Efficient formation of single-copy human artificial chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a ~750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Engineering better artificial chromosomes.

Constructing human artificial chromosomes in yeast avoids unintended multimerization.

Full-length human dystrophin on human artificial chromosome compensates for mouse dystrophin deficiency in a Duchenne muscular dystrophy mouse model.

Dystrophin maintains membrane integrity as a sarcolemmal protein. Dystrophin mutations lead to Duchenne muscular dystrophy, an X-linked recessive disorder. Since dystrophin is one of the largest genes consisting of 79 exons in the human genome, delivering a full-length dystrophin using virus vectors is challenging for gene therapy. Human artificial chromosome is a vector that can load megabase-sized genome without any interference from the host chromosome. Chimeric mice carrying a 2.4-Mb human dystrophin gene-loaded human artificial chromosome (DYS-HAC) was previously generated, and dystrophin expression from DYS-HAC was confirmed in skeletal muscles. Here we investigated whether human dystrophin expression from DYS-HAC rescues the muscle phenotypes seen in dystrophin-deficient mice. Human dystrophin was normally expressed in the sarcolemma of skeletal muscle and heart at expected molecular weights, and it ameliorated histological and functional alterations in dystrophin-deficient mice. These results indicate that the 2.4-Mb gene is enough for dystrophin to be correctly transcribed and translated, improving muscular dystrophy. Therefore, this technique using HAC gives insight into developing new treatments and novel humanized Duchenne muscular dystrophy mouse models with human dystrophin gene mutations.

Assembly of Multiple Full-Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System.

Human artificial chromosomes (HACs) are gene delivery vectors that have been used for decades for gene functional studies. HACs have several advantages over viral-based gene transfer systems, including stable episomal maintenance in a single copy in the cell and the ability to carry up to megabase-sized genomic DNA segments. We have previously developed the alphoidtetO -HAC, which has a single gene acceptor loxP site that allows insertion of an individual gene of interest using Chinese hamster ovary (CHO) hybrid cells. The HAC, along with a DNA segment of interest, can then be transferred from donor CHO cells to various recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we detail a protocol for loading multiple genomic DNA segments or genes into the alphoidtetO -HAC vector using an iterative integration system (IIS) that utilizes recombinases Cre, ΦC31, and ΦBT. This IIS-alphoidtetO -HAC can be used for either serially assembling genomic loci or fragments of a large gene, or for inserting multiple genes into the same artificial chromosome. The insertions are executed iteratively, whereby each round results in the insertion of a new DNA segment of interest. This is accompanied by changes of expression of marker fluorescent proteins, which simplifies screening of correct clones, and changes of selection and counterselection markers, which constitutes an error-proofing mechanism that removes mis-incorporated DNA segments. In addition, the IIS-alphoidtetO -HAC carrying the genes can be eliminated from the cells, offering the possibility to compare the phenotypes of human cells with and without functional copies of the genes of interest. The resulting HAC molecules may be used to investigate biomedically relevant pathways or the regulation of multiple genes, and to potentially engineer synthetic chromosomes with a specific set of genes of interest. The IIS-alphoidtetO -HAC system is expected to be beneficial in creating multiple-gene humanized models with the purpose of understanding complex multi-gene genetic disorders. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Integration of the first DNA segment of interest into the IIS-alphoidteto -HAC Basic Protocol 2: Integration of a second DNA segment of interest into the IIS-alphoidteto -HAC Basic Protocol 3: Integration of a third DNA segment of interest into the IIS-alphoidteto -HAC Support Protocol: Fluorescence in situ hybridization analysis for the circular IIS-alphoidtetO -HAC.

Highly Efficient Microcell-Mediated Transfer of HACs Containing a Genomic Region of Interest into Mammalian Cells.

Human artificial chromosomes (HACs) are considered promising tools for gene delivery, functional analyses, and gene therapy. HACs have the potential to overcome many of the problems caused by the use of viral-based gene transfer systems, such as limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes. The recently developed alphoidtetO -HAC has an advantage over other HAC vectors because it can be easily eliminated from dividing cells by inactivation of its conditional kinetochore. This provides a unique control mechanism to study phenotypes induced by a gene or genes carried on the HAC. The alphoidtetO -HAC has a single gene acceptor loxP site that allows insertion of an individual gene of interest or a cluster of genes of up to several Mb in size in Chinese hamster ovary (CHO) hybrid cells. The HACs carrying chromosomal copies of genes can then be transferred from these donor CHO cells to different recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we describe a detailed protocol for loading a gene of interest into the alphoidtetO -HAC vector and for the subsequent transfer of the HAC to recipient cells using an improved MMCT protocol. The original MMCT protocol includes treatment of donor cells with colcemid to induce micronucleation, wherein the HAC becomes surrounded with a nuclear membrane. That step is followed by disarrangement of the actin cytoskeleton using cytochalasin B to help induce microcell formation. The updated MMCT protocol, described here, features the replacement of colcemid and cytochalasin B with TN16 + griseofulvin and latrunculin B, respectively, and the use of collagen/laminin surface coating to promote attachment of metaphase cells to plates during micronuclei induction. These modifications increase the efficiency of HAC transfer to recipient cells ten fold. The improved MMCT protocol has been successfully tested on several recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Insertion of a BAC containing a gene of interest into a single loxP loading site of alphoidtetO -HAC in hamster CHO cells Basic Protocol 2: Microcell-mediated chromosome transfer from donor hamster CHO cells to mammalian cells.

Pluripotent stem cell-based gene therapy approach: human de novo synthesized chromosomes.

A novel approach in gene therapy was introduced 20 years ago since artificial non-integrative chromosome-based vectors containing gene loci size inserts were engineered. To date, different human artificial chromosomes (HAC) were generated with the use of de novo construction or "top-down" engineering approaches. The HAC-based therapeutic approach includes ex vivo gene transferring and correction of pluripotent stem cells (PSCs) or highly proliferative modified stem cells. The current progress in the technology of induced PSCs, integrating with the HAC technology, resulted in a novel platform of stem cell-based tissue replacement therapy for the treatment of genetic disease. Nowadays, the sophisticated and laborious HAC technology has significantly improved and is now closer to clinical studies. In here, we reviewed the achievements in the technology of de novo synthesized HACs for a chromosome transfer for developing gene therapy tissue replacement models of monogenic human diseases.

H3K9me3 maintenance on a human artificial chromosome is required for segregation but not centromere epigenetic memory.

Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.This article has an associated First Person interview with the first authors of the paper.

Human Cytomegalovirus-Encoded G Protein-Coupled Receptor UL33 Facilitates Virus Dissemination via the Extracellular and Cell-to-Cell Route.

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs. Three of these receptors, UL78, US27 and US28, are known for their roles in HCMV dissemination and latency. Despite importance of its rodent orthologs for viral replication and pathogenesis, such a function is not reported for the HCMV-encoded GPCR UL33. Using the clinical HCMV strain Merlin, we show that UL33 facilitates both cell-associated and cell-free virus transmission. A UL33-deficient virus derivative revealed retarded virus spread, formation of less and smaller plaques, and reduced extracellular progeny during multi-cycle growth analysis in fibroblast cultures compared to parental virus. The growth of UL33-revertant, US28-deficient, and US28-revertant viruses were similar to parental virus under multistep growth conditions. UL33- and US28-deficient Merlin viruses impaired cell-associated virus spread to a similar degree. Thus, the growth defect displayed by the UL33-deficient virus but not the US28-deficient virus reflects UL33's contribution to extracellular transmission. In conclusion, UL33 facilitates cell-associated and cell-free spread of the clinical HCMV strain Merlin in fibroblast cultures.

Lipid Nanoparticle Formulation Increases Efficiency of DNA-Vectored Vaccines/Immunoprophylaxis in Animals Including Transchromosomic Bovines.

The use of nucleic acid as a drug substance for vaccines and other gene-based medicines continues to evolve. Here, we have used a technology originally developed for mRNA in vivo delivery to enhance the immunogenicity of DNA vaccines. We demonstrate that neutralizing antibodies produced in rabbits and nonhuman primates injected with lipid nanoparticle (LNP)-formulated Andes virus or Zika virus DNA vaccines are elevated over unformulated vaccine. Using a plasmid encoding an anti-poxvirus monoclonal antibody (as a reporter of protein expression), we showed that improved immunogenicity is likely due to increased in vivo DNA delivery, resulting in more target protein. Specifically, after four days, up to 30 ng/mL of functional monoclonal antibody were detected in the serum of rabbits injected with the LNP-formulated DNA. We pragmatically applied the technology to the production of human neutralizing antibodies in a transchromosomic (Tc) bovine for use as a passive immunoprophylactic. Production of neutralizing antibody was increased by >10-fold while utilizing 10 times less DNA in the Tc bovine. This work provides a proof-of-concept that LNP formulation of DNA vaccines can be used to produce more potent active vaccines, passive countermeasures (e.g., Tc bovine), and as a means to produce more potent DNA-launched immunotherapies.

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice.

Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/- mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/- iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector-the alphoidtetO-HAC.

Multigene human artificial chromosome vector delivery with herpes simplex virus 1 amplicons.

Gene expression studies and gene therapy require efficient gene delivery into cells. Different technologies by viral and non-viral mechanisms have been used for gene delivery into cells. Small gene vectors transfer across the cell membrane with a relatively high efficiency, but not large genes or entire loci spanning several kilobases, which do not remain intact following introduction. Previously, we developed an efficient delivery system based on herpes virus simplex type 1 (HSV-1) amplicons to transfer large fragments of DNA incorporated in human artificial chromosome (HAC) vectors into the nucleus of human cells. The HSV-1 amplicon lacks the signals for cleavage and replication of its own genome, yet each amplicon has the capacity to incorporate up to 150 kb of exogenous DNA. In this study, we investigated whether the capacity of gene delivery could be increased by simultaneously introducing multiple HSV-1 modified amplicons carrying a gene expressing HAC vector into cells with the aim of generating a single artificial chromosome containing the desired genes. Following co-transduction of two HSV-1 HAC amplicons, artificial chromosomes were successfully generated containing the introduced genes, which were appropriately expressed in different human cell types.

Human artificial chromosome (HAC) for measuring chromosome instability (CIN) and identification of genes required for proper chromosome transmission.

Chromosomal instability (CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole chromosomes. In the vast majority of human tumors the molecular basis of CIN remains unknown. The development of a conceptually simple colony color sectoring assay that measures yeast artificial chromosome (YAC) loss provided a powerful genetic tool to assess the rate of chromosome mis-segregation and also identified 937 yeast genes involved in this process. Similarly, a human artificial chromosome (HAC)-based assay has been recently developed and applied to quantify chromosome mis-segregation events in human cells. This assay allowed identification of novel human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1. The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-based libraries to identify the complete spectrum of CIN genes. This will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.

Applications of bottom-up human artificial chromosomes in cell research and cell engineering.

Chromosome manipulation is a useful technique in biological science. We have constructed human artificial chromosomes (HACs) based on the transfection of centromeric alphoid DNA precursors into cultured human cells. Moreover, HAC-based technology has been developed into a novel gene expression vector tool for introducing large-size genomic DNA. This technique provides natural expression, as well as stable expression without the gene silencing that often occurs with conventional vectors in mammalian cells. Here we review the properties of HACs, and issues regarding the use of HAC technology for basic and applied research.

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission.

One of the hallmarks of cancer is chromosome instability (CIN), which leads to aneuploidy, translocations, and other chromosome aberrations. However, in the vast majority of human tumors the molecular basis of CIN remains unknown, partly because not all genes controlling chromosome transmission have yet been identified. To address this question, we developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses a human artificial chromosome (HAC) expressing the GFP transgene. When this assay was applied to screen an siRNA library of protein kinases, we identified PINK1, TRIO, IRAK1, PNCK, and TAOK1 as potential novel genes whose knockdown induces various mitotic abnormalities and results in chromosome loss. The HAC-based assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, and transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.

Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.

Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Importantly, the phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. AlphoidtetO-HAC-based technology has also been used to develop new drug screening and assessment strategies to manipulate the CIN phenotype in cancer cells. In summary, the alphoidtetO-HAC is proving to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies.

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies.

It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.

Human and mouse artificial chromosome technologies for studies of pharmacokinetics and toxicokinetics.

In the earliest stage of drug discovery/development, various cell-based models and animal models were used for the prediction of human pharmacokinetics and toxicokinetics. Unfortunately, drugs under development are often discontinued because their nonclinical results do not extrapolate to human clinical studies in relation to either safety or efficacy. Therefore, it is important to improve the time- and cost-effectiveness of drug development. This might be achieved by developing new technologies including pharmacokinetics and toxicokinetics models that use human and mouse artificial chromosome vectors (HACs/MACs). HACs/MACs are unique vectors with several advantages: 1) independent maintenance, 2) defined copy number and mitotically stable, 3) no silencing of the transgene, and 4) no limitation of DNA insertion size. This review provides information on the advantages and examples of the utility of various models based on the recent advances in HAC/MAC technologies, including multifunctional cell-based models for assaying drug-drug interactions, bidirectional permeability, and cytotoxicity, as well as fully genetically humanized mouse models. We also discuss the future prospects of these technologies to advance drug discovery. In summary, these technologies offer advantages over current conventional models and should improve the success rate of drug development related to efficacy and safety for humans.

Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System.

The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoidtetO-HAC). We have demonstrated that the IIS-alphoidtetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoidtetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoidtetO-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoidtetO-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.

Development of Caco-2 cells co-expressing CYP3A4 and NADPH-cytochrome P450 reductase using a human artificial chromosome for the prediction of intestinal extraction ratio of CYP3A4 substrates.

The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers. The metabolic activity of CYP3A4 (midazolam 1'-hydroxylase activity) in the CYP3A4-CPR-HAC/Caco-2 cells was constant from 22 to 35 passages, indicating that HAC vectors conferred sufficient and sustained CYP3A4 activity to CYP3A4-CPR-HAC/Caco-2 cells. The strong relationship between the metabolic extraction ratios (ER) obtained from the CYP3A4-CPR-HAC/Caco-2 cells and calculated intestinal extraction ratios in humans (Eg) from reported intestinal availability (Fg) was found for 17 substrates of CYP3A4 (r2 = 0.84). The present study suggests that the CYP3A4-CPR-HAC/Caco-2 cell monolayer can serve as an in vitro tool that facilitates the prediction of intestinal extraction ratio (or availability) in humans.