Search

Indirect modification of animal genomes by interspecific hybridization, cross-breeding, and selection has produced an enormous spectrum of phenotypic diversity over more than 10,000 yr of animal domestication. Using these established technologies, the farming community has successfully increased the yield and efficiency of production in most agricultural species while utilizing land resources that are often unsuitable for other agricultural purposes. Moving forward, animal well-being and agricultural sustainability are moral and economic priorities of consumers and producers alike. Therefore, these considerations will be included in any strategy designed to meet the challenges produced by global climate change and an expanding world population. Improvements in the efficiency and precision of genetic technologies will enable a timely response to meet the multifaceted food requirements of a rapidly increasing world population.

As natural environments deteriorate, genetic improvements to agricultural animals will be required to ensure global food security. Improving livestock production by introducing asexual reproduction (AR) into mainstream animal husbandry can help meet the challenge, but its advantages must be accompanied by social, commercial, and governmental acceptance., Competing Interests: Declaration of interests P.B.H. is a founder of Recombinetics/Acceligen that edits genomes of agricultural animals. The other authors have no interests to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal disorder that causes syndromes characterized by physiological dysfunction in many organs and tissues. Despite the recognizable morphological and behavioral deficits associated with MPS I, neither the underlying alterations in functional neural connectivity nor its restoration following gene therapy have been shown. By employing high-resolution resting-state fMRI (rs-fMRI), we found significant reductions in functional neural connectivity in the limbic areas of the brain that play key roles in learning and memory in MPS I mice, and that adeno-associated virus (AAV)-mediated gene therapy can reestablish most brain connectivity. Using logistic regression in MPS I and treated animals, we identified functional networks with the most alterations. The rs-fMRI and statistical methods should be translatable into clinical evaluation of humans with neurological disorders., (© 2023. Springer Nature Limited.)

There is an urgent need to reform the regulation of transgenic and genome-edited food animals. Now is the time to simplify regulatory safety guidelines based on science before it is too late to have these animals in place to meet societal needs in coming decades., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Genetically modified food animals (GMFAs) are needed to address early the cumulative effects of livestock production on the environment, and to accommodate future food demands. In 2020 China and the USA, the world's two largest economies, embarked on regulatory reforms to boost the commercialization of such animals. However, gaining social acceptance of GMFAs for commercialization remains a global challenge. We propose a framework that focuses on social license for commercialization of GMFAs by defining four classes of improvement using precision genetics: (1) animals equivalent to natural variation to obtain the improved effect of cross-breeding (ENV); (2) animals with an inactivated gene that could occur via natural mutation (ENC-); (3) animals harboring a natural genetic sequence isolated from another species (ENC+); and (4) animals with synthetic sequences encoding novel genes (BNE). Our approach can guide regulators and the public to support orderly commercialization of GMFAs., (© The Author(s) 2021. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd.)

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose- block-2-methylaminoethyl methacrylate) [P(MAG- b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG- b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG- b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG- b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG- b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG- b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG- b-MAEMt) N/P = 5 and P(MAG- b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG- b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG- b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl 3 ) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl 3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl 3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 μg/mL). It is concluded that GdCl 3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative regimens will be required to achieve therapeutic levels of transgene products.

The Sleeping Beauty transposon system has been extensively tested for integration of reporter and therapeutic genes in vitro and in vivo in mice. Dogs were used as a large animal model for human therapy and minimally invasive infusion of DNA solutions. DNA solutions were delivered into the entire liver or the left side of the liver using balloon catheters for temporary occlusion of venous outflow. A peak intravascular pressure between 80 and 140 mmHg supported sufficient DNA delivery in dog liver for detection of secretable reporter proteins. Secretable reporters allowed monitoring of the time course of gene products detectable in the circulation postinfusion. Canine secreted alkaline phosphatase reporter protein levels were measured in plasma, with expression detectable for up to 6 weeks, while expression of canine erythropoietin was detectable for 7-10 days. All animals exhibited a transient increase in blood transaminases that normalized within 10 days; otherwise the treated animals were clinically normal. These results demonstrate the utility of a secreted reporter protein for real-time monitoring of gene expression in the liver in a large animal model but highlight the need for improved delivery in target tissues to support integration and long-term expression of Sleeping Beauty transposons.

Background: T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR., Methods: T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19)., Results: SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients., Conclusions: CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach., Trial Registration: Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036., Funding: National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

Over the past decade, the technology to engineer genetically modified swine has seen many advancements, and because their physiology is remarkably similar to that of humans, swine models of cancer may be extremely valuable for preclinical safety studies as well as toxicity testing of pharmaceuticals prior to the start of human clinical trials. Hence, the benefits of using swine as a large animal model in cancer research and the potential applications and future opportunities of utilizing pigs in cancer modeling are immense. In this review, we discuss how pigs have been and can be used as a biomedical models for cancer research, with an emphasis on current technologies. We have focused on applications of precision genetics that can provide models that mimic human cancer predisposition syndromes. In particular, we describe the advantages of targeted gene-editing using custom endonucleases, specifically TALENs and CRISPRs, and transposon systems, to make novel pig models of cancer with broad preclinical applications.

The liver is an ideal target for nucleic acid therapeutic applications (i.e., siRNA, gene therapy, and genome editing) due to its ability to secrete proteins into the blood. In this work, we present the first synthesis of a novel monomer derived from N-acetyl-d-galactosamine (GalNAc) and its polymerization as a facile route to create multivalent delivery vehicles with exceptional targeting efficiency to asialoglycoprotein receptors (ASGPRs) on liver hepatocytes. A series of cationic diblock GalNAc glycopolymers composed of a GalNAc-derived block of fixed length (n = 62) and cationic 2-aminoethylmethacrylamide (AEMA) blocks of varying lengths (n = 19, 33, and 80) have been synthesized and characterized. In addition, nontargeted control polymers consisting of either glucose or polyethylene glycol-derived neutral blocks with an AEMA cationic block were also created and examined. All polymeric vehicles were able to bind and encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes). The GalNAc-derived polyplexes were colloidally stable and maintained their size over a period of 4 h in reduced-serum cell culture media. The GalNAc-derived homopolymer effectively inhibited the uptake of Cy5-labeled asialofetuin (a natural ligand of ASGPRs) by cultured hepatocyte (HepG2) cells at lower concentrations (IC50 = 20 nM) than monomeric GalNAc (IC50 = 1 mM) and asialofetuin (IC50 = 1 μM), suggesting highly enhanced ASGPR binding due to multivalency. These polymers also showed cell type-specific gene expression in cultured cells, with higher protein expression in ASGPR-presenting HepG2 than HeLa cells, which lack the receptor. Biodistribution studies in mice show higher accumulation of pDNA and GalNAc-derived polymers in the liver compared with the glucose-derived nontargeted control. This study demonstrates the first facile synthesis of a multivalent GalNAc-derived block copolymer architecture that promotes enhanced delivery to liver and offers insights to improve targeted nanomedicines for a variety of applications.

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization to yield pMAT 43 . The pMAT 43 macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT- b -AEMA cationic-block copolymers, pMAT- b -AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT- b -AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT- b -AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo.

Introduction: Mucopolysaccharidoses (MPS) are a family of lysosomal disorders caused by mutations in genes that encode enzymes involved in the catabolism of glycoaminoglycans. These mutations affect multiple organ systems and can be particularly deleterious to the nervous system. At the present time, enzyme replacement therapy and hematopoietic stem-cell therapy are used to treat patients with different forms of these disorders. However, to a great extent, the nervous system is not adequately responsive to current therapeutic approaches., Areas Covered: Recent advances in gene therapy show great promise for treating MPS. This article reviews the current state of the art for routes of delivery in developing genetic therapies for treating the neurologic manifestations of MPS., Expert Opinion: Gene therapy for treating neurological manifestations of MPS can be achieved by intraventricular, intrathecal, intranasal and systemic administrations. The intraventricular route of administration appears to provide the most widespread distribution of gene therapy vectors to the brain. The intrathecal route of delivery results in predominant distribution to the caudal areas of the brain. The systemic route of delivery via intravenous infusion can also achieve widespread delivery to the CNS; however, the distribution to the brain is greatly dependent on the vector system. Intravenous delivery using lentiviral vectors appear to be less effective than adeno-associated viral (AAV) vectors. Moreover, some subtypes of AAV vectors are more effective than others in crossing the blood-brain barrier. In summary, the recent advances in gene vector technology and routes of delivery to the CNS will facilitate the clinical translation of gene therapy for the treatment of the neurological manifestations of MPS.

Most lysosomal storage disorders affect the nervous system as well as other tissues and organs of the body. Previously, the complexities of these diseases, particularly in treating neurologic abnormalities, were too great to surmount. However, based on recent developments there are realistic expectations that effective therapies are coming soon. Gene therapy offers the possibility of affordable, comprehensive treatment associated with these diseases currently not provided by standards of care. With a focus on correction of neurologic disease by systemic gene therapy of mucopolysaccharidoses types I and IIIA, we review some of the major recent advances in viral and non-viral vectors, methods of their delivery and strategies leading to correction of both the nervous and somatic tissues as well as evaluation of functional correction of neurologic manifestations in animal models. We discuss two questions: what systemic gene therapy strategies work best for correction of both somatic and neurologic abnormalities in a lysosomal storage disorder and is there evidence that targeting peripheral tissues (e.g., in the liver) has a future for ameliorating neurologic disease in patients?, (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Viruses have been used to deliver two types of site-specific nucleases into cells for targeted gene editing., (Copyright © 2014, Hackett and Somia.)

The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA-binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA-binding domain remains mostly unstructured but its N-terminal PAI subdomain forms a compact, three-helical structure with a helix-turn-helix motif at higher concentrations of NaCl. Furthermore, we show that the full-length SB DNA-binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA-binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA.

The Sleeping Beauty transposon system, a non-viral, integrating vector that can deliver the alpha-L-iduronidase-encoding gene, is efficient in correcting mucopolysaccharidosis type I in NOD/SCID mice. However, in previous studies we failed to attain reliable long-term alpha-L-iduronidase expression in immunocompetent mice. Here, we focused on achieving sustained high-level expression in immunocompetent C57BL/6 mice. In our standard liver-directed treatment we hydrodynamically infuse mice with plasmids containing a SB transposon-encoding human alpha-L-iduronidase, along with a source of SB transposase. We sought to 1) minimize expression of the therapeutic enzyme in antigen-presenting cells, while avoiding promoter shutdown and gender bias, 2) increase transposition efficiency and 3) improve immunosuppression. By using a liver-specific promoter to drive IDUA expression, the SB100X hyperactive transposase and transient cyclophosphamide immunosuppression we achieved therapeutic-level (>100 wild-type) stabilized expression for 1 year in 50% of C57BL/6 mice. To gain insights into the causes of variability in transgene expression, we quantified the rates of alpha-L-iduronidase activity decay vis-a-vis transposition and transgene maintenance using the data obtained in this and previous studies. Our analyses showed that immune responses are the most important variable to control in order to prevent loss of transgene expression. Cumulatively, our results allow transition to pre-clinical studies of SB-mediated alpha-L-iduronidase expression and correction of mucopolysaccharidosis type I in animal models.

We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.

Investigational therapy can be successfully undertaken using viral- and nonviral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently, the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR(+) T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10(-9). Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene's encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%-50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

Expression-independent gene or polyadenylation [poly(A)] trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed. Although a number of poly(A)-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome, further efforts are needed to avoid the 3'-terminal insertion bias and the splice donor (SD) read-through, and to improve the mutagenicity. Here, we present a Sleeping Beauty (SB) transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding, gene-breaking and large-scale mutagenesis, respectively. The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element (ARE), which greatly reduced the SD read-through events, except that the internal ribosomal entry site (IRES) element was introduced in front of the SD signal to overcome the phenomenon of 3'-bias gene trapping. The breaking cassette consisting of an enhanced splicing acceptor (SA), a poly(A) signal coupled with a transcriptional terminator (TT) effectively disrupted the transcription of trapped genes. Moreover, the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11, which allows the conditional remobilization of a trap insert from a non-coding region. The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells. In addition, the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus, this poly(A)-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals., (Copyright © 2012. Published by Elsevier Ltd.)

Limited curative treatment options exist for patients with advanced B-lymphoid malignancies, and new therapeutic approaches are needed to augment the efficacy of hematopoietic stem-cell transplantation (HSCT). Cellular therapies, such as adoptive transfer of T cells that are being evaluated to target malignant disease, use mechanisms independent of chemo- and radiotherapy with nonoverlapping toxicities. Gene therapy is employed to generate tumor-specific T cells, as specificity can be redirected through enforced expression of a chimeric antigen receptor (CAR) to achieve antigen recognition based on the specificity of a monoclonal antibody. By combining cell and gene therapies, we have opened a new Phase I protocol at the MD Anderson Cancer Center (Houston, TX) to examine the safety and feasibility of administering autologous genetically modified T cells expressing a CD19-specific CAR (capable of signaling through chimeric CD28 and CD3-ζ) into patients with high-risk B-lymphoid malignancies undergoing autologous HSCT. The T cells are genetically modified by nonviral gene transfer of the Sleeping Beauty system and CAR(+) T cells selectively propagated in a CAR-dependent manner on designer artificial antigen-presenting cells. The results of this study will lay the foundation for future protocols including CAR(+) T-cell infusions derived from allogeneic sources.

Gene trapping is a high-throughput approach to elucidate gene functions by disrupting and recapitulating expression of genes in a target genome. A number of transposon-based gene-trapping systems are developed for mutagenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in gene disruption and mutation. Herein, a gene-trapping system mediated by Sleeping Beauty (SB) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped genes and terminate their translation. Once an endogenous gene is captured, the finding cassette independently drives the translation of reporter gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of SB gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 3' bias. The transcription of trapped genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of gene interruption and bio-safety. Thus, this gene-trap vector is an alternative and effective tool for the capture and disruption of endogenous genes in vitro and in vivo.

Transgenic animals are an important source of protein and nutrition for most humans and will play key roles in satisfying the increasing demand for food in an ever-increasing world population. The past decade has experienced a revolution in the development of methods that permit the introduction of specific alterations to complex genomes. This precision will enhance genome-based improvement of farm animals for food production. Precision genetics also will enhance the development of therapeutic biomaterials and models of human disease as resources for the development of advanced patient therapies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The LX-2 cell line has characteristics of hepatic stellate cells (HSCs), which are considered pericytes of the hepatic microcirculatory system. Recent studies have suggested that HSCs might have mesenchymal origin. We have performed an extensive characterization of the LX-2 cells and have compared their features with those of mesenchymal cells. Our data show that LX-2 cells have a phenotype resembling activated HSCs as well as bone marrow-derived mesenchymal stem cells (BM-MSCs). Our immunophenotypic analysis showed that LX-2 cells are positive for activated HSC markers (αSMA, GFAP, nestin and CD271) and classical mesenchymal makers (CD105, CD44, CD29, CD13, CD90, HLA class-I, CD73, CD49e, CD166 and CD146) but negative for the endothelial marker CD31 and endothelial progenitor cell marker CD133 as well as hematopoietic markers (CD45 and CD34). LX-2 cells also express the same transcripts found in immortalized and primary BM-MSCs (vimentin, annexin 5, collagen 1A, NG2 and CD140b), although at different levels. We show that LX-2 cells are capable to differentiate into multilineage mesenchymal cells in vitro and can stimulate new blood vessel formation in vivo. LX-2 cells appear not to possess tumorigenic potential. Thus, the LX-2 cell line behaves as a multipotent cell line with similarity to BM-MSCs. This line should be useful for further studies to elucidate liver regeneration mechanisms and be the foundation for development of hepatic cell-based therapies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.

Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B- T- radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5' rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells.

Sleeping Beauty (SB) transposons have been effective in delivering therapeutic genes to treat certain diseases in mice. Hydrodynamic gene delivery of integrating transposons to 5-20% of the hepatocytes in a mouse results in persistent elevated expression of the therapeutic polypeptides that can be secreted into the blood for activity throughout the animal. An alternative route of delivery is ex vivo transformation with SB transposons of hematopoietic cells, which then can be reintroduced into the animal for treatment of cancer. We discuss issues associated with the scale-up of hydrodynamic delivery to the liver of larger animals as well as ex vivo delivery. Based on our and others' experience with inefficient delivery to larger animals, we hypothesize that impulse, rather than pressure, is a critical determinant of the effectiveness of hydrodynamic delivery. Accordingly, we propose some alterations in delivery strategies that may yield efficacious levels of gene delivery in dogs and swine that will be applicable to humans. To ready hydrodynamic delivery for human application we address a second issue facing transposons used for gene delivery regarding their potential to "re-hop" from one site to another and thereby destabilize the genome. The ability to correct genetic diseases through the infusion of DNA plasmids remains an appealing goal.

Background: The Sleeping Beauty (SB) transposon system can insert defined sequences into chromosomes to direct the extended expression of therapeutic genes. Our goal is to develop the SB system for nonviral complementation of Fanconi anemia (FA), a rare autosomal recessive disorder accompanied by progressive bone marrow failure., Methods: We used a CytoPulse electroporation system (CytoPulse, Glen Burnie, MD, USA) to introduce SB transposons into human lymphoblastoid cells (LCL) derived from both Fanconi anemia type C (FA-C) defective and normal patients. Correction of the FA-C defect was assessed by resistance to mitomycin C, a DNA-crosslinking agent., Results: Culture of both cell types with the antioxidant N-acetyl- l-cysteine improved cell viability after electroporation. Co-delivery of enhanced green fluorescent protein (GFP) transposon with SB100X transposase-encoding plasmid supported a 50- to 90-fold increase in stable GFP expression compared to that observed in the absence of SB100X for normal LCL, but in FA-C defective LCL SB100X enhancement of stable GFP-expression was a more moderate five- to 13-fold. SB-mediated integration and expression of the FA-C gene was demonstrated by the emergence of a mitomycin C-resistant population bearing characteristic transposon-chromosome junction sequences and exhibiting a mitomycin dose response identical to that of normal LCL., Conclusions: The SB transposon system achieved stable expression of therapeutic FA-C genes, complementing the genetic defect in patient-derived cells by nonviral gene transfer., (Copyright © 2011 John Wiley & Sons, Ltd.)

Over the past decade, the Sleeping Beauty (SB) transposon system has been developed as the leading non-viral vector for gene therapy. This vector combines the advantages of viruses and naked DNA. Here we review progress over the last 2 years in vector design, methods of delivery and safety that have supported its use in the clinic. Currently, the SB vector has been validated for ex vivo gene delivery to stem cells, including T-cells for the treatment of lymphoma. Progress in delivery of SB transposons to liver for treatment of various systemic diseases, such as hemophilia and mucopolysaccharidoses types I and VII, has encountered some problems, but even here progress is being made.

The Sleeping Beauty (SB) transposon system can direct integration of DNA sequences into mammalian genomes. The SB system comprises a transposon and transposase that "cuts" the transposon from a plasmid and "pastes" it into a recipient genome. The transposase gene may integrate very rarely and randomly into genomes, which has led to concerns that continued expression might support continued remobilization of transposons and genomic instability. Consequently, we measured the duration of SB11 transposase expression needed for remobilization to determine whether continued expression might be a problem. The SB11 gene was expressed from the plasmid pT2/mCAGGS-Luc//UbC-SB11 that contained a luciferase expression cassette in a hyperactive SB transposon. Mice were imaged and killed at periodic intervals out to 24 weeks. Over the first 2 weeks, the number of plasmids with SB11 genes and SB11 mRNA dropped about 90 and 99.9%, respectively. Expression of the luciferase reporter gene in the transposon declined about 99% and stabilized for 5 months at nearly 1,000-fold above background. In stark contrast, transposition-supporting levels of SB11 mRNA lasted only about 4 days postinfusion. Thus, within the limits of current technology, we show that SB transposons appear to be as stably integrated as their viral counterparts.

DNA transposons are mobile DNA elements that can move from one DNA molecule to another and thereby deliver genetic information into human chromosomes in order to confer a new function or replace a defective gene. This process requires a transposase enzyme. During transposition DD[E/D]-transposases undergo a series of conformational changes. We summarize the structural features of DD[E/D]-transposases for which three-dimensional structures are available and that relate to transposases, which are being developed for use in mammalian cells. Similar to other members of the polynucleotidyl transferase family, the catalytic domains of DD[E/D]-transposases share a common feature: an RNase H-like fold that draws three catalytically active residues, the DDE motif, into close proximity. Beyond this fold, the structures of catalytic domains vary considerably, and the DD[E/D]-transposases display marked structural diversity within their DNA-binding domains. Yet despite such structural variability, essentially the same end result is achieved., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Endothelial tubulogenesis is a crucial step in the formation of functional blood vessels during angiogenesis and vasculogenesis. Here, we use in vivo imaging of living zebrafish embryos expressing fluorescent fusion proteins of beta-Actin, alpha-Catenin, and the ERM family member Moesin1 (Moesin a), to define a novel cord hollowing process that occurs during the initial stages of tubulogenesis in intersegmental vessels (ISVs) in the embryo. We show that the primary lumen elongates along cell junctions between at least two endothelial cells during embryonic angiogenesis. Moesin1-EGFP is enriched around structures that resemble intracellular vacuoles, which fuse with the luminal membrane during expansion of the primary lumen. Analysis of silent heart mutant embryos shows that initial lumen formation in the ISVs is not dependent on blood flow; however, stabilization of a newly formed lumen is dependent upon blood flow. Zebrafish moesin1 knockdown and cell transplantation experiments demonstrate that Moesin1 is required in the endothelial cells of the ISVs for in vivo lumen formation. Our analyses suggest that Moesin1 contributes to the maintenance of apical/basal cell polarity of the ISVs as defined by adherens junctions. Knockdown of the adherens junction protein Ve-cadherin disrupts formation of the apical membrane and lumen in a cell-autonomous manner. We suggest that Ve-cadherin and Moesin1 function to establish and maintain apical/basal polarity during multicellular lumen formation in the ISVs.

Recent results confirm that long-term expression of therapeutic transgenes can be achieved by using a transposon-based system in primary stem cells and in vivo. Transposable elements are natural DNA transfer vehicles that are capable of efficient genomic insertion. The latest generation, Sleeping Beauty transposon-based hyperactive vector (SB100X), is able to address the basic problem of non-viral approaches - that is, low efficiency of stable gene transfer. The combination of transposon-based non-viral gene transfer with the latest improvements of non-viral delivery techniques could provide a long-term therapeutic effect without compromising biosafety. The new challenges of pre-clinical research will focus on further refinement of the technology in large animal models and improving the safety profile of SB vectors by target-selected transgene integration into genomic "safe harbors." The first clinical application of the SB system will help to validate the safety of this approach.

The stable introduction of therapeutic transgenes into human cells can be accomplished using viral and nonviral approaches. Transduction with clinical-grade recombinant viruses offers the potential of efficient gene transfer into primary cells and has a record of therapeutic successes. However, widespread application for gene therapy using viruses can be limited by their initially high cost of manufacture at a limited number of production facilities as well as a propensity for nonrandom patterns of integration. The ex vivo application of transposon-mediated gene transfer now offers an alternative to the use of viral vectors. Clinical-grade DNA plasmids can be prepared at much reduced cost and with lower immunogenicity, and the integration efficiency can be improved by the transient coexpression of a hyperactive transposase. This has facilitated the design of human trials using the Sleeping Beauty (SB) transposon system to introduce a chimeric antigen receptor (CAR) to redirect the specificity of human T cells. This review examines the rationale and safety implications of application of the SB system to genetically modify T cells to be manufactured in compliance with current good manufacturing practice (cGMP) for phase I/II trials.

Two methods of systemic gene delivery have been extensively explored, using the mouse as a model system: hydrodynamic delivery, wherein a DNA solution equivalent in volume to 10% of the mouse weight is injected intravenously in less than 10 sec, and condensation of DNA with polyethylenimine (PEI) for standard intravenous infusion. Our goal in this study was to evaluate quantitatively the kinetics of gene expression, using these two methods for delivery of Sleeping Beauty transposons. Transposons carrying a luciferase expression cassette were injected into mice either hydrodynamically or after condensation with PEI at a PEI nitrogen-to-DNA phosphate ratio of 7. Gene expression in the lungs and liver after hydrodynamic delivery resulted in exponential decay with a half-life of about 35-40 hr between days 1 and 14 postinjection. The decay kinetics of gene expression after PEI-mediated gene delivery were more complex; an initial decay rate of 6 hr was followed by a more gradual loss of activity. Consequently, the liver became the primary site of gene expression about 4 days after injection of PEI-DNA, and by 14 days expression in the liver was 10-fold higher than in the lung. Overall levels of gene expression 2 weeks postinjection were 100- to 1000-fold lower after PEI-mediated delivery compared with hydrodynamic injection. These results provide insight into the relative effectiveness and organ specificity of these two methods of nonviral gene delivery when coupled with the Sleeping Beauty transposon system.

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice. In transposon-treated, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with mucopolysaccharidosis type I (MPS I), plasma IDUA persisted for 18 weeks at levels up to several hundred-fold wild-type (WT) activity, depending on DNA dose and gender. IDUA activity was present in all examined somatic organs, as well as in the brain, and correlated with both glycosaminoglycan (GAG) reduction in these organs and level of expression in the liver, the target of transposon delivery. IDUA activity was higher in the treated males than in females. In females, omission of transposase source resulted in significantly lower IDUA levels and incomplete GAG reduction in some organs, confirming the positive effect of transposition on long-term IDUA expression and correction of the disease. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I in mice, including thickening of the zygomatic arch, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium, implying potential effectiveness of this approach in treatment of human MPS I.

We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.

The genome project of the black legged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5000-10,000 cells and cultures were incubated for 2-3 months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis.

Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient but it requires specialized manufacture of expensive clinical grade recombinant virus. Electrotransfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically meaningful numbers of CAR(+) T cells. We report a new approach to efficiently generating T cells with redirected specificity, introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in memory and effector T cells without drug selection. When coupled with numerical expansion on CD19(+) artificial antigen-presenting cells, this gene transfer method results in rapid outgrowth of CD4(+) and CD8(+) T cells expressing CAR to redirect specificity for CD19(+) tumor cells.

Background: The Sleeping Beauty (SB) transposon system is a non-viral vector system that can integrate precise sequences into chromosomes. We evaluated the SB transposon system as a tool for gene therapy of mucopolysaccharidosis (MPS) types I and VII., Methods: We constructed SB transposon plasmids for high-level expression of human beta-glucuronidase (hGUSB) or alpha-L-iduronidase (hIDUA). Plasmids were delivered with and without SB transposase to mouse liver by rapid, high-volume tail-vein injection. We studied the duration of expressed therapeutic enzyme activity, transgene presence by PCR, lysosomal pathology by toluidine blue staining and cell-mediated immune response histologically and by immunohistochemical staining., Results: Transgene frequency, distribution of transgene and enzyme expression in liver and the level of transgenic enzyme required for amelioration of lysosomal pathology were estimated in MPS I and VII mice. Without immunomodulation, initial GUSB and IDUA activities in plasma reached > 100-fold of wild-type (WT) levels but fell to background within 4 weeks post-injection. In immunomodulated transposon-treated MPS I mice plasma IDUA persisted for over 3 months at up to 100-fold WT activity in one-third of MPS I mice, which was sufficient to reverse lysosomal pathology in the liver and, partially, in distant organs. Histological and immunohistochemical examination of liver sections in IDUA transposon-treated WT mice revealed inflammation 10 days post-injection consisting predominantly of mononuclear cells, some of which were CD4- or CD8-positive., Conclusions: Our results demonstrate the feasibility of achieving prolonged expression of lysosomal enzymes in the liver and reversing MPS disease in adult mice with a single dose of therapeutic SB transposons., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are entirely different; gene therapists hope to avoid altering endogenous gene expression (especially the activation of oncogenes), whereas geneticists do want to alter expression of chromosomal genes. The odds of either outcome depend on a vector's preference to integrate into genes or control regions, and these preferences vary between vectors. Here we discuss the relative strengths of DNA vectors over viral vectors, and review methods to overcome barriers to delivery inherent to DNA vectors. We also review the tendencies of several classes of retroviral and transposon vectors to target DNA sequences, genes, and genetic elements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. The three principle benefits from elucidating factors that affect preferences in integration are as follows: in gene therapy, it allows assessment of the overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; in genomic studies, it allows one to discern random from selected integration events; and in gene therapy as well as functional genomics, it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis.