Evolución de la arquitectura IMS

> o <0 o

g

Day 2 Day 4 Day 6 Day 2 Day 4 Day 6 Day 2 Day 4 Day 6

Intracellular Secreted Total

a- L- fucosidase activity in untransfected cells a- L- fucosidase activity in transfected cells

Figure 5.21 Transfection of Fabry fibroblasts with LID complex containing luciferase cDNA.

Each bar represents the mean of the activities for three wells of cells which were assayed in triplicate.

* denotes that the enzyme activities of the transfected and untransfected cells were significantly different at a 99% level using a student t- test.

tOOOn 7 5 0 - > E

il

I

Day 2 Day 4 Day 6 Day 2 Day 4 Day 6 Day 2 Day 4 Day 6

Intracellular Secreted Total

1 J 3- hexosaminidase activity in untransfected cells L#-l 3- hexosaminidase activity in transfected cells

Figure 5.22 Transfection of Fabry fibroblasts with LID complex containing luciferase cDNA: distribution of p- hexosaminidase.

Each bar represents the mean of the activities for three wells of cells which were assayed in triplicate.

* denotes that the enzyme activities of the transfected and untransfected cells were significantly different at a 99% level using a student t- test.

Chapter 5._______________________________________________ Transfection of patient fibroblasts

Fabry patient fibroblasts have very low levels of a- galactosidase. There were no significant differences in the intracellular, secreted or total a-

galactosidase activity after transfection of Fabry patient fibroblasts with luciferase cDNA. In contrast, small but significant increases of secreted a- L- fucosidase were seen after transfection of Fabry patient fibroblasts with

luciferase cDNA. There were corresponding significant, small decreases in the intracellular a- L- fucosidase. A small decrease in intracellular p-

hexosaminidase was also observed after transfection, with a concomitant small increase in secreted p- hexosaminidase. There were no significant differences in the total p- hexosaminidase activity. The percentage of total p-

hexosaminidase activity secreted increased after transfection of Fabry patient cells with luciferase cDNA.

These results suggest that the changes observed in non- deficient lysosomal enzymes after transfection are the result of transfection and do not depend on the type of enzyme being introduced or its cellular destination. Transfection seems to cause a very small decrease of endogenous intracellular lysosomal enzymic activity and a small increase in secretion. The large

amounts of transgene secreted were much greater than these non- specific changes. These results suggest that there is a problem of targeting all

lysosomal enzymes synthesised after transfection or that there is disruption of the lysosomal system.

5.5. Discussion

The levels of enzymic activity in cultures varied considerably between experiments because of differences in the rates of growth of different cell lines. Therefore comparisons have been made on the basis of changes in the

distribution of enzymic activity for comparable cultures rather than changes in absolute specific activities. This variation between experiments is due to the different cell lines used and differences in protein levels between experiments. To minimise differences between experiments, all experiments on the same cell line transfected with the same LID complex were carried out at the same time. The a- galactosidase activity measured in these experiments was due to both the A and B forms (approximately 95% is in the A form). Therefore, when a-

Chapter 5._______________________________________________ Transfection of patient fibroblasts

galactosidase A was deficient a- galactosidase B would contribute to a measurement of a- galactosidase activity.

Transfection of fibroblasts deficient in an enzyme with the LID- containing cDNA for that enzyme produced overall increases in the target enzyme. This increase was 7- fold for a- L- fucosidase in fucosidosis fibroblasts and 5- fold for a- galactosidase in Fabry fibroblasts. These increases would be enough to correct the cells metabolically if the active enzyme had been

delivered to lysosomes. However, the increased activity was mainly in the secreted fractions and would only correct cells it was taken up by cell surface mannose- 6- phosphate receptors and targeted to the lysosomes. There were very small increases in intracellular activity which, considering transfection generally depleted the intracellular lysosomal enzymes, suggests that small amounts of transgene products may be targeted to the lysosomes. Transfection did not affect the total synthesis of the non- deficient lysosomal enzymes but did cause a slight increase in the fraction that was secreted.

LID complexes containing a- L- fucosidase and a- galactosidase A cDNA were also used to transfect normal cells (both fibroblasts and ECV304 cells). The increases in the total activity of the transgene were greater in normal cells than in the lysosomal storage disease cells. This suggests that the lysosomal storage disease cells may be transfected less efficiently. Transfection of normal fibroblasts with LID- containing a- L- fucosidase or a- galactosidase A cDNA produced increases of 50% in the a- L- fucosidase or a- galactosidase activities in the cultures after 6 days. ECV304 cells were also efficiently transfected with LID- containing a- galactosidase A cDNA demonstrating that endothelial cells could be successfully targeted with this system for Fabry disease. Transfection of normal fibroblasts and ECV304 cells did not affect the total synthesis of endogenous lysosomal enzymes but caused a slight increase in their secretion. This was not compensated for by the intracellular activity of the transgene, as was seen in deficient cells. The fact that this intracellular increase only occurred in deficient fibroblasts suggested that this phenomenon depended on the

original intracellular levels of the enzyme within the cells or that the

phenomenon also occurs in normal cells but is not detected. Since transfection of normal cells with LID- containing luciferase cDNA effected endogenous

Chapter 5._______________________________________________ Transfection of patient fibroblasts

lysosomal enzymes in the same way it is concluded that transfection and not the specific cDNA used causes the increased secretion. The intracellular decreases were balanced by increased secretion. It may be that transfection made the cells more sensitive to serum withdrawal and that this increased the secretion of lysosomal enzymes. Secretion of lysosomal enzymes could also be part of a stress response to transfection. Secondary lysosomes may have released their contents into the medium or all newly synthesised enzymes may have been secreted.

The secreted overexpressed enzyme bound concanavalin- A Sepharose suggesting that it had been glycosylated in the endoplasmic reticulum.

Preliminary experiments were unable to demonstrate uptake of the secreted enzyme into lysosomal storage disease fibroblasts. Re- uptake by other deficient cells would need to be established if this is to be a viable method for gene therapy of lysosomal storage diseases.

5.6. Conclusions

Transfection of fibroblasts from patients with lysosomal storage disease with LID complexes containing cDNA for the appropriate lysosomal enzyme

produces large amounts of the enzyme which are secreted from the cells. Although this enzyme would appear to be glycosylated in the ER it is not known

if it has the mannose- 6- phosphate recognition marker required for correct targeting to the lysosomes. The LID transfection system had small effects on endogenous lysosomal enzymes. The main questions raised are why the transgene products are secreted and whether they can be recaptured by other deficient cells and correct the disease phenotype.

Chapter 6.___________________________________________________________________ Discussion