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.