome is covered with thousands of multimers such as 2000Da polyethylene glycol (PEG), or PEG 2000. 1 % to 2 % of the PEG molecules are covalently bound to the ends of a targeted monoclonal antibody (M A b ) as a molecular Trojan horse (M T H ). M T H binds to love bodies such as transferrin and insulin on the blood-brain barrier and brain cell membranes, and is subsequently triggered in vivo by receptor-mediated cellular delivery of T H L across the blood-brain barrier and receptor-mediated cell-penetration into brain cells.

Author: T. Friedman et al., Translated by W. Qin et al. This experiment is from "Gene Transfer".

Operation method

T H L preparation

Move

T H L preparation Materials

reagents

Instruments

Ultrasonic Scrubber

Hand extruder (e.g., Iiposofast Hand Extruder, Avestin) and polycarbonate filters, two each with pore sizes of 400 nm and IOOnm (50 nm may also be required; see Step 4)

Gel Filtration Analyzer

Glass tube (12m m X 75m m)

Millipore Millex-GV Filter (0.22um)

Laser Force Analyzer

Shake table

Rotary evaporator (optional, see step 4)

Scintillation Counters

Spectrophotometer

Vortex

Methods

1 . Radiometric labeling of M Ab.

The number of molecules of M A b attached to T H L is determined as part of the quality control metrics for each batch of T H L.

a. Labeling of the e-amino group on the lysine residue of M Ab with NSP may also be radiolabeled with [ 125I ] iodine and chloramine T or tetrachlorodiphenylsulfonylurea. The 125I-labeled MAb can be discarded after one month,3 and the H-labeled M Ab can be stored at 20°C for 6 to 12 months.

b. Determine the TCA sedimentation rate of the radiolabeled MAb prior to each THL reaction.

c. Purify by gel filtration chromatography those MACs with a TCA sedimentation rate of less than 95%.

2- Label a portion of the plasmid DNA with 32P radiation by notch panning and measure the amount of DNA encapsulated in the THCL as a quality control indicator for each lot of THCL. The 32P-labeled plasmids are discarded after IO d of storage at 4 °C.

3- The sulfhydryl group of MAB is linked to the maleimide group of D SPE-PEG 2000-maleimide to form a stable thioether bond. Thiolate M A b as follows.

a. Prepare the following solution in a 12 mm X 75 mm glass tube:
3.Omg of MAB (20 nmol)

2uCi3 H labeled M A b

Approx. 200ul 0 . 15m o l/L sodium borate buffer (p H 8.5)/EDTA (0.lm m o l/ L)

b. Add 1200 nm ol Traut reagent.

When the target M Ab is a mouse IgG2a isoform (e.g., 0 X2 6 or 83-14 MAb), the molar ratio to the M Ab is 60:1, and when the target M Ab is a rat IgG (e.g., 8D3 MAb), 40:1 is used as the molar ratio. The purpose is to add 1 to 1.5 more sulfhydryl groups to each MAb.

c. Determine the number of sulfhydryl groups by using the Elmain reagent and spectrophotometer.

If the number of sulfhydryl groups on each MAb is greater than 1.5, intramolecular cross-linking of M Ab may have occurred. 5 per MAb, intramolecular cross-linking of M Ab may have occurred. If the number of sulfhydryl groups on each MAb is less than 1.0, the binding efficiency of M Ab to liposomes will be reduced.

4 . The steps of liposome generation and precipitation are as follows:

a . Prepare the following solution in a 12 mmX75 mm glass tube spiked with Im l chloroform:

18.6/nmol POPC

0.6umol DDAB

0.6umol DSPE-PEG 2000

0.2umol DSPE-PEG 2000-maleimide

b. Completely evaporate the solution under a stream of nitrogen while vortexing continuously to produce a thin liquid film layer.
A rotary evaporator may be substituted. Take care to produce a thin liquid film layer.

c- Add 300ul of 0.05mol/L HEPES (pH 7.0 ) hydrated lipids to make approximately 100 mmol/L.

Hydration and subsequent vortexing should be handled carefully without interruption.

d - The hydrated lipids are placed in an ultrasonic cleaner for 2 m i n , vortexing at maximum speed for l m i n . The diameter of the liposomes was determined by a laser intensity analyzer using the dynamic scattering particle measurement technique. After hydration, vortexing and ultrasonication, the size of the liposomes should be 500 ~ lOOOnm. monitor the size of the liposomes at any time during the process.

e- Add plasmid DNA (100~250ug) and 32P-labeled plasmid DNA of approximately Iu C i to the hydrated lipids.

f. Freeze the mixture for 5 m i n in liquid nitrogen or dry ice or in an ethanol bath at 37°C to dissolve the lipids lOmiri. Repeat this cycle .5 to 7 times.

The plasmid DNA is encapsulated in large liposomes during repeated freeze-thaw cycles. If there are excessive air bubbles in the sample they will interfere with the next step of the precipitation process and should be removed.

g. Add HEPES Buffer (0.05 m o l/L, p H 7.0) so that the final volume of lipids is approximately 500 ul or 40 mmol/L.

h - To reduce the size of the liposomes, filter the lipid/DNA mixture through two stacked layers of 400 nm pore size polycarbonate filter membranes on a manual extruder. Repeat 5 times. Place two stacked IOOnm pore size polycarbonate membranes in the extruder and filter the lipid/DNA mixture 5 times.

The optimal diameter of the liposomes at this point should be 80 to 120 nm, and to reach this diameter it may be necessary to pass the mixture through two layers of membranes with a pore size of 50 nm.

5. Nuclease degradation is performed as follows:

a. Add tryptic endonuclease I and exonuclease III to the liposomes and incubate at 37°C for 60 mIn.

If the precipitation process is done correctly, 40% to 60% of the DNA will be encapsulated in the IOOnm liposomes, but there will still be residues.

This unencapsulated DNA is toxic in vivo and must be removed. This is why the action of nucleases is required (Monnardetal. 1997).

b . The reaction was terminated by the addition of EDTA at a final concentration of 2Ommol/L .

The use of strong anion-exchange columns such as DEAE is not recommended for the removal of external DNA because it does not remove anionic DNA that is electrostatically attached to positively charged lipid residues on the liposome surface. T H L purification with DEAE columns can contain a large amount of unencapsulated DNA, which can lead to in vivo toxicity and inflammatory reactions (Norman etal. 2000).

6. ligation and gel filtration chromatography overnight:

a. Mix nuclease-treated P E G y Ia te d liposomes with thiolated M A b .

b. Seal the solution, add nitrogen, and lightly shake overnight at room temperature.

C- Add the preparatory solution to an I.5c m X 20c m Sepharose C L-4B column equilibrated with 0.05m o l / L H E P E S (p H 7.0). The column is washed with H E P E S buffer at l m l/m i n at room temperature to collect Iml fragments (H u w y l e r et al. 1996).

A The 3 H and 32P radioactivity of each fragment was counted.3 H/32P double isotope scintillation counts were performed, with 3 H counted in a 0-16 k eV window and 32P counted in a 16-1700 k eV window.3 H/32P double isotope scintillation counts were performed.

e- Determine the percent of encapsulated D N A using the radioactivity of 32P at the first or liposome peak of the C L -4B column.

f- The radioactivity of the same peak 3 H is used to calculate the number of individual T H L bound M A b molecules.

T H L peaks at fragment 10, approximately IOml elution volume. Uncovalently bound MAB eluted at about 25 ml, and nuclease-degraded nucleotides eluted at 30 to 35 ml. There is no spillover of 3 H in the 32P channel, but there is about 2% spillover of 32P in the 3 H channel. The spillover should be taken into account when calculating the radioactivity of 3 H and 32P.

7 . Prior to the use of T H L, it was decontaminated by filtration with a 0.22 M m W M i l l i p 0re Millex-G V membrane, which does not alter its structural integrity (Z h a n etal.2002b ). T H L should be used in cultured cells or injected into human animals within 24 h of its generation, preferably on the same day as the elution of the C L-4B column. T H L can be stored at 4°C overnight.

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