Nucleofection of phiC31 Integrase Protein Mediates Sequence-Specific Genomic Integration in Human Cells.

The phage-derived phiC31 integrase is a useful tool for mediating sequence-specific genomic integration in mammalian cells, recombining donor plasmids bearing the attB recognition site with introduced genomic attP sites or endogeneous pseudo-attP sites having partial identity to attP. In most prior studies, phiC31 integrase has been introduced as plasmid DNA or mRNA. The current report examines whether phiC31 integrase functions efficiently in mammalian cells when co-nucleofected as a purified protein, along with attB-containing donor plasmids or PCR fragments.

Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy.

We delivered plasmid DNA encoding therapeutic genes to the muscles of mouse models of limb girdle muscular dystrophy (LGMD) 2A, 2B, and 2D, deficient in calpain3, dysferlin, and alpha-sarcoglycan, respectively. We also delivered the human follistatin gene, which has the potential to increase therapeutic benefit. After intramuscular injection of DNA, electroporation was applied to enhance delivery to muscle fibers.

DNA-Mediated Gene Therapy in a Mouse Model of Limb Girdle Muscular Dystrophy 2B.

Mutations in the gene for dysferlin cause a degenerative disorder of skeletal muscle known as limb girdle muscular dystrophy 2B. To achieve gene delivery of plasmids encoding dysferlin to hind limb muscles of dysferlin knockout mice, we used a vascular injection method that perfused naked plasmid DNA into all major muscle groups of the hind limb. We monitored delivery by luciferase live imaging and western blot, confirming strong dysferlin expression that persisted over the 3-month time course of the experiment.

Use of the DICE (Dual Integrase Cassette Exchange) System.

When constructing transgenic cell lines via plasmid DNA integration, precise targeting to a desired genomic location is advantageous. It is also often advantageous to remove the bacterial backbone, since bacterial elements can lead to the epigenetic silencing of neighboring DNA. The least cumbersome method to remove the plasmid backbone is recombinase-mediated cassette exchange (RMCE).

Knock-in Blunt Ligation Utilizing CRISPR/Cas9.

The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation ( Auer , 2014 ; Byrne , 2015 ). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks ( Jinek , 2012 ) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery ( Geisinger , 2016 ). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells.

Genome Editing Techniques and Their Therapeutic Applications.

Fueled by advances in the field of genetics, the methods available to edit DNA sequences in living cells have continued to develop steadily. These technologies directly impact the fields of gene and cell therapy, where changes in the DNA sequence of target cells offer a route to correct genetic diseases and manipulate disorders like cancer. We review here the expanding menu of genome editing techniques and how they are being applied to therapeutic targets.

Precise Correction of Disease Mutations in Induced Pluripotent Stem Cells Derived From Patients With Limb Girdle Muscular Dystrophy.

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.

In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining.

The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides.

Using phage integrases in a site-specific dual integrase cassette exchange strategy.

ΦC31 integrase, a site-specific large serine recombinase, is a useful tool for genome engineering in a variety of eukaryotic species and cell types. ΦC31 integrase performs efficient recombination between its attB site and either its own placed attP site or a partially mismatched genomic pseudo attP site. Bxb1 integrase, another large serine recombinase, has a similar level of recombinational activity, but recognizes only its own attB and attP sites.

Serine integrase chimeras with activity in E. coli and HeLa cells.

In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases.

Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering.

DICE, an efficient system for iterative genomic editing in human pluripotent stem cells.

To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11, located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes.

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

Background: Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts.

Efficient reversal of phiC31 integrase recombination in mammalian cells.

Over the past decade, the integrase enzyme from phage phiC31 has proven to be a useful genome engineering tool in a wide variety of species, including mammalian cells. The enzyme efficiently mediates recombination between two distinct sequences, attP and attB, producing recombinant product sites, attL and attR. The reaction proceeds exclusively in a unidirectional manner, because integrase is unable to synapse attL and attR.

Site-specific integration with bacteriophage ΦC31 integrase.

Few nonviral techniques exist for efficient and stable eukaryotic gene transfer and fewer still are broadly useful in both cell culture and whole-organism applications. C31 integrase, a site-specific bacteriophage recombinase, is able to catalyze chromosomal transgene insertion under a diverse range of experimental and therapeutic conditions. The enzyme recognizes and catalyzes unidirectional recombination between attachment motifs found in phage and bacterial genomes (attP and attB sites, respectively).

Long-term expression of human coagulation factor VIII in a tolerant mouse model using the φC31 integrase system.

We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the φC31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain.

Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA.

Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications.

Therapeutic applications of the ΦC31 integrase system.

The potential use of the ΦC31 integrase system in gene therapy opens up the possibilities of new treatments for old diseases. ΦC31 integrase mediates the integration of plasmid DNA into the chromsomes of mammalian cells in a sequence-specific manner, resulting in robust, long-term transgene expression. In this article, we review how ΦC31 integrase mediates transgene integration into the genomes of target cells and summarize the recent preclinical applications of the system to gene therapy.

The therapeutic potential of ΦC31 integrase as a gene therapy system.

Introduction: The φC31 integrase system is a phage-derived system that offers the ability to integrate plasmid DNA into the chromosomes at a subset of endogenous preferred locations associated with robust gene expression. Recent progress highlights the unique advantages of this system for in vivo gene therapy and for use in stem cells.

Areas Covered: The φC31 integrase system has been under development for ten years and has been demonstrated to be effective for integration of plasmids in a variety of tissues and organs for gene therapy in animal systems, as well as in isolated human cells.

Long-term phenotypic correction in factor IX knockout mice by using ΦC31 integrase-mediated gene therapy.

Hemophilia B, a hereditary bleeding disorder caused by a deficiency of coagulation factor IX (FIX), is an excellent candidate for gene therapy. However, to date, success in hemophilia gene therapy clinical trials has been limited due to failure to achieve or sustain therapeutic levels of factor expression. The ΦC31 integrase system efficiently integrates plasmid DNA carrying a transgene and an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, sustained transgene expression.

Site-specific integration of transgene targeting an endogenous lox-like site in early mouse embryos.

Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (ψlox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/ψlox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals.

Impact of hydrodynamic injection and phiC31 integrase on tumor latency in a mouse model of MYC-induced hepatocellular carcinoma.

Background: Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety.

Kinetics and longevity of ΦC31 integrase in mouse liver and cultured cells.

The ΦC31 integrase system provides genomic integration of plasmid DNA that may be useful in gene therapy. For example, the ΦC31 system has been used in combination with hydrodynamic injection to achieve long-term expression of factor IX in mouse liver. However, a concern is that prolonged expression of ΦC31 integrase within cells could potentially stimulate chromosome rearrangements or an immune response.