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Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene autotemcel) consists of autologous transplantation of a patient's hematopoietic stem cells transduced with the BB305 lentiviral vector that encodes the β

We present a computer program to calculate the quantised rotational and hyperfine energy levels of diatomic molecules in the presence of dc electric, dc magnetic, and off-resonant optical fields. Our program is applicable to the bialkali molecules used in ongoing state-of-the-art experiments with ultracold molecular gases. We include functions for the calculation of space-fixed electric dipole moments, magnetic moments and transition dipole moments. Program summary Program Title: Diatomic-Py CPC Library link to program files: https://doi.org/10.17632/3yfxnh5bn5.1 Developer's repository link: https://doi.org/10.5281/zenodo.6632148 Licensing provisions: BSD 3-clause Programming language: Python ≥ 3.7 Nature of problem: Calculation of the rotational and hyperfine structure of molecules in the presence of dc magnetic, dc electric, and off-resonant laser fields. Solution method: A matrix representation of the Hamiltonian is constructed in the uncoupled basis set. Eigenstates and eigenenergies are calculated by numerical diagonalization of the Hamiltonian. Additional comments including restrictions and unusual features: Restricted to calculating the Stark and Zeeman shifts with co-axial electric and magnetic fields.

Quantum states with long-lived coherence are essential for quantum computation, simulation and metrology. The nuclear spin states of ultracold molecules prepared in the singlet rovibrational ground state are an excellent candidate for encoding and storing quantum information. However, it is important to understand all sources of decoherence for these qubits, and then eliminate them, in order to reach the longest possible coherence times. Here, we fully characterise the dominant mechanisms for decoherence of a storage qubit in an optically trapped ultracold gas of RbCs molecules using high-resolution Ramsey spectroscopy. Guided by a detailed understanding of the hyperfine structure of the molecule, we tune the magnetic field to where a pair of hyperfine states have the same magnetic moment. These states form a qubit, which is insensitive to variations in magnetic field. Our experiments reveal an unexpected differential tensor light shift between the states, caused by weak mixing of rotational states. We demonstrate how this light shift can be eliminated by setting the angle between the linearly polarised trap light and the applied magnetic field to a magic angle of $\arccos{(1/\sqrt{3})}\approx55^{\circ}$. This leads to a coherence time exceeding 6.9 s (90% confidence level). Our results unlock the potential of ultracold molecules as a platform for quantum computation., Comment: 19 pages, 12 figures

We explore coherent multi-photon processes in $^{87}$Rb$^{133}$Cs molecules using 3-level lambda and ladder configurations of rotational and hyperfine states, and discuss their relevance to future applications in quantum computation and quantum simulation. In the lambda configuration, we demonstrate the driving of population between two hyperfine levels of the rotational ground state via a two-photon Raman transition. Such pairs of states may be used in the future as a quantum memory, and we measure a Ramsey coherence time for a superposition of these states of 58(9) ms. In the ladder configuration, we show that we can generate and coherently populate microwave dressed states via the observation of an Autler-Townes doublet. We demonstrate that we can control the strength of this dressing by varying the intensity of the microwave coupling field. Finally, we perform spectroscopy of the rotational states of $^{87}$Rb$^{133}$Cs up to $N=6$, highlighting the potential of ultracold molecules for quantum simulation in synthetic dimensions. By fitting the measured transition frequencies we determine a new value of the centrifugal distortion coefficient $D_v=h\times207.3(2)~$Hz.

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD. Zinc finger protein transcription factors are developed for the selective silencing of the mutant huntingtin gene in human neurons in vitro and multiple animal models of Huntington’s disease in vivo while preserving expression of the wild-type allele.

Understanding ultracold collisions involving molecules is of fundamental importance for current experiments, where inelastic collisions typically limit the lifetime of molecular ensembles in optical traps. Here we present a broad study of optically trapped ultracold RbCs molecules in collisions with one another, in reactive collisions with Rb atoms, and in nonreactive collisions with Cs atoms. For experiments with RbCs alone, we show that by modulating the intensity of the optical trap, such that the molecules spend 75% of each modulation cycle in the dark, we partially suppress collisional loss of the molecules. This is evidence for optical excitation of molecule pairs mediated via sticky collisions. We find that the suppression is less effective for molecules not prepared in the spin-stretched hyperfine ground state. This may be due either to longer lifetimes for complexes or to laser-free decay pathways. For atom-molecule mixtures, RbCs+Rb and RbCs+Cs, we demonstrate that the rate of collisional loss of molecules scales linearly with the density of atoms. This indicates that, in both cases, the loss of molecules is rate-limited by two-body atom-molecule processes. For both mixtures, we measure loss rates that are below the thermally averaged universal limit., Comment: 21 pages, 9 figures

We investigate the effects of static electric and magnetic fields on the differential ac Stark shifts for microwave transitions in ultracold bosonic $^{87}$Rb$^{133}$Cs molecules, for light of wavelength $\lambda = 1064~\mathrm{nm}$. Near this wavelength we observe unexpected two-photon transitions that may cause trap loss. We measure the ac Stark effect in external magnetic and electric fields, using microwave spectroscopy of the first rotational transition. We quantify the isotropic and anisotropic parts of the molecular polarizability at this wavelength. We demonstrate that a modest electric field can decouple the nuclear spins from the rotational angular momentum, greatly simplifying the ac Stark effect. We use this simplification to control the ac Stark shift using the polarization angle of the trapping laser.

We show that the lifetime of ultracold ground-state ^{87}Rb^{133}Cs molecules in an optical trap is limited by fast optical excitation of long-lived two-body collision complexes. We partially suppress this loss mechanism by applying square-wave modulation to the trap intensity, such that the molecules spend 75% of each modulation cycle in the dark. By varying the modulation frequency, we show that the lifetime of the collision complex is 0.53±0.06 ms in the dark. We find that the rate of optical excitation of the collision complex is 3_{-2}^{+4}×10^{3} W^{-1} cm^{2} s^{-1} for λ=1550 nm, leading to a lifetime of

We show that the lifetime of ultracold ground-state ^{87}Rb^{133}Cs molecules in an optical trap is limited by fast optical excitation of long-lived two-body collision complexes. We partially suppress this loss mechanism by applying square-wave modulation to the trap intensity, such that the molecules spend 75% of each modulation cycle in the dark. By varying the modulation frequency, we show that the lifetime of the collision complex is 0.53±0.06 ms in the dark. We find that the rate of optical excitation of the collision complex is 3_{-2}^{+4}×10^{3} W^{-1} cm^{2} s^{-1} for λ=1550 nm, leading to a lifetime of100 ns for typical trap intensities. These results explain the two-body loss observed in experiments on nonreactive bialkali molecules.

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system.

Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Supplementation with PGE2 increased lentiviral vector (LVV) transduction of CD34+ cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34+ cells from multiple normal human donors and from patients with β-thalassemia or sickle cell disease. Notably, PGE2 increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE2 improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE2 may be useful in clinical gene therapy applications using lentivirally modified HSPCs., Heffner et al. performed a small-molecule screen and identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Their data suggest that PGE2-mediated improvements in transduction efficiency may aid in clinical gene therapy applications using lentivirally modified HSPCs.

Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.

Background Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown. Results Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription–splicing territories and changes to pre-mRNA splicing are observed. Conclusion We have produced the first H2A.B knockout mouse using the TALEN approach. Electronic supplementary material The online version of this article (10.1186/s13059-019-1633-3) contains supplementary material, which is available to authorized users.

We present the production of ultracold 87 RbCs molecules in the electronic, rovibrational and hyperfine ground state, using stimulated Raman adiabatic passage to transfer the molecules from a weakly bound Feshbach state. We measure one-way transfer efficiencies of 92(1)% and fully characterise the strengths and linewidths of the transitions used. We model the transfer, including a Monte Carlo simulation of the laser noise, and find this matches well with both the transfer efficiency and our previous measurements of the laser linewidth and frequency drifts.

Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether the disruption of the C-C chemokine receptor 5 (CCR5) locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early posttransplant, and 3% to 5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multilineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome-editing therapies for HIV and potentially other diseases as well.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

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.

Primary immunodeficiencies, including Wiskott-Aldrich syndrome (WAS), are a main target for genome-editing strategies using specific nucleases (SNs) because a small number of corrected hematopoietic stem cells could cure patients. In this work, we have designed various WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific zinc finger nucleases (ZFNs), using K-562 cells as a cellular model and plasmid nucleofection or integration-deficient lentiviral vectors (IDLVs) for delivery. The various CRISPR/Cas9 and ZFN SNs showed similar efficiency when using plasmid nucleofection for delivery. However, dual IDLVs expressing ZFNs were more efficient than dual IDLVs expressing Cas9 and guide RNA or all-in-one IDLVs, expressing Cas9 and guide RNA in the same vector. The specificity of heterodimeric ZFNs and CRISPR/Cas9, measured by increments in γ-H2AX focus formation in WAS-edited cells, was similar for both, and both outperformed homodimeric ZFNs independently of the delivery system used. Interestingly, we show that delivery of SNs, using IDLVs, is more efficient and less genotoxic than plasmid nucleofection. We also show the similar behavior of heterodimeric ZFNs and CRISPR/Cas9 for homology-directed gene knock-in strategies, with 88 and 83% of the donors inserted in the WAS locus, respectively, whereas when using homodimeric ZFNs only 45% of the insertions were on target. In summary, our data indicate that CRISPR/Cas9 and heterodimeric ZFNs are both good alternatives to further develop SN-based gene therapy strategies for WAS. However, IDLV delivery of WAS-specific heterodimeric ZFNs was the best option of all systems compared in this study.

We investigate the effect of far-off-resonant trapping light on ultracold bosonic 87Rb133Cs molecules. We use kHz-precision microwave spectroscopy to measure the differential AC~Stark shifts between the ground and first excited rotational levels of the molecule with hyperfine-state resolution. We demonstrate through both experiment and theory that coupling between neighboring hyperfine states manifests in rich structure with many avoided crossings. This coupling may be tuned by rotating the polarization of the linearly polarized trapping light. A combination of spectroscopic and parametric heating measurements allows complete characterization of the molecule polarizability at a wavelength of 1550~nm in both the ground and first excited rotational states.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress 99% of HD-causing alleles over a wide dose range while preserving expression of 86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis. Keywords: embryonic stem cells; human genome engineering; shelterin; telomerase telomere maintenance, National Institutes of Health (U.S.) (Grant R37-CA084198), National Institutes of Health (U.S.) (Grant RO1-CA087869), National Institutes of Health (U.S.) (Grant RO1-HD045022)

SummaryDistal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis, with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5(Δ32/Δ32)) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5(Δ32/Δ32) donors, we reasoned that engineered autologous CD34(+) hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.

Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans., Author Summary HIV-1 infection persists and requires life-long therapy. Approaches to prevent viral replication in the absence of treatment will likely require effective antiviral immune responses, but this goal has been confounded by HIV-1’s ability to target CD4 T cells that coordinate adaptive immunity. We describe a novel approach to confer HIV-resistance to CD4 T cells using peptides from the HIV-1 gp41 heptad repeat-2 (HR2) domain to inhibit infection. By linking a 34 amino acid HR2 peptide to the amino terminus of CCR5 or CXCR4 we were able to use the physiologic trafficking of these coreceptors to deliver the inhibitory peptide to sites of viral fusion where they exerted potent, specific and broad resistance irrespective of viral clade or tropism. This effect was highly dependent on the positioning of the peptide and most effective when conjugated to CXCR4. In vitro and in humanized mice, primary CD4 T cells were protected and expanded following HIV-1 infection. This work represents a proof of concept that T cells can be genetically engineered to resist infection in vivo and provides a rationale to explore this approach as a novel cell-based therapeutic in strategies to augment antiviral immune responses that target viral reservoirs and for long-term control of HIV-1.

We demonstrate coherent control of the rotational and hyperfine state of ultracold, chemically stable $^{87}\mathrm{Rb}^{133}\mathrm{Cs}$ molecules with external microwave fields. We create a sample of $\ensuremath{\sim}2000$ molecules in the lowest hyperfine level of the rovibronic ground state $N=0$. We measure the transition frequencies to eight different hyperfine levels of the $N=1$ state at two magnetic fields $\ensuremath{\sim}23$ G apart. We determine accurate values of rotational and hyperfine coupling constants that agree well with previous calculations. We observe Rabi oscillations on each transition, allowing complete population transfer to a selected hyperfine level of $N=1$. Subsequent application of a second microwave pulse allows the transfer of molecules back to a different hyperfine level of $N=0$.

We report the binding energy of $^{87}\mathrm{Rb}^{133}\mathrm{Cs}$ molecules in their rovibrational ground state measured using an offset-free optical frequency comb based on difference frequency generation technology. We create molecules in the absolute ground state using stimulated Raman adiabatic passage (STIRAP) with a transfer efficiency of 88%. By measuring the absolute frequencies of our STIRAP lasers, we find the energy-level difference from an initial weakly bound Feshbach state to the rovibrational ground state with a resolution of $\ensuremath{\sim}5$ kHz over an energy-level difference of more than $114\phantom{\rule{0.28em}{0ex}}\mathrm{T}\mathrm{Hz}$; this lets us discern the hyperfine splitting of the ground state. Combined with theoretical models of the Feshbach-state binding energies and ground-state hyperfine structure, we determine a zero-field binding energy of $h\ifmmode\times\else\texttimes\fi{}114\phantom{\rule{0.16em}{0ex}}268\phantom{\rule{0.16em}{0ex}}135.24(4)(3)\phantom{\rule{0.28em}{0ex}}\mathrm{M}\mathrm{Hz}$. To our knowledge, this is the most accurate determination to date of the dissociation energy of a molecule.

We present the production of ultracold

Malaria afflicts over 200 million people worldwide and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum pathogenesis, including drug resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite, using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene deletion parasites with unprecedented speed (two weeks), both with and without direct selection. ZFNs engineered against the endogenous parasite gene pfcrt, responsible for chloroquine treatment escape, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. The efficiency, versatility and precision of this method will enable a diverse array of genome editing approaches to interrogate this human pathogen.

Selective inhibition of disease-related proteins underpins the majority of successful drug–target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.

SummaryChromatin loops juxtapose distal enhancers with active promoters, but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and β-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the β-globin promoter in GATA1 null erythroblasts, in which the β-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the β-globin promoter substantially activated β-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as a critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.PaperFlick

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4(+) T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4(+) T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4(+) T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4(+) T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4(+) T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4(+) T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4(+) T cells during HIV-1 infection.

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR beta- and alpha-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

Zinc-finger nucleases (ZFNs) are chimeric endonucleases created by fusing the cleavage domain from the type IIS restriction enzyme FokI to a designed zinc-finger protein (ZFP). In order to generate a DNA double strand break (DSB) at a predetermined genomic site, two engineered ZFNs must be designed to bind DNA with the appropriate orientation and spacing (i.e., on opposite sides of the DNA with 5 or 6 bp of sequence separating them) (Carroll 2008; Cathomen and Joung 2008). This architecture (analogous to that depicted in Fig. 1A) permits the two FokI cleavage domains to heterodimerize—a prerequisite for subsequent cleavage of the targeted DNA (Bitinaite et al. 1998). Figure 1. Mutation of the catalytic domain of a ZFN monomer generates a ZFN heterodimer with nicking activity. (A) Illustration of the ZFN heterodimer architecture for single-stranded break (SSB) generation. The FokI catalytic domain of ZFN (left, blue) is mutated ... Repair of the ZFN-induced site-specific DSB provides the molecular basis for gene disruption, gene correction, or gene addition—the different potential outcomes of ZFN-mediated genome editing (for review, see Urnov et al. 2010). Specifically, a given DSB may be repaired via either the nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways (Kowalczykowski 2000; Sung and Klein 2006; Wyman and Kanaar 2006; Brugmans et al. 2007). HDR utilizes a homologous donor sequence as template for the conservative repair of the DNA break. Provision of a suitably designed donor DNA molecule can, therefore, specify gene addition or gene correction at the ZFN-induced DSB. NHEJ, on the other hand, simply catalyzes the rejoining of the two DNA ends—a process that can result in the deletion or insertion of nucleotides at the repair junction. DNA repair via NHEJ is, therefore, mutagenic. To date, ZFN-induced DNA repair via HDR or NHEJ has been utilized to target modifications to the genomes of numerous species (Le Provost et al. 2010) including Caenorhabditis elegans (Morton et al. 2006), Drosophila melanogaster (Bibikova et al. 2002), silkworms (Takasu et al. 2010), zebrafish (Doyon et al. 2008; Meng et al. 2008), sea urchins (Ochiai et al. 2010), Arabidopsis thaliana (Osakabe et al. 2010; Zhang et al. 2010), tobacco (Maeder et al. 2008; Cai et al. 2009; Townsend et al. 2009), corn (Shukla et al. 2009), mice (Carbery et al. 2010; Meyer et al. 2010), and rats (Geurts et al. 2009). ZFNs have also been used to efficiently engineer a diverse range of mammalian cell types (Urnov et al. 2010), including human stem cells, with subsequent retention of full potency and normal growth characteristics (Hockemeyer et al. 2009; Zou et al. 2009; Holt et al. 2010). Moreover, the therapeutic potential of ZFNs is currently under evaluation in clinical trials (clinicaltrials.gov identifiers NCT00842634, NCT01252641, NCT01044654). Given the markedly different genome editing outcomes mediated by NHEJ and HDR, it would be desirable to have control over the choice of pathway used and thus specify the outcome of repair. In practice, for gene disruption this can already be achieved, since NHEJ is the predominant pathway in mammalian cells (Lieber 2008) and codelivery of donor DNA is unnecessary. For gene correction and addition, however, NHEJ events represent competition to the desired homology-directed outcome at the target locus. One potential strategy for enforcing DNA repair pathway bias would be the targeted introduction of a DNA single strand break (SSB) or nick. Like the DSB, a DNA SSB/nick can theoretically stimulate homology-directed repair pathways (Holliday 1964; Meselson and Radding 1975; Radding 1982). For example, SSBs induced by the bacteriophage fd gene II protein (gIIp) stimulated HDR in yeast (Galli and Schiestl 1998), while nicks generated by specific mutants of the RAG proteins—critical components of the V(D)J recombination system—are repaired via initiation of homologous recombination (Lee et al. 2004). Moreover, experiments using a nick-inducing variant of the I-Anil LAGLIDADG homing endonuclease showed that the SSB/nicks induced by these engineered enzymes are also repaired via HDR (Smith et al. 2009). Importantly, and in contrast to a DSB, a DNA nick or SSB is not a bona fide substrate for repair via the NHEJ pathway. Thus, a targeted SSB has the potential to restrict repair to the homology-directed pathway. Here, we describe the engineering of a zinc finger nickase (ZFNickase) by mutation of a residue critical for FokI cleavage activity in one monomer of the ZFN heterodimer, thus allowing the ZFNs to heterodimerize on DNA but restricting cleavage to a single DNA strand. We show that targeting of these ZFNickases to the endogenous CCR5 locus stimulates gene correction and gene addition via HDR at frequencies of between ∼1%–8% in both transformed and primary human lines. Importantly, we show that the CCR5 ZFNickase-induced gene addition occurs without a marked increase in detectable mutagenesis by the error-prone NHEJ repair pathway. Specific outcomes of either gene addition/correction or gene disruption may, therefore, be modulated via the targeted introduction of a single- or double-strand break, respectively. Moreover, beyond the targeted event itself, elimination of DNA repair via NHEJ has the potential to eradicate mutagenesis at putative off-target cleavage sites, further improving the fidelity of genome editing.

Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site-specific endonucleases. Two genomic sites within the MBS85 and chemokine (C-C motif) receptor 5 (CCR5) genes (AAVS1 and CCR5 zinc-finger nuclease (CCR5-ZFN) sites, respectively) have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells (HSCs). Matrix chromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptional inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and HSCs. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a ZFN was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.