RESUMEN
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.
Asunto(s)
ADN , Técnicas de Transferencia de Gen , Ensayo de Materiales , Plásmidos , Polielectrolitos , Animales , ADN/química , ADN/farmacocinética , ADN/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Metacrilatos/química , Metacrilatos/farmacología , Ratones , Plásmidos/química , Plásmidos/farmacocinética , Plásmidos/farmacología , Polielectrolitos/química , Polielectrolitos/farmacocinética , Polielectrolitos/farmacología , Distribución TisularRESUMEN
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.
Asunto(s)
Acetilgalactosamina/química , Portadores de Fármacos/química , Hígado/metabolismo , Plásmidos/administración & dosificación , Poliaminas/química , Animales , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , ADN/administración & dosificación , Portadores de Fármacos/efectos adversos , Portadores de Fármacos/síntesis química , Etilaminas/química , Terapia Genética/métodos , Células HeLa , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/efectos de los fármacos , Metacrilatos/química , Ratones , Ratones Endogámicos C57BL , PolielectrolitosRESUMEN
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.
Asunto(s)
Cruzamiento/métodos , Desoxirribonucleasas/metabolismo , Técnicas de Transferencia de Gen , Variación Genética , Genética de Población , Ganado/genética , Animales , Análisis Mutacional de ADN , Secuencias Invertidas Repetidas/genética , Mutagénesis , Tasa de Mutación , Oligonucleótidos/genética , Plásmidos/genéticaRESUMEN
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?
Asunto(s)
Terapia Genética , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/terapia , Animales , Barrera Hematoencefálica , Modelos Animales de Enfermedad , Vectores Genéticos , Humanos , Lisosomas/genética , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/terapia , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/terapiaRESUMEN
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.
Asunto(s)
Crianza de Animales Domésticos , Ganado , Animales , Ganado/genética , Ambiente , Reproducción AsexuadaRESUMEN
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.
Asunto(s)
Elementos Transponibles de ADN/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Animales , Hemofilia A/terapia , Humanos , Mucopolisacaridosis/terapia , Transposasas/genéticaRESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/metabolismo , Células Endoteliales/metabolismo , Proteínas de Microfilamentos/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Uniones Adherentes/metabolismo , Animales , Antígenos CD/genética , Cadherinas/genética , Polaridad Celular , Células Endoteliales/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Neovascularización Fisiológica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Pez Cebra/genética , Proteína de la Zonula Occludens-1RESUMEN
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.
Asunto(s)
Animales Modificados Genéticamente , Bovinos/genética , Reprogramación Celular , Desoxirribonucleasas/metabolismo , Ingeniería Genética/veterinaria , Genoma , Ribonucleasas/metabolismo , Sus scrofa/genética , Factores de Transcripción/metabolismo , Animales , Desoxirribonucleasas/genética , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen/veterinaria , Genotipo , Fenotipo , Ribonucleasas/genética , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Mucopolisacaridosis I , Humanos , Animales , Ratones , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/terapia , Encéfalo/diagnóstico por imagen , Terapia Genética/métodos , Mapeo Encefálico/métodos , Imagen por Resonancia MagnéticaRESUMEN
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.
Asunto(s)
Elementos Transponibles de ADN , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Transposasas/genética , Animales , Células Madre Hematopoyéticas/metabolismo , Humanos , Mutagénesis Insercional , TransgenesRESUMEN
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.
Asunto(s)
Inocuidad de los Alimentos , Genoma , Animales , Animales Modificados GenéticamenteRESUMEN
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.
Asunto(s)
Elementos Transponibles de ADN/genética , Anemia de Fanconi/genética , Vectores Genéticos/genética , Adulto , Línea Celular , Supervivencia Celular/genética , Preescolar , Electroporación , Anemia de Fanconi/terapia , Femenino , Regulación de la Expresión Génica , Orden Génico , Técnicas de Transferencia de Gen , Terapia Genética , Recombinación Homóloga , Humanos , Transducción Genética , Transgenes , Adulto JovenRESUMEN
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.
Asunto(s)
Citidina Desaminasa/genética , Elementos Transponibles de ADN/genética , Técnicas de Transferencia de Gen , Porcinos/genética , Desaminasa APOBEC-3G , Animales , Biomarcadores , Cruzamiento , Genoma , Humanos , Integrasas/genética , Porcinos/embriología , Transgenes , Transposasas/genética , Transposasas/metabolismoRESUMEN
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.
Asunto(s)
Línea Celular , Células Estrelladas Hepáticas , Células Madre Mesenquimatosas , Animales , Antígenos CD/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular , Trasplante de Células , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones SCID , Células Madre Multipotentes/citología , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Neovascularización Patológica , OsteogénesisRESUMEN
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.
Asunto(s)
Elementos Transponibles de ADN/genética , Terapia Genética/métodos , Vectores Genéticos , Receptores de Antígenos de Linfocitos T/genética , Transducción Genética/métodos , Transposasas/metabolismo , Animales , Antígenos CD19/inmunología , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Humanos , Ratones , Plásmidos , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/inmunología , Linfocitos T/trasplante , Transposasas/genéticaRESUMEN
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.
Asunto(s)
Elementos Transponibles de ADN/genética , ADN/genética , Hígado/metabolismo , Transposasas/metabolismo , Animales , Western Blotting , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Transposasas/genéticaRESUMEN
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.