Dmitry Guschin

Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases

Homozygosity for the naturally occurring Δ32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted ∼50% of CCR5 alleles in a pool of primary human CD4+ T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4+ T cells had lower viral loads and higher CD4+ T-cell counts than mice engrafted with wild-type CD4+ T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4+ T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN–modified autologous CD4+ T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.

An improved zinc-finger nuclease architecture for highly specific genome editing

Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations

Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinsons disease by modifying the underlying point mutations in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies.

A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6.

The protein tyrosine kinases JAK1, JAK2 and Tyk2 and STATs (signal transducers and activators of transcription) 1 and 3 are activated in response to interleukin‐6 (IL‐6) in human fibrosarcoma cells. In mutant cells lacking JAK1, JAK2 or Tyk2, the absence of one kinase does not prevent activation of the others; activation does not, therefore, involve a sequential three‐kinase cascade. In the absence of JAK1, the phosphorylation of the gp130 subunit of the IL‐6 receptor and the activation of STATs 1 and 3 are greatly reduced. JAK1 is also necessary for the induction of IRF1 mRNA, thus establishing a requirement for the JAK/STAT pathway in the IL‐6 response. JAK2 and Tyk2 although activated cannot, in the absence of JAK1, efficiently mediate activation of STATs 1 and 3. A kinase‐negative mutant of JAK2 can, however, inhibit such activation, and ancillary roles for JAK2 and Tyk2 are not excluded. A major role for JAK1 and the nonequivalence of JAK1 and JAK2 in the IL‐6 response pathway are, nevertheless, clearly established for these cells.

Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR−/− cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.

A Rapid and General Assay for Monitoring Endogenous Gene Modification

The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.

A JAK1/JAK2 chimera can sustain alpha and gamma interferon responses.

Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.

Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes.

The activation of Janus protein tyrosine kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins by interleukin (IL)‐2, the T cell antigen receptor (TCR) and interferon (IFN) alpha was explored in human peripheral blood‐derived T cells and the leukemic T cell line Kit225. An IL‐2‐induced increase in JAK1 and JAK3, but not JAK2 or Tyk2, tyrosine phosphorylation was observed. In contrast, no induction of tyrosine phosphorylation of JAKs was detected upon stimulation of the TCR. IFN alpha induced the tyrosine phosphorylation of JAK1 and Tyk2, but not JAK2 or JAK3. IFN alpha activated STAT1, STAT2 and STAT3 in T cells, but no detectable activation of these STATs was induced by IL‐2. However, IL‐2 regulates the DNA binding and tyrosine phosphorylation of two STAT‐like protein complexes which do not include STAT1, STAT2 or STAT3. STAT4 is not activated by IL‐2. The activation of STAT5 cannot be excluded, so the IL‐2‐activated complexes most probably include at least one novel STAT. No STAT activity was detected in TCR‐stimulated lymphocytes, indicating that the JAK/STAT pathway defined in this study constitutes an IL‐2R‐mediated signaling event which is not shared by the TCR. Finally, in other cell types the correlation between JAK1 activation and the induction of STAT1 has suggested that JAK1 may activate STAT1. The observation that IL‐2 and IFN alpha activate JAK1 to a comparable degree, but only IFN alpha activates STAT1, indicates that JAK1 activation is not the only determining factor for STAT1 activation. Moreover, the data show that JAK1 stimulation is also not sufficient for STAT3 activation.

Translating dosage compensation to trisomy 21

Down’s syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down’s syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a ‘chromosome 21 Barr body’. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of ‘chromosome therapy’.

Distinct requirements for chromatin assembly in transcriptional repression by thyroid hormone receptor and histone deacetylase

Histone deacetylase and chromatin assembly contribute to the control of transcription of the Xenopus TRβA gene promoter by the heterodimer of Xenopus thyroid hormone receptor and 9‐cis retinoic acid receptor (TR‐RXR). Addition of the histone deacetylase inhibitor Trichostatin A (TSA) relieves repression of transcription due to chromatin assembly following microinjection of templates into Xenopus oocyte nuclei, and eliminates regulation of transcription by TR‐RXR. Expression of Xenopus RPD3p, the catalytic subunit of histone deacetylase, represses the TRβA promoter, but only after efficient assembly of the template into nucleosomes. In contrast, the unliganded TR‐RXR represses templates only partially assembled into nucleosomes; addition of TSA also relieves this transcriptional repression. This result indicates the distinct requirements for chromatin assembly in mediating transcriptional repression by the deacetylase alone, compared with those needed in the presence of unliganded TR‐RXR. In addition, whereas hormone‐bound TR‐RXR targets chromatin disruption as assayed through changes in minichromosome topology and loss of a regular nucleosomal ladder on micrococcal nuclease digestion, addition of TSA relieves transcriptional repression but does not disrupt chromatin. Thus, TR‐RXR can facilitate transcriptional repression in the absence of hormone through mechanisms in addition to recruitment of deacetylase, and disrupts chromatin structure through mechanisms in addition to the inhibition or release of deacetylase.