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.)

Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by AAV serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome editing technologies in HSPCs.

This report describes the identification, cloning, and molecular analysis of UME6 (CAR80/CARGRI), a key transcriptional regulator of early meiotic gene expression. Loss of UME6 function results in the accumulation of fully derepressed levels (70-to 100-fold increase above basal level) of early meiotic transcripts during vegetative growth. In contrast, mutations in five previously identified UME loci (UME1 to UME5), result in low to moderate derepression (2-to 10-fold increase) of early meiotic genes. The behavior of insertion and deletion alleles indicates that UME6 is dispensable for mitotic division but is required for meiosis and spore germination. Despite the high level of meiotic gene expression during vegetative growth, the generation times of ume6 mutant haploid and diploid cells are only slightly reduced. However, both ascus formation and spore viability are affected more severely. The UME6 gene encodes a 91-kD protein that contains a C6 zinc cluster motif similar to the DNA-binding domain of GAL4. The integrity of this domain is required for UME6 function. It has been reported recently that a mutation in CAR80 fails to complement an insertion allele of UME6. CAR80 is a gene required for nitrogen repression of the arginine catabolic enzymes. Here, through sequence analysis, we demonstrate that UME6 and CAR80 are identical. Analyses of UME6 mRNA during both nitrogen starvation and meiotic development indicate that its transcription is constitutive, suggesting that regulation of UME6 activity occurs at a post-transcriptional level.

Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10-to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.Adeno-associated virus type 2 (AAV-2) is a parvovirus belonging to the Dependovirus genus (16). AAV-2 has a linear, single-stranded DNA genome containing 4,679 nucleotides that encodes two genes called rep and cap. The rep gene encodes four proteins that play roles in viral DNA replication, DNA packaging, and the regulation of transcription and splicing. The cap gene encodes three proteins (VP1, VP2, and VP3) that package viral DNA and determine viral tropism. The external surface of the capsid is also the target for neutralizing antibodies. To date, about 180 different AAV capsids have been identified. The three capsid proteins are produced from the same translational reading frame by means of alternative splicing and translation initiation sites.The C termini, each of which is typically about 530 amino acids long, are identical and represent VP3. VP3 is the most abundant capsid protein, representing about 80% of the protein in an assembled capsid. VP2 is typically about 65 amino acids longer than VP3 at the amino terminus and is dispensable for most AAV-2 functions (51). VP1 is typically about 135 amino acids longer than VP2 at the amino terminus. This N-terminal domain is located inside the capsid (26) and encodes the phospholipase A2, which is required f...

We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose-and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 ؋ 10 12 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases.