Pei-Qi Liu

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.

Correction of the sickle-cell disease mutation in human hematopoietic stem/progenitor cells

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.

Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases†

Mammalian cells with multi‐gene knockouts could be of considerable utility in research, drug discovery, and cell‐based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi‐locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105.

Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors.

Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2–H19 domain is a hallmark of cancer and Beckwith–Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for ‘transcription therapy’ of human disease.

Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF–generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

Cell Lines for Drug Discovery: Elevating Target-Protein Levels Using Engineered Transcription Factors:

Drug discovery requires high-quality, high-throughput bioassays for lead identification and optimization. These assays are usually based on immortalized cell lines, which express the selected drug target either naturally or as a consequence of transfection with the cDNA encoding the target. Natural untransfected cell lines often fail to achieve the levels of expression required to provide assays of sufficient quality with a high enough signal-to-noise ratio. Unfortunately, the use of cDNA is increasingly restricted, as the sequences for more and more genes become subject to patent restrictions. To overcome these limitations, the authors demonstrate that engineered transcription factors with Cys2-His2 zinc finger DNA-binding domains can be used to effectively activate an endogenous gene of interest without the use of isolated cDNA of the target gene. Using this approach, the authors have generated a cell line that provides a high-quality and pharmacologically validated G-protein-coupled receptor bioassay. In principle, this technology is applicable to any gene of pharmaceutical importance in any cell type. (Journal of Biomolecular Screening 2004:44-51)

Pharmacological analysis of CCK2 receptors up-regulated using engineered transcription factors.

Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA50 = 9.05+/-0.11, pentagastrin pA50 = 9.11+/-0.13). In addition, binding studies were conducted using [125I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pK(D) = 10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds.

641. Highly Efficient, ZFN-Driven Knockout of Surface Expression of the T-Cell Receptor and HLA Class I Proteins in Human T-Cells for Enhancing Allogeneic Adoptive Cell Therapies

While adoptive transfer of T-cells modified to express a chimeric antigen receptor or tumor antigen specific T-cell receptor (TCR) has shown great promise for the treatment of malignant cancers, most current clinical applications are limited by the use of autologous T-cell products. Targeting of the TCR and HLA Class I genes in primary T-cells thus represent attractive targets for genome editing in order to produce universal T cells from allogeneic donors. Elimination of the native TCR and HLA class I proteins on T-cells would, respectively, reduce the risk of graft-versus-host disease and host-versus-graft clearance mediated by the adaptive immune system. We have developed clinical grade zinc finger nuclease (ZFN) reagents that can efficiently target the T-cell receptor alpha constant (TRAC) and beta-2-microglobulin (B2M) loci. ZFN encoding mRNAs were introduced into purified T-cells by electroporation. Without selection, T-cells treated with TRAC specific ZFNs showed modification of up to 89% of alleles as gauged by deep sequencing. Flow analysis showed 91% of treated cells were negative for CD3 expression. Results from ZFN mRNA dose titration studies showed that the level of TRAC gene modification by deep sequencing was highly correlated with the percentage of cells that stain negative for CD3 expression by FACS (Spearman rho = 0.96; p < 0.0001). B2M is a subunit in all HLA class I molecules, and represents a conserved target for eliminating HLA class I presentation in cells from different donors. Analysis of T-cells transfected with mRNAs encoding B2M specific ZFNs showed up to 94% of alleles were modified, as determined by deep sequencing. FACS analysis showed that 89% of treated cells were negative for expression of HLA-A, B, C. Similar to TRAC modified cells, a marked correlation was observed between the percentage of B2M alleles modified and percentage of cells lacking surface expression of HLA-A,B, and C (Spearman rho = 0.57; p < 0.0001). ZFN mediated genome editing was well tolerated. T-cells treated with TRAC or B2M specific ZFNs showed similar viability and growth characteristics as mock transfected cells. Together, these highly efficient ZFN reagents permit the highly efficient double knockout of TCR and HLA class I surface expression in primary human T-cells for potential use in the development of allogeneic cell therapies.

54. Genome Editing of Primary Human CD34+ Hematopoietic Stem Cells Enables a Safe Harbor Targeted Gene Addition Therapeutic Strategy for Chronic Granulomatous Disease

Many monogenic recessive diseases of blood can, in principle, be cured by transfer of functional therapeutic transgene to the genome of the hematopoietic stem cell (HSC) – a strategy proven successful for multiple rare diseases using current integrating vector gene therapy. With a focus on X-linked chronic granulomatous disease (X-CGD), we report a directed approach orthogonal to randomly integrating retrovector gene therapy: the highly specific targeted placement of the curative transgene into a validated safe harbor locus in human HSCs via human genome editing with zinc finger nucleases (ZFNs) and donor insert delivery using an AAV6 vector.We describe here an integrated targeted delivery platform customized for targeted addition to human HSCs using a cGMP-compliant electroporation system compatible with clinical scale production. Using next-generation, highly optimized ZFNs against the AAVS1/PPP1R12C gene locus, we optimized conditions for addition of the fluorescent Venus cDNA into human peripheral blood G-CSF mobilized CD34+ HSCs. Venus expression in manipulated human HSCs in vitro reached >50% efficiency, while earlier experiments demonstrated persistence of gene-modified cells in NSG mice with 12-15% Venus+ human CD45+ cells retrieved from transplanted mouse bone marrow and overall human HSC engraftment levels of >15%. Targeted integration (TI) rates achieved in human CD45+ cells from mouse bone marrow were 28-57%. Similar levels of Venus+ (~10%) are observed in spleen and peripheral blood CD45+ cells, indicating differentiation of gene-modified CD34 HSCs into circulating blood cells.X-CGD patients suffer from severe bacterial and fungal infections with excessive inflammation due to a defect in the gp91phox subunit of phagocyte oxidase. To extend the results above to CGD, we therefore used the same approach to target addition of the relevant curative transgene, gp91phox, into the AAVS1 safe harbor locus of HSCs from patients with X-linked CGD. In vitro levels of gp91phox expression in gene-modified patient CD34+ HSCs population achieve 12-16% gp91phox expression by flow cytometric analysis, with an NSG xenograft study demonstrating 3-5% of the engrafted human CD45+ cells expressing gp91phox. Of note, the MND-driven gp91phox expression from the safe harbor locus in human neutrophils differentiating from CD34+ cells transplanted into the NSG mouse model parallels wildtype gp91phox levels produced at the native locus.Our studies demonstrate the feasibility of targeted addition of different genes at the AAVS1 safe harbor site of the genome in human HSCs at an unprecedented efficiency and specificity; we demonstrate the efficient correction of the enzymatic defect in neutrophils arising from patient-derived HSCs in vivo. In sum with the advances in GMP-scale cell processing for genome editing, and the charted regulatory path for ZFNs to the clinic provided by ongoing trials in HIV, our studies represent the foundation for a rapid translation of ZFN-driven targeted addition as a clinical modality for X-linked CGD.

659. Development of Designed Zinc Finger Protein Transcriptional Regulators for Use in Multi-Factor Therapeutic Angiogenesis

Gene therapy-mediated revascularization strategies offer considerable promise in the treatment of cardiovascular disease. To date, over two dozen studies have reported that gene-based delivery of angiogenic factors can enhance blood flow and/or tissue function in preclinical models of limb and heart ischemia. However, although several programs have advanced into clinical trials, it has been noted that natural angiogenesis proceeds via the coordinate action of multiple factors; therefore strategies utilizing two or more angiogenic factor genes may realize enhanced therapeutic benefit1. In consideration of this possibility we are developing a panel of designed zinc finger protein (ZFP) transcriptional activators for regulation of multiple different angiogenic factor genes. In previous studies we had described the development of pro-angiogenic ZFPs that function via activation of VEGF-A2,3,4. Here we have extended this approach to develop regulators of three additional factors: Ang-1, FGF-5 and VEGF-C. These studies involved: (i) DNase I mapping of accessible locus regions; (ii) design and assembly of ZFPs targeting sites within accessible and/or functional promoter regions; and (iii) ZFP screening for activation of gene expression. By this approach, we have identified ZFPs that activate Ang-1, FGF-5 and VEGF-C by up to 14-fold in cells from both human and mouse. Furthermore, by targeting a sequence common to the promoters of VEGF-A and VEGF-C we have produced a ZFP that coordinately activates both of these genes. The use of designed ZFPs to regulate a patients own endogenous genetic loci offers a number of advantages over cDNA-based gene therapy approaches, including the capacity to upregulate all splice variants of a therapeutic gene2,3,4, the option for ligand-regulatable protein expression using a single introduced transgene5, and transgene size economy (a critical feature for multi-factor therapies with vectors of limited insert capacity). We anticipate that these ZFPs will provide powerful reagents for the development of multi-factor strategies for therapeutic angiogenesis.