TARGETING INTERFERON-a TO THE LIVER: APOLIPOPROTEIN A-I AS A SCAFFOLD FOR PROTEIN DELIVERY - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 427

gene was introduced into the hepatocytes using an adenoas-
sociated virus (AAV) type 2. This virus infects the liver and
allows the long-term expression of the transgene in

molecules such as liposoluble vitamins. The protein com-
ponents of the lipoproteins are formed by the apolipopro-
teins. These proteins are involved in the assembly of the
lipoproteins and they act as coactivators of enzymes and as
ligands of receptors for their cellular uptake.
Apolipoproteins are a heterogeneous family with differ-
ent sequences and sizes. They are classified as Apo As (A-I,
A-II, A-IV, and A-V), apo Bs (B-48 and B-100), apo Cs (C-I,
C-II, and C-III), apo D, apo E, apo J, apo F, apo J, apo H, and
apo M [33]. Apolipoprotein A-I is the major protein con-
stituent of the high-density lipoprotein (HDL) particles.
HDLs play a central role in reverse cholesterol transport,
the process by which excess cholesterol in peripheral tissues
is esterified in plasma and transported to the liver for
excretion (Figure 29.2). This function of HDLs is believed
to explain the strong inverse correlation between plasma
HDL levels and coronary heart disease [34]. Apo A-I is the
preferential acceptor of cell cholesterol [35], acting as
cofactor for the lecithin cholesterol acyltransferase
(LCAT) enzyme [36] and as a ligand for the putative
HDL receptor: scavenger receptor class B type I (SR-BI)
[37,38].
Apo A-I is synthesized both in the liver and in the
intestine as pre-pro-apo A-I, and is then processed to the
mature form of 243 residues [39]. Then, functional inter-
actions between apo A-I and ABCA1 are necessary for the
initial lipidation of apo A-I. Lipidated apo A-I proceeds to
form discoidal HDL particles that can be converted to
spherical particles by the action of LCAT. Discoidal and
spherical HDL can interact functionally with SR-BI and
these interactions lead to selective lipid uptake and net efflux

10% of
total hepatocytes. However, most of the woodchucks elim-
inated the transgene after 2 months and, therefore the
antiviral efficacy of this intervention was limited. Nowa-
days, after several failures in clinical trials, it is clear that the
inflammation limits the long-term expression of transgenes.
So, long-term expression of an inflammatory cytokine is a
challenge [31]. The only woodchuck with sustained expres-
sion of IFN-a exemplified other limitations of the IFN-a
gene therapy. Three months after AAV infection, this animal
had to be sacrificed because of the development of severe
blood cytopenia and liver steatosis. Sustained liver expres-
sion of IFN-a proved lethal for the animal.

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29.1.4 Apolipoprotein A-I as a Scaffold for Peptide and
Protein Delivery

Lipids are insoluble and they need to be carried as lip-
oproteins to be transported in plasma. Lipoproteins consist
of a hydrophobic nucleus of nonpolar lipids (triglycerides
and esterified cholesterol), enveloped within a hydrophilic
surface containing amphipathic lipids (phospholipids and
nonesterified cholesterol) [32].
Lipoproteins are classified on the basis of the hydrated
density, a function of the lipid/protein contents. This enables
the lipoproteins to be separated by ultracentrifugation.
The main functions of lipoproteins are (i) transport of
exogenous and endogenous triglycerides, (ii) transport and
removal of cholesterol, and (iii) transport of hydrophobic

FIGURE 29.2 HDL metabolism. Apo A-I is synthesized both in liver and intestine. After lipidation
steps, discoidal HDLs are converted to spherical HDLs by LCAT, and these HDLs can interact with
SR-BI to promote cholesterol efflux. HDL, high-density lipoprotein; LDL, low-density lipoprotein;
Apo, apolipoprotein; CE, cholesterol ester; TG, triglyceride; LCAT, lecithin cholesterol acyltrans-
ferase; SR-BI, scavenger receptor class B type I.