Aspects of Gene Therapy Products Using Current Genome-Editing Technology in Japan.

The development of genome-editing technology could lead to breakthrough gene therapy. Genome editing has made it possible to easily knock out or modify a target gene, while current gene therapy using a virus vector or plasmid hampering modification with respect to gene replacement therapies. Clinical development using these genome-editing tools is progressing rapidly. However, it is also becoming clear that there is a possibility of unintended gene sequence modification or deletion, or the insertion of undesired genes, or the selection of cells with abnormalities in the cancer suppressor gene p53; these unwanted actions are not possible with current gene therapy. The Science Board of the Pharmaceuticals and Medical Devices Agency of Japan has compiled a report on the expected aspects of such genome-editing technology and the risks associated with it. This article summarizes the history of that discussion and compares the key concepts with information provided by other regulatory authorities.

Dual usage of a stage-specific fluorescent reporter system based on a helper-dependent adenoviral vector to visualize osteogenic differentiation.

We developed a reporter system that can be used in a dual manner in visualizing mature osteoblast formation. The system is based on a helper-dependent adenoviral vector (HDAdV), in which a fluorescent protein, Venus, is expressed under the control of the 19-kb human osteocalcin (OC) genomic locus. By infecting human and murine primary osteoblast (POB) cultures with this reporter vector, the cells forming bone-like nodules were specifically visualized by the reporter. In addition, the same vector was utilized to efficiently knock-in the reporter into the endogenous OC gene of human induced pluripotent stem cells (iPSCs), by homologous recombination. Neural crest-like cells (NCLCs) derived from the knock-in reporter iPSCs were differentiated into osteoblasts forming bone-like nodules and could be visualized by the expression of the fluorescent reporter. Living mature osteoblasts were then isolated from the murine mixed POB culture by fluorescence-activated cell sorting (FACS), and their mRNA expression profile was analyzed. Our study presents unique utility of reporter HDAdVs in stem cell biology and related applications.

The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds. RESULTS: We generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient's iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5 weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient's iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes. CONCLUSION: The use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches.

Gene targeting in human-induced pluripotent stem cells with adenoviral vectors.

Helper-dependent adenoviral vector (HDAdV), which is also called gutless AdV, has been used to deliver donor DNA for gene targeting in human pluripotent stem cells. Surprisingly, the targeting efficacies, both per chromosomal integration (drug-resistant colony) and per treated cells, are much higher than those by standard electroporation and equivalent to those by utilizing artificial nucleases, such as TAL effector nucleases (Aizawa et al., Mol Ther 20:424-431, 2012; Suzuki, Proc Natl Acad Sci U S A 105:13781-13786, 2008). Importantly, gene targeting with HDAdVs was equally efficient in transcriptionally inactive loci in human ES/iPS cells. Therefore, multiple gene-targeted clones can be obtained from human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) cultured in one 100-mm dish. For virus-mediated gene targeting, it is not required to introduce artificial double-strand breaks. By using electroporation for gene targeting, target cells should be expanded to 106-107 cells. In contrast, as an advantage of virus-mediated method, DNA delivery efficiency is high even in a smaller number of cells, resulting in minimizing the number of passages/cell divisions before performing gene targeting. The characteristics suggest that HDAdV-mediated gene targeting has potential advantages for manipulation of chromosomes of pluripotent stem cells for therapeutic applications.

Genome sequence of a novel virus of the species human adenovirus d associated with acute gastroenteritis.

A novel virus of the species human adenovirus D, HAdV-67 (P-New/H9/F25), was first isolated from diarrheal feces of six children in Dhaka City, Bangladesh. The genome of this novel virus may be composed of multiple recombinations among HAdV-9, HAdV-25, HAdV-26, HAdV-33, HAdV-46, and an unknown human adenovirus D which was an origin of HAdV-67.

Albumin gene targeting in human embryonic stem cells and induced pluripotent stem cells with helper-dependent adenoviral vector to monitor hepatic differentiation.

Although progresses in developing differentiation procedures have been achieved, it remains challenging to generate hES/iPS cell-derived mature hepatocytes. We performed knock-in of a monomeric Kusabira orange (mKO1) cassette in the albumin (ALB) gene, in human embryonic stem (hES) cells and induced pluripotent stem (hiPS) cells, with the use of the helper-dependent adenovirus vector (HDAdV). Upon induction into the hepatic lineages, these knock-in hES/iPS cells differentiated into cells that displayed several known hepatic functions. The mKO1 knock-in (ALB/mKo1) hES/hiPS cells were used to visualize hepatic differentiation in vitro. mKO1 reporter expression recapitulated endogenous ALB transcriptional activity. ALB/mKo1 [Hi] population isolated by flow cytometry was confirmed to be enriched with ALB mRNA. Expression profile analyses revealed that characteristic hepatocyte genes and genes related to drug metabolism and many aspects of liver function were highly enriched in the ALB/mKo1 [Hi] population. Our data demonstrate that ALB/mKo1 knock-in hES/iPS cells are valuable resources for monitoring in vitro hepatic differentiation, isolation and analyses of hES and hiPS cells-derived hepatic cells that actively transcribing ALB. These knock-in hES/iPS cell lines could provide further insights into the mechanism of hepatic differentiation and molecular signatures of the hepatic cells derived from hES/iPS cells.

Novel human adenovirus strain, Bangladesh.

We report a novel human adenovirus D (HAdV-65) isolated from feces of 4 children in Bangladesh who had acute gastroenteritis. Corresponding genes of HAdV-65 were related to a hexon gene of HAdV-10, penton base genes of HAdV-37 and HAdV-58, and a fiber gene of HAdV-9. This novel virus may be a serious threat to public health.

Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors.

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

Convergence of Notch and beta-catenin signaling induces arterial fate in vascular progenitors.

Molecular mechanisms controlling arterial-venous specification have not been fully elucidated. Previously, we established an embryonic stem cell differentiation system and demonstrated that activation of cAMP signaling together with VEGF induces arterial endothelial cells (ECs) from Flk1(+) vascular progenitor cells. Here, we show novel arterial specification machinery regulated by Notch and beta-catenin signaling. Notch and GSK3beta-mediated beta-catenin signaling were activated downstream of cAMP through phosphatidylinositol-3 kinase. Forced activation of Notch and beta-catenin with VEGF completely reconstituted cAMP-elicited arterial EC induction, and synergistically enhanced target gene promoter activity in vitro and arterial gene expression during in vivo angiogenesis. A protein complex with RBP-J, the intracellular domain of Notch, and beta-catenin was formed on RBP-J binding sites of arterial genes in arterial, but not venous ECs. This molecular machinery for arterial specification leads to an integrated and more comprehensive understanding of vascular signaling.

Introduction of a foreign gene into zebrafish and medaka cells using adenoviral vectors.

Viral vectors represent a tractable system that can efficiently introduce an exogenous gene into different target cells and are thus a potentially powerful genetic manipulation tool. In our current study, we investigated the infection efficiency of mammalian virus vectors, adenoviruses (Ads), adeno-associated viruses, and lentiviruses to the Sertoli cell line and the newly established cell line from a single embryo in zebrafish. Among the viral vectors tested, Ads showed the highest infection efficiency of 10(7)-10(8) green fluorescent protein-transducing units (gtu)/mL in zebrafish cells. In addition, the adenoviral vector was also infected at 10(5) gtu/mL in the medaka testicular somatic cell line that was established from the testes of p53-deficient mutant. Further, we found that Ads could successfully infect cultured male zebrafish germ cells. Our results thus indicate that the adenoviral vector could be used as a chromosomally nonintegrating vector system in zebrafish.

Gene targeting in human pluripotent stem cells with adeno-associated virus vectors.

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20-87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

BACKGROUND: Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. METHODS: We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. RESULTS: Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. CONCLUSIONS: The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.

Insufficient accumulation of viral late mRNAs restricts the replicative cycle of human adenovirus type 37 in A549 cells.

Most serotypes of human adenoviruses, except subgroup C viruses such as serotype 2 (Ad2) and Ad5, can grow only to low titers in vitro in human cell lines. To analyze the properties of the replicative cycles of poorly growing Ad serotypes, we compared Ad37 and Ad5 in human A549 cells. In comparison with Ad5, Ad37 showed a slower development of viral-induced cytopathic effects and prolonged cell viability. Furthermore, the maximum yields of progeny viruses were reduced by almost 100-fold. Although the amount of viral DNA and E1A mRNA in infected cells reached comparable levels between these two serotypes, the accumulation of viral late mRNAs and structural proteins in Ad37-infected cells was severely reduced. Overall, our study explains critical steps in the replicative cycle of Ad, which cause serotype-specific restrictions in permissive human cell lines.

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors.

Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was approximately 100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only approximately 1% with other gene delivery methods. Furthermore, in combination with negative selection, approximately 45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells.

Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors.

For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse ES cells. The efficiency of HR was compared between four types of AdVs that contained various lengths of homologies at the Hprt locus and with various multiplicities of infections. The frequency of HR with helper-dependent AdVs (HD AdVs) with an 18.6-kb homology reached 0.2% per transduced cell at a multiplicity of infection of 10 genomes per cell. Detection of random integration at DNA levels by PCR revealed extremely high efficiency of 5% per cell. We also isolated and characterized chromosomal sites where HD AdVs integrated in a random manner. In contrast to retroviral, lentiviral, and adeno-associated viral vectors, which tend to integrate into genes, the integration sites of AdV was distributed randomly inside and outside genes. These findings suggest that HR mediated by HD AdVs is efficient and relatively safe and might be a new viable option for ex vivo gene therapy as well as a tool for chromosomal manipulation of a variety of stem cells.

Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector.

Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Delta) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected approximately 50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Delta Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Delta Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism.

An HSV amplicon-based helper system for helper-dependent adenoviral vectors.

To produce a helper virus-free stock of helper-dependent adenoviral vectors (HDAdVs), we have developed a new helper system in which adenoviral genes for propagation of HDAdVs are delivered into producer cells by a herpes simplex virus-1 (HSV) amplicon-adenovirus hybrid. The hybrid amplicon was constructed to carry the E1 gene (HA-E1) or the entire adenoviral genome except E1 (HA-Ad). E1 expression from the HSV amplicon successfully complemented propagation of an E1-deleted adenoviral vector in a human glioma cell line. HDAdVs were propagated in 293 cells infected with HA-Ad. In addition, HDAdVs were rescued and propagated in a glioma cell line superinfected with both HA-E1 and HA-Ad amplicons, although relatively low titers of HSV amplicon resulted in low propagation efficiency of HDAdVs. Since the HSV amplicon can be easily and completely inactivated by chloroform extraction and/or heat treatment from the HDAdV stock, this helper system might be an alternative method to produce helper virus-free HDAdVs.

A new hybrid system capable of efficient lentiviral vector production and stable gene transfer mediated by a single helper-dependent adenoviral vector.

To achieve efficient and sustained gene expression, we developed a new lentivirus/adenovirus hybrid vector (LA vector) that encodes sequences required for production of a human immunodeficiency virus-based lentiviral vector (i.e., a lentiviral vector, a gag/pol/rev expression cassette, a tetracycline-inducible envelope cassette, and the tetracycline-inducible transcriptional activator cassette) in a single helper-dependent adenovirus vector backbone. Via either transfection or infection, human cell lines transduced with the LA vector produced a lentiviral vector in a doxycycline-dependent manner at titers up to 10(5) to 10(6) green fluorescent protein transducing units per ml, which are comparable to the titers obtained by conventional multiple plasmid transfection methods. Efficient spread and persistent expression of the transgene were observed in cells maintained in long-term culture that had been infected with the LA vector. Furthermore, when cocultured with adherent cells infected with the LA vector, the human T-cell leukemia cell line was successfully transduced with a marker gene. This LA vector possesses the advantages of efficient gene transfer from an adenoviral vector and stable integration from a lentiviral vector; therefore, it might have potential for a variety of gene therapy applications.

Helper-dependent adenoviral vectors efficiently express transgenes in human dendritic cells but still stimulate antiviral immune responses.

Adenoviral (AdV) vectors can be used to transduce a wide range of human cells and tissues. However, pre-existing immunity to AdV, and enhancement of this immunity after repeated administration, limits their clinical application. This may be especially relevant when vectors are loaded into APCs. Helper-dependent AdV (Hd-AdV), in which viral coding regions are replaced by human stuffer DNA, offers a new approach for limiting antiviral responses. To evaluate their immunogenicity, human dendritic cells (DCs) were infected with either an Hd-AdV or a conventional replication-deficient E1-deleted AdV (E1-AdV) and were evaluated for their capacity to stimulate antiviral T cell responses. Hd-AdV proved to be 50- to 275-fold more effective than E1-AdV at expressing the lacZ transgene in human DCs. PCR demonstrated similar transduction efficiencies, but RT-PCR revealed much higher expression of transgene mRNA after transduction with Hd-AdV. Functionally, DCs transduced with Hd-AdV stimulated the proliferation of autologous T cells to the same level as DCs transduced with E1-AdV. Identical viral-specific T cell responder frequencies were observed and T cells stimulated with either type of AdV-transduced DC lysed viral-infected target cells. Disrupting transcription of vector-based genes had no effect on T cell activation, suggesting that responses against both vectors were directed against preformed components of the viral capsid. We conclude that Hd-AdV vectors can be used to obtain higher transgene expression in human DCs but that they still evoke a vector-related immune response similar to that generated by E1-AdV.

Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase.

Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and beta-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-beta-gal) and "gutless" AdV (AdV-CMV-PnNOS; AdV-CMV-beta-gal) vectors, and injected into the penis of adult (beta-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of beta-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-beta-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 10(6) viral particles (vp) of AdV-CMV-beta-gal, and with 10(7) vp beta-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (10(7) vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads.