Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Essential nucleotide- and protein-dependent functions of Actb/ß-actin.

The highly similar cytoplasmic ß- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked ß-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, ß-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.

sdf1 Expression reveals a source of perivascular-derived mesenchymal stem cells in zebrafish.

There is accumulating evidence that mesenchymal stem cells (MSCs) have their origin as perivascular cells (PVCs) in vivo, but precisely identifying them has been a challenge, as they have no single definitive marker and are rare. We have developed a fluorescent transgenic vertebrate model in which PVC can be visualized in vivo based upon sdf1 expression in the zebrafish. Prospective isolation and culture of sdf1(DsRed) PVC demonstrated properties consistent with MSC including prototypical cell surface marker expression; mesodermal differentiation into adipogenic, osteogenic, and chondrogenic lineages; and the ability to support hematopoietic cells. Global proteomic studies performed by two-dimensional liquid chromatography and tandem mass spectrometry revealed a high degree of similarity to human MSC (hMSC) and discovery of novel markers (CD99, CD151, and MYOF) that were previously unknown to be expressed by hMSC. Dynamic in vivo imaging during fin regeneration showed that PVC may arise from undifferentiated mesenchyme providing evidence of a PVC-MSC relationship. This is the first model, established in zebrafish, in which MSC can be visualized in vivo and will allow us to better understand their function in a native environment.

Deletion of exons 2 and 3 from Actb and cell immortalization lead to widespread, ß-actin independent alterations in gene expression associated with cell cycle control.

The cytoplasmic actin proteins, ß- and γ-actin, are 99% identical but thought to perform non-redundant functions. The nucleotide coding regions of cytoplasmic actin genes, Actb and Actg1, are 89% identical. Knockout (KO) of Actb by Cre-mediated deletion of first coding exons 2 and 3 in mice is embryonic lethal and fibroblasts derived from KO embryos (MEFs) fail to proliferate. In contrast, Actg1 KO MEFs display with a much milder defect in cell proliferation and Actg1 KO mice are viable, but present with increased perinatal lethality. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the ß-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and mass spectrometry reveal largescale changes to the transcriptome, proteome, and phosphoproteome in cells lacking Actb but not those only lacking ß-actin protein. Pathway analysis of genes and proteins differentially expressed upon Actb KO suggest widespread dysregulation of genes involved in the cell cycle that may explain the severe defect in proliferation.

The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness.

The gene coding for centrosomal protein 290 (CEP290), a large multidomain protein, is the most frequently mutated gene underlying the non-syndromic blinding disorder Leber's congenital amaurosis (LCA). CEP290 has also been implicated in several cilia-related syndromic disorders including Meckel-Gruber syndrome, Joubert syndrome, Senor-Loken syndrome and Bardet-Biedl syndrome (BBS). In this study, we characterize the developmental and functional roles of cep290 in zebrafish. An antisense oligonucleotide [Morpholino (MO)], designed to generate an altered cep290 splice product that models the most common LCA mutation, was used for gene knockdown. We show that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of bbs genes in zebrafish. Consistent with a role in cilia function, the cep290 MO-injected embryos exhibited a curved body axis. Patients with LCA caused by mutations in CEP290 have reduced visual perception, although they present with a fully laminated retina. Similarly, the histological examination of retinas from cep290 MO-injected zebrafish revealed no gross lamination defects, yet the embryos had a statistically significant reduction in visual function. Finally, we demonstrate that the vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data reveal that a specific region of the CEP290 protein is sufficient to restore visual function and this region may be a viable gene therapy target for LCA patients with mutations in CEP290.

Stromal cell-derived factor-1 and hematopoietic cell homing in an adult zebrafish model of hematopoietic cell transplantation.

In mammals, stromal cell-derived factor-1 (SDF-1) promotes hematopoietic cell mobilization and migration. Although the zebrafish, Danio rerio, is an emerging model for studying hematopoietic cell transplantation (HCT), the role of SDF-1 in the adult zebrafish has yet to be determined. We sought to characterize sdf-1 expression and function in the adult zebrafish in the context of HCT. In situ hybridization of adult zebrafish organs shows sdf-1 expression in kidney tubules, gills, and skin. Radiation up-regulates sdf-1 expression in kidney to nearly 4-fold after 40 Gy. Assays indicate that zebrafish hematopoietic cells migrate toward sdf-1, with a migration ratio approaching 1.5 in vitro. A sdf-1a:DsRed2 transgenic zebrafish allows in vivo detection of sdf-1a expression in the adult zebrafish. Matings with transgenic reporters localized sdf-1a expression to the putative hematopoietic cell niche in proximal and distal renal tubules and collecting ducts. Importantly, transplant of hematopoietic cells into myelosuppressed recipients indicated migration of hematopoietic cells to sdf-1a-expressing sites in the kidney and skin. We conclude that sdf-1 expression and function in the adult zebrafish have important similarities to mammals, and this sdf-1 transgenic vertebrate will be useful in characterizing the hematopoietic cell niche and its interactions with hematopoietic cells.

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle.

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or ßcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.

Relative importance of ßcyto- and γcyto-actin in primary mouse embryonic fibroblasts.

The highly homologous ß (ßcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine ßcyto- and γcyto-isoform--specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary ßcyto- or ß/γcyto-actin double-knockout fibroblasts. Gene targeting of ßcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of ßcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of ßcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin.

A model of glucose-6-phosphate dehydrogenase deficiency in the zebrafish.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common genetic defect and enzymopathy worldwide, affecting approximately 400 million people and causing acute hemolysis in persons exposed to prooxidant compounds such as menthol, naphthalene, antimalarial drugs, and fava beans. Mouse models have not been useful because of a lack of significant response to oxidative challenge. We turned to zebrafish (Danio rerio) embryos, which develop ex utero and are transparent, allowing visualization of hemolysis. We designed morpholinos to zebrafish g6pd that were effective in reducing gene expression as shown by Western blot and G6PD enzyme activity, resulting in a brisk hemolysis and pericardial edema secondary to anemia. Titration of the g6pd knockdown allowed us to generate embryos that displayed no overt phenotype until exposed to the prooxidant compounds 1-naphthol, menthol, or primaquine, after which they developed hemolysis and pericardial edema within 48-72 hours. We were also able to show that g6pd morphants displayed significant levels of increased oxidative stress compared with controls. We anticipate that this will be a useful model of G6PD deficiency to study hemolysis as well as oxidative stress that occurs after exposure to prooxidants, similar to what occurs in G6PD-deficient persons.

Zebrafish stromal cells have endothelial properties and support hematopoietic cells.

The goal of this study was to determine if we could establish a mesenchymal stromal line from zebrafish that would support hematopoietic cells. Such a coculture system would be a great benefit to study of the hematopoietic cell-stromal cell interaction in both in vitro and in vivo environments. Zebrafish stromal cells (ZStrC) were isolated from the "mesenchymal" tissue of the caudal tail and expanded in a specialized growth media. ZStrC were evaluated for phenotype, gene expression, and ability to maintain zebrafish marrow cells in coculture experiments. ZStrC showed mesenchymal and endothelial gene expression. Although ZStrC lacked the ability to differentiate into classic mesenchymal stromal cell lineages (i.e., osteocytes, adipocytes, chondrocytes), they did have the capacity for endotube formation on Matrigel and low-density lipoprotein uptake. ZStrC supported marrow cells for >2 weeks in vitro. Importantly, marrow cells were shown to retain homing ability in adoptive transfer experiments. ZStrC were also shown to improve hematopoietic recovery after sublethal irradiation after adoptive transfer. As the zebrafish model grows in popularity and importance in the study of hematopoiesis, new tools to aid in our understanding of the hematopoietic cell-stromal cell interaction are required. ZStrC represent an additional tool in the study of hematopoiesis and will be useful in understanding the factors that mediate the stromal cell-hematopoietic cell interactions that are important in hematopoietic cell maintenance.