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. We describe preparation of phiC31 integrase protein and evidence that it can mediate genomic integration in human 293 cells, including PCR evidence for integration at an endogenous pseudo-attP site. This work demonstrates for the first time the ability of 605- and 613-amino-acid versions of phiC31 integrase protein to mediate efficient, site-specific integration into the genome of human cells when co-nucleofected with full-sizedattB-containing donor plasmids or linear 2.5-kb PCR fragments. This protein-mediated approach may be especially useful for integration of exogenous sequences into valuable therapeutic target cells, such as hematopoietic stem cells or T cells, that are sensitive to introduced DNA.

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. When plasmids encoding the human calpain3 or dysferlin cDNA sequences were injected into quadriceps muscles of LGMD2A and LGMD2B mouse models, respectively, in 3-month studies, robust levels of calpain3 and dysferlin proteins were detected. We observed a statistically significant decrease in Evans blue dye penetration in LGMD2B mouse muscles after delivery of the dysferlin gene, consistent with repair of the muscle membrane defect in these mice. The therapeutic value of delivery of the genes for alpha-sarcoglycan and follistatin was documented by significant drops in Evans blue dye penetration in gastrocnemius muscles of LGMD2D mice. These results indicated for the first time that a combined gene therapy involving both alpha-sarcoglycan and follistatin would be valuable for LGMD2D patients. We suggest that this non-viral gene delivery method should be explored for its translational potential in patients.

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. Co-delivery of the follistatin gene, which may promote muscle growth, was monitored by ELISA. Immunohistochemistry documented the presence of dysferlin in muscle fibers in treated limbs, and PCR confirmed the presence of plasmid DNA. Because dysferlin is involved in repair of the sarcolemmal membrane, dysferlin loss leads to fragile sarcolemmal membranes that can be detected by permeability to Evan's blue dye. We showed that after gene therapy with a plasmid encoding both dysferlin and follistatin, statistically significant reduction in Evan's blue dye permeability was present in hamstring muscles. These results suggest that vascular delivery of plasmids carrying these therapeutic genes may lead to simple and effective approaches for improving the clinical condition of limb girdle muscular dystrophy 2B.

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). RMCE is accomplished by arranging recombinase sites in the genome and in a donor plasmid such that a recombinase can both integrate the donor plasmid and excise its bacterial backbone. When a single recombinase is used for RMCE, recombination between undesired pairings of the sites can lead to a significant number of unwanted cell lines. To reduce the frequency with which these side products occur, several variants of RMCE that increase desired outcomes have been developed. Nevertheless, an important feature lacking from these improved RMCE methods is that none have fully utilized the recombinases that have been demonstrated to be the most robust and stringent at performing genomic integration in plants and animals, i.e., the phiC31 and Bxb1 phage integrases. To address this need, we have developed an RMCE protocol using these two serine integrases that we call dual integrase cassette exchange (DICE). Our DICE system provides a means to achieve high-precision DNA integration at a desired location and is especially well suited for repeated recombination into the same locus. In this chapter, we provide our most current protocols for using DICE in feeder-free human-induced pluripotent stem cells .

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 et al., 2014 ; Byrne et al., 2015 ). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks ( Jinek et al., 2012 ) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery ( Geisinger et al., 2016 ). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.

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.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.

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. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

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. Previously, we have used these integrases sequentially to integrate plasmid DNA into the genome. This approach relied on placing a landing pad attP for Bxb1 integrase in the genome by using phiC31 integrase-mediated recombination at a genomic pseudo attP site. In this chapter, we present a protocol for using these integrases simultaneously to facilitate cassette exchange at a predefined location. This approach permits greater control and accuracy over integration. We also present a general method for using polymerase chain reaction assays to verify that the desired cassette exchange occurred successfully.

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. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{-1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

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. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.

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. Recipient cell lines were established by site-specific placement of a 'landing pad' cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson's disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.

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. METHODS AND RESULTS: We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. CONCLUSIONS: The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

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. To date, use of phiC31 integrase has been limited to attP × attB recombination. The factor needed for the reverse reaction--the excisionase or recombination directionality factor (RDF)--was identified recently and shown to function in vitro and in bacterial cells. To determine whether the phiC31 RDF could also function in mammalian cells, we cloned and tested several vectors that permit assessment of phiC31 RDF activity in mammalian environments. In the human and mouse cell lines tested (HeLa, HEK293, and NIH3T3), we observed robust RDF activity, using plasmid and/or genomic assays. This work is the first to demonstrate attL-attR serine integrase activity in mammalian cells and validates phiC31 RDF as a new tool that will enable future studies to take advantage of phiC31 integrase recombination in the forward or reverse direction.

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). Use of C31 integrase for gene transfer requires that an attB sequence be cloned into a transgene-bearing plasmid. When this modified plasmid is introduced into cells alongside integrase-expressing plasmid, C31 integrase is able to catalyze insertion of the transgene plasmid into one of a limited pool of sites in the target genome that show sequence similarity to wild-type attP. Efficient delivery of C31 integrase and attB donor plasmid to the tissue or cells of interest remains the most challenging aspect of the system. Unlike viral methods of genome manipulation, use of C31 integrase almost always requires an additional method of stimulating cellular DNA uptake. However, the relative simplicity of the plasmid-based system means that nearly any proven method of introducing exogenous DNA into cells can be used with C31 integrase. This protocol describes the use of C31 integrase in mammalian cell culture for the creation of clonal lines showing robust and stable expression of an experimental transgene.

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. Various plasmids encoding φC31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Long-term expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type φC31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of φC31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and φC31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The φC31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia A.

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. These applications encompass in vivo studies in liver and lung, as well as increasing ex vivo uses of the system, including in neural and muscle stem cells, in cord-lining epithelial cells, and for the production of induced pluripotent stem cells. The safety of the ΦC31 integrase system for gene therapy is evaluated, and its ability to provide treatments for hemophilia is discussed. We conclude that gene therapy strategies utilizing ΦC31 integrase offer great promise for the development of treatments in the future.

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. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.

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. We focus on work with the φC31 integrase system during the past 12-18 months. This work has centered on a series of papers involving in vivo delivery of the integrase system to the liver and a variety of studies demonstrating the utility of the integrase system in stem cells. EXPERT OPINION: We conclude that the φC31 integrase system has significant potential for liver gene therapy, if effective DNA delivery methods for large mammals become available. The φC31 integrase system displays an outstanding fit for use in pluripotent stem cells, and this area is expected to be the subject of intense development.