BACKGROUND CCR5 is the major coreceptor for human immunodeficiency virus (HIV). 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. METHODS We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (−1.81 cells per day) was significantly less than the decline in unmodified cells (−7.25 cells per day) (P = 0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.)

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

We have targeted the activation of an endogenous chromosomal locus including the human erythropoietin gene using synthetic transcription factors. These transcription factors are targeted to particular DNA sequences in the 5-flanking region of the erythropoietin gene through engineering of a zinc finger DNA binding domain. The DNA binding domain is linked to a VP16 transcriptional activation domain. We find that these synthetic transcription factors invariably activate transiently transfected templates in which sequences within the 5 flank of the erythropoietin gene are fused to a luciferase reporter. The efficiency of activation under these circumstances at a defined site is dependent on DNA binding affinity. In contrast, only a subset of these same zinc finger proteins is able to activate the endogenous chromosomal locus. The activity of these proteins is influenced by their capacity to gain access to their recognition elements within the chromatin infrastructure. Zinc finger transcription factors will provide a powerful tool to probe the determinants of chromatin accessibility and remodeling within endogenous chromosomal loci.The enormous progress in our understanding of gene control in eukaryotes using model systems presents substantial opportunities to apply this knowledge for therapeutic benefit in man. The rational design and engineering of components of the transcriptional machinery provide a powerful means to test conventional paradigms for the roles of protein-DNA and proteinprotein interactions in gene regulation. These designer transcription factors may also provide novel means of regulating endogenous chromosomal loci for a variety of beneficial purposes. Over the past decade, the primary structural determinants of DNA recognition by zinc fingers of the Cys 2 -His 2 type have been elucidated (1-8). Designer transcriptional regulators containing three or more zinc finger domains have been used in isolation (9, 10) or following linkage to transcriptional activation (9, 11-13) or repression domains (12, 13). These novel proteins control the transcription of reporter genes both transiently transfected into human cells (9, 11-13) and endogenous chromosomal loci (9, 13). Exactly how these regulatory functions are exerted remains to be resolved. An important issue in considering transcription factor function in eukaryotes is the capacity of the regulator to gain access to specific sites in chromatin and recruit transcriptional co-activators and co-repressors that modify the chromatin environment (14). These issues have been investigated for the archetypal Cys 2 -His 2 zinc finger protein and transcriptional regulator TFIIIA 1 (15-26). There is general agreement that the nucleosome can impede recognition of specific promoter elements by TFIIIA (15,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and that modification of histone-DNA interactions through nucleosome repositioning (18,19), histone depletion (20, 21), and removal of the histone tails (22, 23) can promote TFIIIA binding to a nucleosomal infrastructure. Ac...

Zinc-finger protein transcription factors (ZFP TFs)D efects in transcriptional regulation underlie numerous disease states, most notably cancer (1). A major goal of current strategies for correcting such defects is to achieve sufficient specificity of action (2). Designed zinc-finger protein transcription factors (ZFP TFs) emulate natural transcriptional control mechanisms and therefore provide an attractive tool for precisely regulating gene expression. Accurate control of gene expression is important for understanding gene function (target validation) and for developing therapeutics to treat disease (3). We and others have used engineered ZFP TFs to either activate or repress a variety of endogenous gene targets (4-11). For these proteins, or any other gene-regulation technology, to succeed as tools in drug discovery or direct agents in the clinic, their specificity of action within the genome must be precise, a challenging criterion to meet given the size and complexity of the human genome. Recent studies with small interfering RNA (12, 13) and antisense DNA͞RNA (14) have illuminated the magnitude of the task of achieving single-gene specificity in regulating the human genome.We focus here on the use of ZFP TFs in the area of oncology and specifically on the emerging role of checkpoint kinase 2 (CHK2). CHK2 acts as a key integrator of DNA-damage signals regulating cell-cycle progression, DNA repair, and cell death by phosphorylating a variety of substrates, including the p53 tumor suppressor protein (15, 16). Here we show that a designed ZFP TF targeted to a unique 18-bp recognition sequence in the promoter of the CHK2 gene binds the intended site within chromatin and represses CHK2 transcription in vivo. Moreover, repression of CHK2 by this engineered ZFP TF occurs with remarkable specificity, while simultaneously reducing CHK2 protein to levels that functionally ablate the action of this kinase. Finally, we show that constitutive expression of the ZFP TF in telomerase-immortalized, untransformed human fibroblasts provides stable repression of the CHK2 gene and results in loss of DNA-damage-induced CHK2-dependent phosphorylation of p53 on Ser-20. These data demonstrate that ZFP TFs can be exquisitely specific, yet potent repressors of gene expression and, therefore, are potentially powerful reagents for target validation and therapeutic interventions in vivo.