Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

SUMMARYThe liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I-CreI homing endonuclease. A single-chain endonuclease, comprising a pair of I-CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium-mediated transformation of immature embryos, and transgenic T 0 plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T 0 plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant.

pus oocytes contained 7-11 zinc ions per protein, 21 led to the proposal that each 30-amino acid sequence bound one zinc ion through the conserved cysteine and histidine residues. This hypothesis was supported by limited proteolysis studies that yielded fragments differing in length by about 3 kDa, 21 suggesting that these proposed metal-binding units form individually folded, structurally stable domains.The nature of the metal binding sites in TFIIIA was directly probed by X-ray absorption spectroscopic methods. EXAFS analysis of the TFIIIA-5S RNA complex isolated from immature Xenopus oocytes was consistent with the proposed coordination with two sulfur atoms at 2.30 Å and two nitrogen atoms at 2.00 Å. 23 These distances matched reasonably closely those observed for zinc model complexes prepared by Koch and co-workers. 24,25 Zinc Finger Domain Peptides with Naturally Occurring SequencesThe zinc finger hypothesis suggested a reductionist approach to the characterization of these domains. Derek Jantz was born in Littleton, Colorado, and graduated from the University of Colorado at Boulder (B.A. 1996). He received his Ph.D. from the Johns Hopkins University School of Medicine in 2003 under the direction of Jeremy Berg. His research concerned the DNA-binding characteristics of designed Cys 2 His 2 zinc finger proteins. He is presently working as a Postdoctoral Fellow with Homme Hellinga at Duke University Medical Center, focusing on computational protein design. He was selected as graduate student speaker at his graduation from Johns Hopkins, where he described graduate school as "the best period of indeterminate length of my life". Barbara T. Amann was born and raised in Swarthmore, Pennsylvania. She received her B.A. in chemistry from Mt. Holyoke College in 1983. She developed an interest in bioinorganic chemistry at the Pennsylvania State University, where she received her Ph.D. working with Bill Horrocks on lanthanide substitution of the calcium-binding protein calmodulin. In 1988, she joined Jeremy Berg's laboratory as a Postdoctoral Fellow in the Chemistry Department of Johns Hopkins University. She moved with the Berg laboratory to the Department of Biophysics and Biophysical Chemistry at Johns Hopkins School of Medicine and became a Research Associate. In this position, she has studied a range of zinc-binding domains, primarily by NMR, and has run the NMR facility of the department. She has enjoyed teaching other researchers NMR methods and assisting them with their projects. She loves spending time with her husband, two children, dog, and snake. Gregory J. Gatto, Jr., was raised in East Brunswick, New Jersey. He received his A.B. in chemistry in 1994 from Princeton University, where he worked in the laboratory of Martin F. Semmelhack. In the spring of 2003, he received his M.D. and Ph.D. degrees from Johns Hopkins University School of Medicine. There, he worked in the laboratory of Jeremy Berg on the structural biology of designed zinc finger proteins and the molecular details of peroxisomal targeting sig...

Clinical translation of in vivo genome editing to treat human genetic diseases requires thorough preclinical studies in relevant animal models to assess safety and efficacy. A promising approach to treat hypercholesterolemia is inactivating the secreted protein PCSK9, an antagonist of the LDL receptor. Here we show that single infusions in six non-human primates of adeno-associated virus vector expressing an engineered meganuclease targeting PCSK9 results in dose-dependent disruption of PCSK9 in liver, as well as a stable reduction in circulating PCSK9 and serum cholesterol. Animals experienced transient, asymptomatic elevations of serum transaminases owing to the formation of T cells against the transgene product. Vector DNA and meganuclease expression declined rapidly, leaving stable populations of genome-edited hepatocytes. A second-generation PCSK9-specific meganuclease showed reduced off-target cleavage. These studies demonstrate efficient, physiologically relevant in vivo editing in non-human primates, and highlight safety considerations for clinical translation.

SUMMARYThe I-CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double-strand breaks and enhancing DNA recombination reactions in maize cells. The DNA-binding properties of this protein were re-designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single-chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium-mediated transformation, the cleavage resulted in mutations at a co-delivered extra-chromosomal ms26-site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26-site in 5.8% of transgenic T 0 plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of doublestrand break repair generated by non-homologous end joining. One of 21 mutagenized T 0 plants carried two mutated alleles of the MS26 gene. As expected, the bi-allelic mutant T 0 plant and the T 1 progeny homozygous for the ms26 mutant alleles were male-sterile. This paper described the second maize chromosomal locus (liguless-1 being the first one) mutagenized by a re-designed I-CreI-based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.