TY  - UNPB
TI  - Protein and cell therapy for lecithin-cholesterol acyltransferase (LCAT) deficiency
N2  - Lecithin-cholesterol acyltransferase (LCAT) is an enzyme principally secreted by the
liver into the circulation where it esterifies cholesterol and plays a key role in high-
density lipoprotein (HDL) metabolism. In familial and acquired (liver disease) LCAT
deficiency, the failure to esterify cholesterol causes many cellular and metabolic
disturbances. Here, I describe the purification of recombinant LCAT and assess two
approaches to treat LCAT deficiency. Human LCAT cDNA was cloned into a
selectable expression vector and used to generate a stably?transfected Chinese
hamster ovary (CHO) cells secreting human LCAT tagged with 6 histidine residues.
Productive clones were selected, monitoring LCAT activity by a modification of a
radioactive enzymic assay for plasma, and the enzyme purified from culture medium
by immobilised cobalt affinity chromatography. The pure LCAT, as judged by SDS-
PAGE, was used to raise monoclonal antibodies in LCAT knockout mice for future
development of a sensitive immunoassay. For therapy, I evaluated injection of pure
LCAT into the peritoneal cavity of LCAT knockout mice using single and repeat
dose regimes. LCAT activity was measurable in plasma post-injection and the
percentage of esterified cholesterol increased, while agarose gel electrophoresis
confirmed a rise in HDL levels. In a second approach, I encapsulated the
recombinant CHO cells in biocompatible and semipermeable alginate-polylysine
microcapsules using a syringe pump extrusion method. A study in vitro showed that,
after an initial lag phase, LCAT was secreted for over 90 days with the capsules
remaining intact. These microencapsulated cells were implanted into peritoneal
cavities of LCAT-deficient mice. LCAT activity was detected in mice plasma one
week post-implantation; the relative amount of esterified cholesterol was increased
and lipoprotein profile was improved. I conclude that injection of recombinant
enzyme or of encapsulated LCAT-secreting cells are feasible therapies for familial
and acquired LCAT deficiency.
N1  - Unpublished
ID  - discovery14474
EP  - 217
UR  - https://discovery.ucl.ac.uk/id/eprint/14474/
PB  - UCL (University College London)
M1  - Doctoral
A1  - Low, J.K.
AV  - public
Y1  - 2009/01//
ER  -