Nanomedicines for Systemic Delivery of RNAi Therapeutics - RNA Interference from Biology to Therapeutics - page 134

Two studies from the same group presented novel strategies to target leucocytes
for treating viral infections. scFvCD7Cys is a single-chain antibody against CD7 (a
surface antigen present on the majority of human T cells) that was modified to
include a Cys residue for conjugation to a 9 Arg peptide (Fig. 6.2f ). This conjugate
was used for targeted delivery of CCR5 (a chemokine receptor that functions as a
co-receptor for HIV) and Vif/Tat (HIV replication proteins)-siRNA payloads into T
cells, and has been demonstrated to suppress HIV infection in humanised mice
without inducing toxicity in the target cells [ 73 ]. A similar approach for treating
dengue virus infected cells employed DC3 (12-mer peptide that targets dendritic
cells)-9dR for targeting, with TNF-a (which plays a major role in dengue pathogen-
esis) or specific highly conserved sequence in the viral envelope. These complexes
significantly reduced virus-induced production of TNF-a and succeeded to sup-
press the viral replication in monocytes-derived dendritic cells and macrophages
in vitro. In vivo, treatment of mice with intravenous injection of DC3-9dR-
complexes containing TNF-a-siRNA effectively suppressed this cytokine produc-
tion by dendritic cells [ 74 ] .
Another approach for targeting leucocytes is based on leucocytes' integrins,
which are the largest family of cell adhesion molecules that mediate cell-cell and
cell-matrix interactions [ 75 ]. Antibody-protamine fusion proteins utilise the lym-
phocyte function-associated antigen-1 (LFA-1) integrin, a pan leucocyte cell surface
marker for selective targeting. The use of LFA-1 for targeting leucocytes is sup-
ported by its exclusive expression on leucocytes, its constitutive internalisation and
recycling activity and its ability to undergo activation-dependent conformational
changes. Using antibody-protamine fusion proteins, RNAi payloads could be
directed into leucocytes both in vitro and in vivo. Importantly, neither lymphocytes
activation nor interferon response induction was observed. Furthermore, by target-
ing these fusion proteins to the high affinity conformation of LFA-1 that character-
ises activated lymphocytes, the ability to induce more selective gene silencing was
demonstrated, which unlike most immunosuppressive therapies, could provide a
way to overcome the unwanted immune stimulation without global immunosup-
pressive effects on bystander immune cells. Additionally, due to the prevalence of
aberrant affinity modulation of integrins in a variety of leucocyte-implication dis-
eases [ 76, 77 ], targeting the high-affinity conformation of LFA-1 seems to be very
promising therapeutic strategy [ 78, 79 ] .
In order to increase payload and achieve more robust targeted gene silencing, a
different strategy was generated. Integrin-targeted and stabilised nanoparticles
(I-tsNP) that successfully deliver siRNAs into specific leucocytes subset involved in
gut inflammation were developed. Using this system, cyclin D1, a regulator protein
of the entry into, and the progression throughout the cell cycle, was identified as a
potential new therapeutic target for treating inflammation. The I-tsNPs were devel-
oped from neutral charged lipids of about 80 nm in diameter liposomes that were
loaded with siRNAs pre-condensed with human recombinant protamine. The parti-
cles were coated with hyaluronan (HA) for stabilisation during siRNA entrapment
and for prolonging the circulation time in vivo. The targeting of the particles to sub-
sets of leucocytes was achieved by attaching a monoclonal antibody against b 7 inte-
grin (which is highly expressed in gut mononuclear leucocytes) to the HA-coated