Search

Describes the cloning and functional characterization of a calcium and magnesium permeable divalent cation channel, LTRPC7, a new member of the LTRPC family of putative ion channels. Role of LTRPC7 in cell physiology; Analysis of the permeation characteristics of the mediated current of LTRPC7; Analysis of LTRPC7 function.

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cγ (PLCγ) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P3 regulates receptor-dependent calcium signals through the function of Tec kinases. [ABSTRACT FROM AUTHOR]

Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.

An abstract of the article "Engineering and Development of I-AniI Homing Endonucleases for Gene Correction Applications," by Andrew M. Scharenberg and colleagues is presented.

Gene editing is a rapidly developing area of biotechnology in which the nucleotide sequence of the genome of living cells is precisely changed. The use of genome-editing technologies to modify various types of blood cells, including hematopoietic stem cells, has emerged as an important field of therapeutic development for hematopoietic disease. Although these technologies offer the potential for generation of transformative therapies for patients suffering from myriad disorders of hematopoiesis, their application for therapeutic modification of primary human cells is still in its infancy. Consequently, development of ethical and regulatory frameworks that ensure their safe and effective use is an increasingly important consideration. Here, we review a number of issues that have the potential to impact the clinical implementation of genome-editing technologies, and suggest paths forward for resolving them such that new therapies can be safely and rapidly translated to the clinic. [ABSTRACT FROM AUTHOR]

Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper- immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter--dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LGlocus in primary human T cells using a combination of a transcription activator-like effector nuclease--induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3'-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome. [ABSTRACT FROM AUTHOR]

Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for "genome editing" in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing. [ABSTRACT FROM AUTHOR]

Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the centre of a 20-base-pair (bp) DNA target site, with the amino (N)-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured carboxy (C)-terminal domain interacting with the right (+) side. Here we show that, despite the approximate twofold symmetry of the enzyme–DNA complex, there is almost complete segregation of interactions responsible for substrate binding to the (-) side of the interface and interactions responsible for transition-state stabilization to the (+) side. Although single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (-) DNA half-site almost exclusively increase the dissociation constant (KD) and the Michaelis constant under single-turnover conditions (KM*), and those in the (+) half-site primarily decrease the turnover number (kcat*). The reduction of activity produced by mutations on the (-) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the use of enzyme–substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (-) side do so by modulating KM*, whereas redesigns with altered specificities on the (+) side modulate kcat*. Our results illustrate how classical enzymology and modern protein design can each inform the other. [ABSTRACT FROM AUTHOR]

TRPM7 is a polypeptide with intrinsic ion channel and protein kinase domains whose targeted deletion causes cells to experience growth arrest within 24 hr and eventually die. Here, we show that while TRPM7''s kinase domain is not essential for activation of its channel, a functional coupling exists such that structural alterations of the kinase domain alter the sensitivity of channel activation to Mg2+. Investigation of the relationship between Mg2+ and the cell biological role of TRPM7 revealed that TRPM7-deficient cells become Mg2+ deficient, that both the viability and proliferation of TRPM7-deficient cells are rescued by supplementation of extracellular Mg2+, and that the capacity of heterologously expressed TRPM7 mutants to complement TRPM7 deficiency correlates with their sensitivity to Mg2+. Overall, our results indicate that TRPM7 has a central role in Mg2+ homeostasis as a Mg2+ uptake pathway regulated through a functional coupling between its channel and kinase domains. [Copyright &y& Elsevier]

Trace metal ions such as Zn[sup 2+], Fe[sup 2+], Cu[sup 2+], Mn[sup 2+], and Co[sup 2+] are required coihctors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca[sup 2+]- and Mg[sup 2+]-permeable cation channel, whose activity is regulated by intracellular Mg[sup 2+] and Mg[sup 2+]·ATP and have designated native TRPMT-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn[sup 2+] and Ni[sup 2+], which both permeate TRPM7 up to four times better than Ca[sup 2+]. Similarly, native MagNuM currents are also able to support Zn[sup 2+] entry. Furthermore, TRPM7 allows other essential metals such as Mn[sup 2+] and Co[sup 2+] to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd[sup 2+], Ba[sup 2+], and Sr[sup 2+]. Equimolar replacement studies substituting 10 mM Ca[sup 2+] with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn[sup 2+] ≈ Ni[sup 2+] » Ba[sup 2+] > Co[sup 2+] > Mg[sup 2+] ≥ Mn[sup 2+] ≥ Sr[sup 2+] ≥ Cd[sup 2+] ≥ Ca[sup 2+], while trivalent ions such as La[sup 3+] and Gd[sup 3-] are not measurably permeable. With the exception of Mg[sup 2+], which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn[sup 2+], Co[sub 2+], or Ni[sub 2+] suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca[sub 2+] and Mg[sup 2+], suggesting that TRPM7 represents a novel ion-channel mechanism for cellular... [ABSTRACT FROM AUTHOR]

Examines the calcium-activated nonselective cation channel for cellular responses. Characterization of molecular gene encoding in Drosophila melanogaster; Evaluation on systematic cloning strategy for mammalian isoforms; Details of electrophysiological characterizations in various cell types.

Presents a study which showed that an amino acid protein and a homologous domain near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific free ADP-ribose pyrophosphatases (ADPR). Analysis of the permeation selectivity of LTRPC2; Effect of ADPR on LTRPC 2 function; Results of the study.

Abstract: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Engineered T cells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of in vivo cytokine support can lead to poor engraftment of transferred T cells, including regulatory T cells (T reg ). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary T cells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ T cells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the T reg master regulator FOXP3. Following editing of CD4+ T cells, CISC+ engineered T reg (CISC EngTreg) were selectively expanded using rapamycin and maintained T reg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, in vivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both in vitro enrichment and in vivo engraftment and activation, features likely beneficial across multiple gene-edited T cell applications., Competing Interests: Declaration of interests D.J.R. is a scientific co-founder and Scientific Advisory Board (SAB) member of GentiBio, Inc., and scientific co-founder and SAB member of BeBiopharma, Inc. A.M.S. is a scientific co-founder and SAB member of GentiBio, Inc., and scientific co-founder and CEO of Umoja Biopharma. D.J.R. received past and current funding from GentiBio, Inc., for related work. D.J.R., A.M.S., P.J.C., and K.S. are inventors on patents describing methods for generating antigen-specific engineered regulatory T cells and/or use of the CISC platform. G.I.U. was a previous employee of Casebia Therapeutics and a current employee of GentiBio, Inc., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation., Methods: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice., Results: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy., Conclusions: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing., Competing Interests: Competing interests: All authors hold equity with Umoja Biopharma., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases., (© 2021. The Author(s).)

Thymic regulatory T cells (tT regs ) are potent inhibitors of autoreactive immune responses, and loss of tT reg function results in fatal autoimmune disease. Defects in tT reg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of T reg as cell therapies to establish immune tolerance. Because tT regs are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous T reg -like cells from bulk CD4 + T cells. We used homology-directed repair (HDR)-based gene editing to enforce expression of FOXP3, the master transcription factor for tT reg Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edT regs , manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tT reg Both human and murine edT regs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edT reg with robust in vitro and in vivo functional activity. Last, edT reg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edT reg production may permit broad future clinical application., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34 + mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12-14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ -/- Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The treatment or cure of HIV infection by cell and gene therapy has been a goal for decades. Recent advances in both gene editing and chimeric antigen receptor (CAR) technology have created new therapeutic possibilities for a variety of diseases. Broadly neutralizing monoclonal antibodies (bNAbs) with specificity for the HIV envelope glycoprotein provide a promising means of targeting HIV-infected cells. Here we show that primary human T cells engineered to express anti-HIV CARs based on bNAbs (HIVCAR) show specific activation and killing of HIV-infected versus uninfected cells in the absence of HIV replication. We also show that homology-directed recombination of the HIVCAR gene expression cassette into the CCR5 locus enhances suppression of replicating virus compared with HIVCAR expression alone. This work demonstrates that HIV immunotherapy utilizing potent bNAb-based single-chain variable fragments fused to second-generation CAR signaling domains, delivered directly into the CCR5 locus of T cells by homology-directed gene editing, is feasible and effective. This strategy has the potential to target HIV-infected cells in HIV-infected individuals, which might help in the effort to cure HIV., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)