Material and methods 2.6.2 Peptide component of LID complexes

2.6.2.1. Peptide synthesis and purification

Peptides containing an RGD sequence and a polylysine domain were used in LID complexes for transfection. The RGD domain binds to integrins on the cell surface and mediates internalisation of the complex. The poly-lysine domain binds to DNA by electrostatic interaction of the positive lysines with the negative phosphate backbone of DNA. This might also allow a degree of condensation of the DNA.

Peptide K16 was a gift from Dr. A. Miller, Department of Chemistry, Imperial College, London. Peptide 6 was synthesized and purified in the Peptide Synthesis Laboratory of the Imperial Cancer Research Fund, London as described by Hart et al., (1998). Peptide 1, peptide 5 peptide 9 and 11 were synthesized and purified by Zinsser Analytic. Details of the peptides are in Table 2.7.

The peptides were diluted in OptiMEM medium (Gibco-BRL) to 0.1 mg/ml and incubated overnight at 4 X , exposed to air to allow oxidation of cysteine residues to cyclise the RGD domain. The solution was then stored at -70°C in 1.5ml aliquots prior to use for transfections.

Peptide name

Peptide sequence Molecular

weight

Net charge

Reference

K16 [K]i6 2079 +16

Peptide 1 [KJieGACRGDMFGCA 3124 +16 Hart et a!., 1994,1995

Peptide 5 [KJieGACDCRGDCFCA 3271 +15 Koivunen et a!., 1995;

Hart et a!., 1997

Peptide 6 [K]i6GACRRETAWACG 3331 +17 Koivunen et a!., 1995

Peptide 7 [K]i6GAGPEILDVPST 3206 +14

Peptide 9 [K]i5GACRRETAWACG

K GACRRETAWAGG

4594 +18

Peptide 11 [KjieGACRGEMFGCA 3151 +16

Chapter 2. Material and methods

Calculations of charge and molecular composition of transfection complex

The charge ratios of LID complexes were calculated using the data in Table 2.8. The number of moles of negative charge per mole of plasmid DNA was calculated on the basis of 1 negatively charged phosphate group being

attached to each base, multiplied by 2 to take into account both strands of DNA e.g. 1pg of pGL2 plasmid DNA (6 046bp, Mr=3.92 x 10®) contains 0.26 pmol of plasmid and 3.1 nmol (0.26 x 10 x 12 092) of negative charge. In the peptides aspartic acid and glutamic acids contribute one negative charge whereas lysine and arginine each contribute one positive charge e.g. each mole of peptide 1 contributes 16mol of positive charge.

Moiecuie Molecular weight Net charge Peptide K16 2 079.84 +16 Peptide 1 3 124.45 +16 Peptide 5 32 7 1 .1 7 +15 Peptide 6 3 331.5 +17 Peptide 7 3 331.5 +14 Peptide 11 3151.3 +16 N-[1-((2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) 669.5 +1

Dioleoyl phosphatidylethanolamine (DOPE) 744 0

2,3-dioleyloxy-N(sperminecarboxamido)ethyl)-N, N-dimethyl-1 - propanaminium trifluoroacetate (DOSPA)

1461 +5

(Tfx) 1048.5 0

pGL2 plasmid (6 046bp)

3 .9 2 x 1 0 * -12 092

Table 2.8 Formula weight and charge of components used in transfection compiexes.

2.6.3. Liposome component of LID complexes

The liposomes used routinely in this study were Lipofectin (Life

Technologies), Lipofectamine (Life Technologies) and Tfx (Mirus). Lipofectin is a formulation of 1:1 N- [1-((2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE).

Chapter 2.___________________________________________________ Material and methods

Lipofectamine is a formulation of 2,3-dioieyioxy-N(sperminecarboxamido)ethy!)- N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and DOPE. These formulations were stored at 4°C. It has been proposed that the liposome destabilises endosomes containing the LID complexes, allowing them to escape into the cytosol.

2.7. Transfection of cells with LID complexes

2.7.1. Preparation of cells for transfection with LID complexes

Two normal cell lines were used in experiments to optimise the transfection system for delivery of genes to fibroblasts. Optimisation experiments were generally repeated on the second cell line to verify the results.

LID complexes containing a- L- fucosidase cDNA were used to transfect cells from fucosidosis and Fabry patients as well as normal controls. LID

complexes containing a- galactosidase A cDNA were used to transfect cell lines from Fabry patients, normal controls and ECV304 cells.

Fibroblasts and ECV304 cells to be transfected with LID complexes containing reporter cDNA were seeded into 24- well plates (Helena

Biosciences) at a density of 5 xIO* cells per well for transfection with LID complexes containing lysosomal cDNA the fibroblasts or ECV304 cells were seeded into 6- well plates (Helena Biosciences) at a density of 25 x10* cells per well. The cells were allowed to grow overnight at 37°C in an atmosphere of 5%

CO2.

2.7.2. Preparation of LID complexes

The plasmid was diluted in an Eppendorf tube so that there was Ipg of plasmid in lOOpI or 5pg of plasmid in 500pl of OptiMEM serum- free medium (Gibco life technologies) per well of cells for transfection with the reporter cDNA and lysosomal cDNA, respectively. For reporter cDNA transfections, the

transfection complex was prepared by putting 0.75pl Lipofectin in an eppendorf tube to which was added 40pl of peptide solution followed by lOOpI of plasmid DNA per well to be transfected. The solutions were mixed thoroughly after each addition. The ratio of the components was Ipg plasmid: 4pg peptide: 0.75pg Lipofectin. For lysosomal cDNA transfections, the transfection complex was

Chapter 2.___________________________________________________ Material and methods

prepared by adding first 3.75pl Lipofectin then 200pl peptide solution and finally 500pl of plasmid DNA solution per well to be transfected to an Eppendorf tube, with mixing at each step. The ratio of the components is the same in all

experiments. The complexes were incubated at room temperature for 30 min. The volume was then made up to 500pl (reporter cDNA transfections) or 2500pl (lysosomal cDNA transfections) per well to be transfected with OptiMEM and the solution mixed gently.

2.7.3. Transfection

The culture medium was removed from the cells to be transfected and 500pl (reporter cDNA transfections) or 2 SOOpI (test cDNA transfections) of transfection complex solution was added to each well. The plates were then incubated for 6 h at 37“C in an atmosphere of 5% CO2. The transfection

medium was removed and replaced with 0.5ml normal growth medium (reporter cDNA transfections) or 2ml OptiMEM (Gibco life technologies) low serum

medium (lysosomal cDNA transfections) 0.25ml or 1ml of the appropriate medium was added to the medium already in the well every 2 days until the cells were harvested.

2.7.4. Analysis of transfected cells and medium

LAC Z histochemical staining

The medium was removed from the cells and they were washed twice with PBS, before fixing by incubation in 0.5% (v/v) glutaraldehyde in PBS for 15 min at room temperature. They were then washed 3 times for 5 min with PBS at room temperature, p- Galactosidase activity in cultured cells was assessed by staining with X-gal solution (Section 2.3.2.1).

Luciferase assays

Reporter Lysis Buffer (RLB, Promega) was diluted to make a 1 x stock. Cells were grown in 24- well plates (approximately 5pg protein per well). Growth medium was removed from the cells to be assayed and the cells were washed twice with PBS. lOOpI of RLB was added to each well and incubated at 4°C for 15 min to lyse the cells. The cells were then scraped off the cell surface and the

Chapter 2.___________________________________________________ Material and methods

dish tilted to concentrate the cell suspension at the lower edge of the well (taking care to scrape off all visible cell debris). The cell lysate was transferred to an Eppendorf tube, vortexed briefly and incubated at -70°C for 90 min. The samples were then thawed to room temperature, vortexed briefly and

centrifuged at 12 000 x g for 15 min at 4°C. The supernatant was transferred to a fresh tube and allowed to equilibrate at room temperature before being

assayed (Section 2.3.2.2).

Lysosomal enzyme assays

Cells were trypsinized in initial experiments but gently scraping the cells off the dish was found to be more efficient. The cells were scraped gently off the dish in 200pl of sterile PBS using a Gilson tip. The dish was tilted to decant the released cells to the lower edge of the well. The cell extract was transferred to an Eppendorf tube, vortexed briefly and centrifuged at 12 000 x g for 15 min. The cells were then washed in 200pl of sterile PBS and sonicated in lOOpI of water for 10s at an amplitude of 6 micons in an MSE ultrasonicator. The sonicate was vortexed and centrifuged at 2000 x g for 3 min and the pellet of cell debris discarded. Cell extracts were stored on ice and the lysosomal enzymes were assayed (section 2.3.3).

After transfection (see section 2.7.3) cells were grown in OptiMEM low serum medium (Gibco life technologies) and fresh medium was added to that already in the well every 2 days. At certain time points over a week, the spent medium was removed and centrifuged at 1 000 x g for 5 min and the cells were harvested as described above. The supernatant was placed on ice and the lysosomal enzymes were assayed as described in section 2.3.3.

Chapter 3.__________________________________________ Characterisation of fucosidosis patients