1 Introduction
As of today, there is no cure for infection with human immunodeficiency
virus type 1 (HIV-1). The available antiretroviral therapies (ART)
target different steps in the HIV replication cycle. Although ART
manages to reduce HIV burden below detection limit as long as the
medication is taken, it does not eradicate the virus. Inhibition of HIV
entry has been considered a promising strategy that was explored in
numerous ways. In particular, targeting CCR5, the co-receptor of
prevalent HIV-1 strains, has been pursued using CCR5 inhibitors or
blocking antibodies [1]. But again, even when combined with other
ARTs, they do not eradicate HIV.
On the other hand, the knowledge that a naturally occurring 32
nucleotide deletion (∆32) mutation in CCR5 that abrogates CCR5
protein expression leads to resistance to R5-tropic HIV-1 strains
[2] [3], has spurred some encouraging approaches [4]. People
with a homozygous Δ32 mutation are clinically unremarkable and conduct a
normal life [5]. The Δ32 allele can only be found in the Caucasian
population and follows a specific distribution pattern in Europe. It was
proposed that the higher allelic frequency of this mutation in Northern
Europe of about 10% was due to smallpox infections in the Middle Ages:
individuals lacking CCR5 had a higher chance of survival, leading to a
strong selection for the Δ32 mutation in a few decades [6]. In 2007,
an HIV-positive patient, who later was referred to as the Berlin
Patient, was transplanted with an allogeneic stem cell graft of an
HLA-matched donor homozygous for the CCR5 Δ32 mutation after
diagnosis of acute myeloid leukemia (AML). After stopping ART, the
patient was closely monitored for virus titers. As of today, i.e. 13
years later, no viral RNA or proviral DNA has been detected, so that he
was declared the first patient to be cured from HIV [7-9]. Following
the principle of the Berlin patient, recently two more patients, known
as the London [10, 11] and the Düsseldorf [12] patients, might
be the next patients to be declared as cured. These observations made it
clear that it is possible to transfer HIV resistance from a donor to a
patient. However, due to the potential severe side effects that
allogenic transplantations encompass and the difficulty to find matching
homozygous Δ32 donors, this strategy is not considered a universal
approach to treat patients affected by HIV.
Alternatively, CCR5 gene editing in autologous cells may
represent a potent approach to provide patients with cells that are
resistant to HIV [13]. To this end, genome editing with programmable
nucleases, in particular zinc-finger nucleases (ZFN) and CRISPR-Cas
nucleases, have been used in several preclinical studies to disrupt theCCR5 gene in T cells (reviewed in [14, 15]). Moreover, in a
clinical trial that enrolled 12 patients, a single dose ofCCR5 -edited autologous CD4+ T cells were administered. Although
the effect lasted only transiently, the blood level of HIV provirus
decreased in most patients and the infusion of CCR5 -modified
autologous CD4+ T cells proved to be safe [16].
In our study we sought to engineer HIV-1 resistant CD4+ T cells in a
manner compatible with good manufacturing practice (GMP). We aimed at
disrupting the CCR5 locus in those clinically relevant cell type
by transferring mRNA that code for a highly effective transcription
activation-like effector nuclease (TALEN) using a GMP compliant
electroporation device. We have previously shown that TALENs can be
employed to disrupt CCR5 in human cell lines with high
specificity [17, 18]. Here, we show that improvement of the TALEN
platform in combination with novel RNA transfer technologies [19]
enabled us to knockout CCR5 in CD4+ T cells with high activity
and high specificity. The manufactured T cells were HIV resistant,
genetically stable, and maintained their proliferation capacity as well
as their ability to respond to exogenous stimuli.