Efficient ZFN-Mediated Stop Codon Integration into the CCR5 Locus in Hematopoietic Stem Cells: A Possible Source for Intrabone Marrow Cell Transplantation

Introduction

E x vivo genome manipulation of patients' cells before therapeutic replacement can currently be achieved using available genome editing tools and recent advances in cell therapy. ^{1 –3} Optimistically, this combination approach can provide more therapeutic options for various incurable diseases, including human immunodeficiency virus-1 (HIV) infection. Although antiretroviral drugs (ARVs) can achieve effective viral reduction, HIV can re-emerge from the latent reservoir, which means that ARV treatments must be continued for the lifetime of the patient. ^4,5

Chemokine receptor 5 (CCR5) is an important coreceptor that allows HIV to enter targeted cells. Natural dysfunction of CCR5 due to a homozygous 32-bp deletion in CCR5 is known to impart some level of resistance to HIV infection and to slow the rate of progression. ^6,7 This variant is believed to have naturally been selected among Caucasian populations of Eastern Europe as a result of pandemics of vaccinia and/or small pox. ⁸ This was demonstrated by HIV eradication in a patient with chronic HIV who received a CCR5-defective bone marrow transplantation (BMT) for the treatment of acute myeloid leukemia. ^9,10

Subsequent attempts to replicate BMT using CCR5delta32 donors as a strategy to cure HIV have, however, been unsuccessful. Following this discovery, researchers have attempted to find ways to provide patients with their own lymphocytes, modified to resist re-emerging virus; disruption of CCR5 has been attempted using various genome editing techniques and in various cells. ^{11 –24} Most of the work relies on an error-prone DNA repair method, nonhomologous end joining, that tends to generate uncontrollable insertion/deletion mutations.

In 2015, we demonstrated the possibility to integrate a stop codon into the CCR5 locus via zinc finger nuclease (ZFN)-mediated homologous recombination (HR) in bone marrow-derived mesenchymal stem cells, which can directly be converted to hematopoietic stem cell (HSCs)-like cells. ²⁵ However, to date, direct cell conversion to HSCs has not been achieved in clinical practice. ^26,27 In this study, we tested the efficacy of HR-mediated stop codon insertion into bone marrow HSCs, which are better suited to self-transplantation.

Materials and Methods

Genome editing and crude bone marrow expansion

CCR5-specific ZFN was purchased from Sigma Aldrich (St. Louis, MO). This ZFN was designed to target the third exon of CCR5. ^11,25 A pair of ZFN mRNAs were synthesized using the MassageMax T7 mRNA Transcription and Poly(A) Polymerase Tailing Kit (Epicentre; Madison, WI). The d-stop donor that carried the universal stop codon “TAGATAGTTAG” flanking the region −733 bp upstream of the left-hand ZFN-binding site to 1,038 bp downstream of the right-hand ZFN-binding site has previously been described. ²⁵ Nucleated cells were isolated from 3 to 5 mL of heparinized bone marrow and were transfected with ZFN mRNA and d-stop donor plasmid. Cells were plated on Matrigel-coated flasks and cultured in CD34⁺ culture medium (Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and 100 units/mL penicillin and G-streptomycin (Gibco; Grand Island, NY), 50 ng/mL Flt3, 50 ng/mL thrombopoietin, and 50 ng/mL stem cell factor (R&D Systems)) for 5 days. Flow cytometry for CD34 was performed at day 0 and 5. CD34-positive cells were isolated from expansion cells at day 5 using a magnetic-activated cell sorting system (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol (Fig. 1a.).

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FIG. 1.

Experimental protocol and PCR results. (a) Schematic of our experimental protocol. (b) Representation of the flow cytometry analysis (from patient number 7). Blue areas: day 0 isotype control; green line: CD34⁺ cells at day 0; magenta line: CD34⁺ cells at 5 days after nucleoporation and culture. (c) Representative screening/nested PCR of samples from two patients with chronic HIV infection (patient number 6 and 8). Lane 1: markers; lane 2: no template; lane 3: normal subject peripheral blood cells; lane 4; d-stop donor; lane 5: DNA from expansion cells. PCR, polymerase chain reaction. HIV, human immunodeficiency virus-1. Color images available online at www.liebertpub.com/aid

Results

Discussion

Although the debate on the optimal latent viral eradication mechanism continues, allogenic BMT of CCR5 -defective HSCs is the only treatment shown to cure an HIV infection. Genome editing of patients' own cells is a potential way to source CCR5-defective HSCs without immunologic incompatibility barriers. However, simple cell replacement with HIV-resistant cells might not lead to a complete cure, ^28,29 because in chronic infection, the virus has already integrated its DNA into the genome of latent cells. Recently the “shock and kill” concept, in which the latent integrated provirus is intentionally activated, has been postulated as a strategy for reservoir eradication. ^{5,30 –32} As part of this strategy, virus-resistant cells may provide benefit by reducing de novo infection during proviral activation. In addition, HIV-resistant cell replacement also has potential among uninfected persons as a prophylactic treatment against HIV-1. These maneuvers theoretically can prevent primary HIV-1 infection among uninfected persons who subsequently become exposed. In this work, we showed the efficacy of ZFN-mediated stop codon integration into CCR5 loci in potential patients' own cells, using a simple protocol. Without the need for cell isolation or complex cell stimulation, the edited cells survived and showed a considerable proliferation rate. At day 5 of culture expansion, we observed that ∼50 cells per 10⁵ CD34⁺ cells harbored a stop codon at the CCR5 locus. This number may be relatively low compared to the required dose for entire bone marrow regeneration in clinical practice, however, clonal generation of edited self-cells is different to bone marrow replacement. ³³ One interesting approach is to directly inject these cells back to the bone marrow. Recent studies suggested that direct cell infusion into the marrow, that is, intrabone marrow injection, showed effective engraftment at lower doses. ^{34 –38} Our protocol requires only mRNA and a promoter-less plasmid. None of the materials in the process was contaminated by bovine or other animal substances. The overall bone marrow aspiration and editing process required less than 30 min, allowing the edited crude bone marrow cells to be injected back to the bone marrow in the same setting as the aspiration. In addition, the process may be repeated on multiple rounds and visits to generate the desired amount of CCR5-defective cells.

In summary, we reported a method for ZFN-mediated stop-codon integration into CCR5 loci of HIV-positive patients' bone marrow. Cells in the edited CD34⁺ fraction survived with a substantial proliferation rate. We therefore postulate that these CD34⁺ cells should expand if they are immediately injected back into the bone marrow. This approach might be beneficial for HIV treatment.