Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice.

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4+ and CD8+ T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers.

Silent IL2RG Gene Editing in Human Pluripotent Stem Cells.

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.

The effects of polymorphisms on human gene targeting.

DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce gene targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human gene targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific targeting or to improve unfavorable vector designs that introduce deletions.

Thermo-catalytic decomposition of formaldehyde: a novel approach to produce mesoporous ZnO for enhanced photocatalytic activities.

A novel method, thermo-catalytic decomposition of formaldehyde, is used to synthesize mesoporous ZnO crystals with enhanced photocatalytic activities. The mechanism of the mesoporous formation is investigated by synthesizing a series of samples at various systems and characterizing them with FT-IR, EDS, XRD, SEM, and TEM. The results show that formaldehyde can be adsorbed on the crystal planes of ZnO during the crystal growth and can then be catalytically decomposed into CO, CO2 and H2 during a sintering process. Because of the formation and the escape of these gases, which act as templates, the crystalline particles of ZnO are forced to rearrange consistently, and pores are formed in the internal crystal. Also, porous TiO2 crystals have been obtained via the same approach. Photocatalytic tests indicate that a porous ZnO crystal has higher activity than that of a nonporous one.

Fabrication of ordered ZnO/TiO2 heterostructures via a templating technique.

Two kinds of ordered ZnO/TiO2 heterostructures were fabricated via a facile approach. The architecture of the TiO2 substrate could be controlled by alternating the filling forms of the template, and the morphology of the secondary ZnO nanostructure could be further tuned by adjusting the parameters of the hydrothermal reaction. Then two different morphologies of ZnO/TiO2 heteroarchitectures with ZnO nanorods and nanoplates growing on TiO2 shells and bowls were successfully achieved, respectively.

ANCCA, an estrogen-regulated AAA+ ATPase coactivator for ERalpha, is required for coregulator occupancy and chromatin modification.

AAA+ proteins play crucial roles in diverse biological processes via their ATPase-driven remodeling of macromolecular complexes. Here we report our identification of an evolutionarily conserved AAA+ protein, ANCCA/pro2000, endowed with a bromodomain that is strongly induced by estrogen in human breast cancer cells and is a direct target of protooncogene ACTR/AIB1/SRC-3. We found that ANCCA associates directly with estrogen-bound estrogen receptor (ER) alpha and ACTR. It is selectively recruited, upon estrogen stimulation, to a subset of ERalpha target genes including cyclin D1, c-myc, and E2F1 and is required for their estrogen-induced expression as well as breast cancer cell proliferation. Further studies indicate that ANCCA binds and hydrolyzes ATP and is critical for recruitment of coregulator CBP and histone hyperacetylation at the ER target chromatin. Moreover, mutations at the ATP binding motifs rendered ANCCA defective as a coactivator in mediating estrogen induction of gene expression. Together, our findings reveal an unexpected layer of regulatory mechanism in hormone signaling mediated by ANCCA and suggest that hormone-induced assembly of transcriptional coregulator complexes at chromatin is a process facilitated by AAA+ ATPase proteins.

Proto-oncogene ACTR/AIB1 promotes cancer cell invasion by up-regulating specific matrix metalloproteinase expression.

Overexpression of ACTR/AIB1 is frequently found in different cancers with distant metastasis. To address its possible involvement in tumor metastasis, we performed invasion assays to examine the effect of ACTR alteration on the invasiveness of breast cancer cells (MDA-MB-231 or T-47D) and found that high levels of ACTR are required for their strong invasiveness. Molecular analysis indicates that ACTR functions as a coactivator of AP-1 to up-regulate the expression of matrix metalloproteinases such as MMP-7 and MMP-10 and reduce cell adhesion to specific extracellular matrix proteins. These novel findings provide a mechanistic link between ACTR and MMPs, and suggest that ACTR may also play an important role in cancer progression by facilitating tumor invasion.

A genome-wide map of adeno-associated virus-mediated human gene targeting.

To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. We used an adeno-associated virus vector to target identical loci introduced as transcriptionally active retroviral vectors. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats or DNase I-hypersensitive sites. Targeted sites were preferentially located within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. We determined the impact of DNA replication by mapping replication forks, which revealed a preference for recombination at target loci transcribed toward an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells and demonstrate a strong recombinogenic effect of colliding polymerases.

Trisomy correction in Down syndrome induced pluripotent stem cells.

Human trisomies can alter cellular phenotypes and produce congenital abnormalities such as Down syndrome (DS). Here we have generated induced pluripotent stem cells (iPSCs) from DS fibroblasts and introduced a TKNEO transgene into one copy of chromosome 21 by gene targeting. When selecting against TKNEO, spontaneous chromosome loss was the most common cause for survival, with a frequency of ~10(-4), while point mutations, epigenetic silencing, and TKNEO deletions occurred at lower frequencies in this unbiased comparison of inactivating mutations. Mitotic recombination events resulting in extended loss of heterozygosity were not observed in DS iPSCs. The derived, disomic cells proliferated faster and produced more endothelia in vivo than their otherwise isogenic trisomic counterparts, but in vitro hematopoietic differentiation was not consistently altered. Our study describes a targeted removal of a human trisomy, which could prove useful in both clinical and research applications.