ABSTRACT:
Gene editing, who allows for specific locations in the genome to be targeted and altered by deleting, adding or substituting nucleotides, is currently the subject of important academic and policy discussions. With the advent of efficient tools, the plausibility of using gene editing safely in humans for either somatic or germ line gene editing is being considered seriously. Beyond safety issues, living gene editing in humans does raise ethical, legal and social issues, however, it is suggested to be less challenging to existing ethical and legal frameworks; indeed somatic gene editing is already applied in (pre-) clinical trials. In contrast, the notion of altering the germ line or embryo such that alterations could be heritable in humans raises a large number of ELSI; it is currently debated whether it should even be allowed in the context of basic research. Even greater ELSI debates address the potential use of germ line or embryo gene editing for clinical purposes, which, at the moment is not being conducted and is prohibited in several jurisdictions. We investigated whether site-specific modification of the gene (“gene editing”) — in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN) — is safe.
INTRODUCTION:
The ability to edit the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity (Scherer and Davis, 1979). The challenge of genome editing is the ability to generate a single double-strand break at a specific point in the DNA molecule. Various agents, including meganucleases, oligonucleotides that form DNA triplexes, and peptide nucleic acids, have been tested and shown to be limited by inefficiency (Ashworth et al., 2006; Chin et al., 2009; Kim et al., 2009). Another class of reagent, the zinc-finger nucleases (ZFNs), have proved flexibility for genome editing, and the use of ZFNs is now well established in a number of model organisms and in human cells (Carroll D, 2011; Urnov et al., 2010).
ZFNs are well suited for genome engineering because they combine the DNA recognition specificity of zinc-finger proteins (ZFPs) with the robust but restrained enzymatic activity of the cleavage domain of the restriction enzyme FokI (a nuclease) (Carroll D, 2011; Urnov et al., 2010). ZFPs, which provide DNA-binding specificity, contain a tandem array of Cys2His2 zinc fingers, each recognizing approximately 3 base pairs of DNA (Miller et al., 1985). By comparison, the bacterial type IIS restriction endonuclease, FokI, has no sequence specificity and must dimerize to cut the DNA (Bitinaite at al., 1998). After the ZFN-mediated double-strand cut, the DNA can be repaired by either homologous recombination or non-homologous end joining. Homologous recombination repairs the break while preserving the original DNA sequence. However, these cells are susceptible to recutting by ZFNs. In contrast, non-homologous end joining can result in random insertion or deletion of nucleotides at the break site, resulting in permanent disruption of the primary DNA sequence. Therefore, non-homologous end joining can be exploited to mutate a specific gene, leading to its functional knockout (Carroll D, 2011; Urnov et al., 2010).
The design of a ZFN pair consisting of two 4-finger proteins that bind to a target site within the human chemokine (C-C motif) receptor 5 genes (CCR5) was reported previously (Perez et al., 2008). In preclinical tests, CCR5-modified CD4 T cells expanded and functioned normally in response to mitogens, were protected from human immunodeficiency virus (HIV) infection, and reduced HIV RNA levels in a humanized mouse model (involving xenotransplantation) of HIV infection (Perez et al., 2008).
We selected CCR5, which encodes a co-receptor for HIV entry, (Alkhatib et al., 1996; Deng et al., 1996) for several reasons. First, its destortion seemed likely to increase the survival of CD4 T cells; persons homozygous for a 32-bp deletion (delta32/delta32) in CCR5 are resistant to HIV infection (Liu et al., 1996). In vitro, CD4 T cells from such persons are highly resistant to infection with CCR5-using strains of HIV, which are the dominant strains in vivo (Samson et al., 1996). Moreover, persons who are heterozygous for CCR5 delta32 and who have HIV infection have a slower progression to the acquired immunodeficiency syndrome (Eugen-Olsen et al., 1997; Cohen at al., 1997). Furthermore, the effectiveness of blocking or inhibiting CCR5 with the use of small-molecule inhibitors has been shown in humans (Gulick et al., 2008). Finally, one person who underwent allogeneic transplantation with progenitor cells homozygous for the CCR5-delta32 deletion has remained off antiviral therapy for more than 4 years, with undetectable HIV RNA and proviral DNA in the blood, bone marrow, and rectal mucosa (Hutter et al., 2009; Allers et al., 2011). Although the mechanism responsible for the apparent cure associated with this procedure remains to be established, acquired CCR5 deficiency is one possibility (Deeks and McCune JM, 2010). Here we report the partial induction of acquired genetic resistance to HIV infection after targeted gene disruption (i.e., the infusion of autologous CD4 T cells modified at CCR5 by a ZFN).
METHODS:
We enrolled 12 patients in two case series (cohort 1 and cohort 2), each with 6 patients (Table 1).
Table 1. Patient Demographics and Cell Manufacturing.
The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy (HAART). Patients were infused with SB-728-T (Sangamo BioSciences), consisting of autologous CD4-enriched T cells that have been modified at the CCR5 gene locus by ZFNs. The investigational ZFN was donated by Sangamo BioSciences, which had no role in any aspect of the study design, the writing of the manuscript, or the decision to submit the manuscript for publication; the ZFN-modified cells were manufactured at the University of Pennsylvania. The primary objective of the study was to assess the safety and side-effect profile of a single dose of autologous CD4-enriched T cells modified at CCR5 by ZFNs. Secondary objectives included the assessment of increases in the CD4 T-cell count, persistence of the modified cells, homing to gut mucosa, and effects on viral load. All patients provided written informed consent. All the authors vouch for the accuracy and completeness of the data and the fidelity of the study to the protocol (Pablo Tebas et al., 2014).
RESULTS:
One serious adverse event occurred in a single patient from cohort 2. Fever, chills, joint pain, and back pain developed in the patient and precipitated a visit to the emergency department within 24 hours after infusion of the study drug. We attributed the symptoms to a transfusion reaction related to the study drug (Pablo Tebas et al., 2014).
CCR5-Modified CD4 T Cells:
CCR5-modified CD4 T cells could be tracked after infusion because of the creation of a five-nucleotide (pentamer) duplication that occurred in approximately 25% of the modified cells (Perez et al., 2008). Therefore, the total number of gene-modified cells is calculated by multiplying the number of cells with the pentamer duplication by four. After infusion, we observed an increase in the number of CCR5-modified circulating CD4 T cells (Figure 2A), with peak levels observed at week 1 (range among the 12 patients, 30 to 1106 cells per cubic millimeter). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted a median of 8.8% of peripheral-blood mononuclear cells (PBMCs) and 13.9% of the CD4 T cells in the vascular compartment. The number of CCR5-modified CD4 T cells in the circulation constituted a similar percentage of the circulating CD4 T cells and PBMCs in the participants with and in those without adequate CD4 T-cell recovery after HAART. The time to peak level (known as Tmax) of gene-modified cells ranged from 3 to 14 days (median, 7). The number of gene-modified cells in the vascular compartment decreased moderately, with an estimated mean half-life of 48 weeks at a median follow-up of 64 weeks (range, 24 to 142). The gene-modified T cells could be detected in all patients at all subsequent time points examined during the long-term follow-up study, the longest to date being 42 months in the first patient, at which time CCR5-modified CD4 T cells were present at a concentration of 13 cells per cubic millimeter, representing 0.6% of circulating PBMCs and 1.7% of circulating CD4 T cells, respectively (Pablo Tebas et al., 2014).
Figure 2. CCR5-Modified CD4 T Cells in the Circulation and Mucosal Tissues.
Trafficking of CCR5-Modified CD4 T Cells to Rectal Mucosa
In humans, the vascular compartment contains 1 to 2% of the T-cell mass, whereas the mucosal tissues are the largest lymphoid reservoir, containing at least 50% of the T-cell mass (Douek et al., 2009). In this study, CCR5-modified CD4 T cells were detected in all rectal-biopsy specimens. One patient in cohort 1 declined to undergo the scheduled biopsies; the remaining 11 participants underwent biopsies on two or more occasions. A total of 30 of 33 scheduled biopsies were performed. Gene-modified cells constituted a median of 0.8% of rectal mononuclear cells on day 21 and varied from 0.4% to 0.2% thereafter (Figure 2B).
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