|
1
|
Tissue and cell-type-specific transduction using rAAV vectors in lung diseases. J Mol Med (Berl) 2021; 99:1057-1071. [PMID: 34021360 DOI: 10.1007/s00109-021-02086-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2020] [Revised: 04/20/2021] [Accepted: 04/26/2021] [Indexed: 10/21/2022]
Abstract
Gene therapy of genetically determined diseases, including some pathologies of the respiratory system, requires an efficient method for transgene delivery. Recombinant adeno-associated viral (rAAV) vectors are well studied and employed in gene therapy, as they are relatively simple and low immunogenic and able to efficiently transduce eukaryotic cells. To date, many natural and artificial (with modified capsids) AAV serotypes have been isolated, demonstrating preferential tropism toward different tissues and cells in accordance with the prevalent receptors on the cell surface. However, rAAV-mediated delivery is not strictly specific due to wide tropism of some viral serotypes. Thus, the development of the methods allowing modulating specificity of these vectors could be beneficial in some cases. This review describes various approaches for retargeting rAAV to respiratory cells, for example, using different types of capsid modifications and regulation of a transgene expression by tissue-specific promoters. Part of the review is devoted to the issues of transduction of stem and progenitor lung cells using AAV, which is a complicated task today.
Collapse
|
|
2
|
Cappella M, Elouej S, Biferi MG. The Potential of Induced Pluripotent Stem Cells to Test Gene Therapy Approaches for Neuromuscular and Motor Neuron Disorders. Front Cell Dev Biol 2021; 9:662837. [PMID: 33937264 PMCID: PMC8080375 DOI: 10.3389/fcell.2021.662837] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Accepted: 03/22/2021] [Indexed: 12/11/2022] Open
Abstract
The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) represents a major advance for the development of human disease models. The emerging of this technique fostered the concept of "disease in a dish," which consists into the generation of patient-specific models in vitro. Currently, iPSCs are used to study pathological molecular mechanisms caused by genetic mutations and they are considered a reliable model for high-throughput drug screenings. Importantly, precision-medicine approaches to treat monogenic disorders exploit iPSCs potential for the selection and validation of lead candidates. For example, antisense oligonucleotides (ASOs) were tested with promising results in myoblasts or motor neurons differentiated from iPSCs of patients affected by either Duchenne muscular dystrophy or Amyotrophic lateral sclerosis. However, the use of iPSCs needs additional optimization to ensure translational success of the innovative strategies based on gene delivery through adeno associated viral vectors (AAV) for these diseases. Indeed, to establish an efficient transduction of iPSCs with AAV, several aspects should be optimized, including viral vector serotype, viral concentration and timing of transduction. This review will outline the use of iPSCs as a model for the development and testing of gene therapies for neuromuscular and motor neuron disorders. It will then discuss the advantages for the use of this versatile tool for gene therapy, along with the challenges associated with the viral vector transduction of iPSCs.
Collapse
Affiliation(s)
- Marisa Cappella
- Sorbonne University, INSERM, Institute of Myology, Center of Research in Myology, Paris, France
| | - Sahar Elouej
- Sorbonne University, INSERM, Institute of Myology, Center of Research in Myology, Paris, France
| | - Maria Grazia Biferi
- Sorbonne University, INSERM, Institute of Myology, Center of Research in Myology, Paris, France
| |
Collapse
|
|
3
|
Brown N, Song L, Kollu NR, Hirsch ML. Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes? Hum Gene Ther 2018; 28:450-463. [PMID: 28490211 DOI: 10.1089/hum.2017.038] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the stage for future elucidation and eventual therapeutic applications.
Collapse
Affiliation(s)
- Nolan Brown
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Liujiang Song
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Nageswara R Kollu
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Matthew L Hirsch
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| |
Collapse
|
|
4
|
Ide K, Mitsui K, Irie R, Matsushita Y, Ijichi N, Toyodome S, Kosai KI. A Novel Construction of Lentiviral Vectors for Eliminating Tumorigenic Cells from Pluripotent Stem Cells. Stem Cells 2018; 36:230-239. [DOI: 10.1002/stem.2725] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/30/2023]
Abstract
Abstract
The risk of tumor formation poses a challenge for human pluripotent stem cell (hPSC)-based transplantation therapy. Specific and total elimination of tumorigenic hPSCs by suicide genes (SGs) has not been achieved because no methodology currently exists for testing multiple candidate transgene constructs. Here, we present a novel method for efficient generation of tumorigenic cell-targeting lentiviral vectors (TC-LVs) with diverse promoters upstream of a fluorescent protein and SGs. Our two-plasmid system achieved rapid and simultaneous construction of different TC-LVs with different promoters. Ganciclovir (GCV) exerted remarkable cytotoxicity in herpes simplex virus thymidine kinase-transduced hPSCs, and high specificity for undifferentiated cells was achieved using the survivin promoter (TC-LV.Surv). Moreover, GCV treatment completely abolished teratoma formation by TC-LV.Surv-infected hPSCs transplanted into mice, without harmful effects. Thus, TC-LV can efficiently identify the best promoter and SG for specific and complete elimination of tumorigenic hPSCs, facilitating the development of safe regenerative medicine.
Collapse
Affiliation(s)
- Kanako Ide
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Kaoru Mitsui
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
- Center for Innovative Therapy Research and Application, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Rie Irie
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
- Center for Innovative Therapy Research and Application, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Yohei Matsushita
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Nobuhiro Ijichi
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
- Center for Innovative Therapy Research and Application, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Soichiro Toyodome
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Ken-ichiro Kosai
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
- Center for Innovative Therapy Research and Application, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
- Center for Clinical and Translational Research, Kagoshima University Hospital, Kagoshima, Japan
| |
Collapse
|
|
5
|
Pashaiasl M, Khodadadi K, Kayvanjoo AH, Pashaei-asl R, Ebrahimie E, Ebrahimi M. Unravelling evolution of Nanog, the key transcription factor involved in self-renewal of undifferentiated embryonic stem cells, by pattern recognition in nucleotide and tandem repeats characteristics. Gene 2016; 578:194-204. [DOI: 10.1016/j.gene.2015.12.023] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2015] [Revised: 12/10/2015] [Accepted: 12/10/2015] [Indexed: 12/27/2022]
|
|
6
|
Silent IL2RG Gene Editing in Human Pluripotent Stem Cells. Mol Ther 2015; 24:582-91. [PMID: 26444081 DOI: 10.1038/mt.2015.190] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2015] [Accepted: 09/30/2015] [Indexed: 12/19/2022] Open
Abstract
Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.
Collapse
|
|
7
|
Genome Engineering Using Adeno-associated Virus: Basic and Clinical Research Applications. Mol Ther 2015; 24:458-64. [PMID: 26373345 DOI: 10.1038/mt.2015.151] [Citation(s) in RCA: 77] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2015] [Accepted: 07/21/2015] [Indexed: 12/18/2022] Open
Abstract
In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.
Collapse
|
|
8
|
Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs. Mol Ther 2015; 24:812-22. [PMID: 26316390 PMCID: PMC4886927 DOI: 10.1038/mt.2015.154] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2014] [Accepted: 08/14/2015] [Indexed: 12/11/2022] Open
Abstract
Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.
Collapse
|
|
9
|
Focosi D, Maggi F, Ceccherini-Nelli L, Pistello M. Cell therapies for treatment of human immunodeficiency virus infection. Rev Med Virol 2015; 25:156-74. [PMID: 25727480 DOI: 10.1002/rmv.1831] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2014] [Revised: 01/30/2015] [Accepted: 02/05/2015] [Indexed: 12/15/2022]
Abstract
After the serendipitous discovery of HIV eradication in the "Berlin patient", interest has grown in curing HIV infection by replacing the patient's replication-competent blood cells with infection-resistant ones. At the same time, induced pluripotent stem cell technologies and genetic engineering have boosted cell therapy transfer into the clinic. Currently available cell therapy approaches to attempt to cure HIV infection include the following: (1) Transplantation of autologous or allogeneic cells spontaneously resistant or edited to resist HIV infection; (2) Transplantation of autologous T-lymphocytes spontaneously targeting or redirected against HIV; and (3) Transplantation of autologous cells engineered to work as anti-HIV antibody factories. We review here the preliminary results and potential for future applications of these approaches.
Collapse
Affiliation(s)
- Daniele Focosi
- Retrovirus Center and Virology Section, Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Pisa, Italy
| | | | | | | |
Collapse
|
|
10
|
Li M, Suzuki K, Kim NY, Liu GH, Izpisua Belmonte JC. A cut above the rest: targeted genome editing technologies in human pluripotent stem cells. J Biol Chem 2013; 289:4594-9. [PMID: 24362028 DOI: 10.1074/jbc.r113.488247] [Citation(s) in RCA: 99] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) offer unprecedented opportunities to study cellular differentiation and model human diseases. The ability to precisely modify any genomic sequence holds the key to realizing the full potential of hPSCs. Thanks to the rapid development of novel genome editing technologies driven by the enormous interest in the hPSC field, genome editing in hPSCs has evolved from being a daunting task a few years ago to a routine procedure in most laboratories. Here, we provide an overview of the mainstream genome editing tools, including zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases, and helper-dependent adenoviral vectors. We discuss the features and limitations of these technologies, as well as how these factors influence the utility of these tools in basic research and therapies.
Collapse
Affiliation(s)
- Mo Li
- From the Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037 and
| | | | | | | | | |
Collapse
|
|
11
|
Ramamoorthi K, Curtis D, Asuri P. Advances in homology directed genetic engineering of human pluripotent and adult stem cells. World J Stem Cells 2013; 5:98-105. [PMID: 24179598 PMCID: PMC3812527 DOI: 10.4252/wjsc.v5.i4.98] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/26/2013] [Revised: 09/06/2013] [Accepted: 09/17/2013] [Indexed: 02/06/2023] Open
Abstract
The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.
Collapse
|
|
12
|
Rahman SH, Bobis-Wozowicz S, Chatterjee D, Gellhaus K, Pars K, Heilbronn R, Jacobs R, Cathomen T. The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting. Hum Gene Ther 2013; 24:67-77. [PMID: 23072634 PMCID: PMC3555098 DOI: 10.1089/hum.2012.168] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2012] [Accepted: 10/08/2012] [Indexed: 01/28/2023] Open
Abstract
Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord-derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3'-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3'-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications.
Collapse
Affiliation(s)
- Shamim H Rahman
- Laboratory of Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany.
| | | | | | | | | | | | | | | |
Collapse
|
|
13
|
Targeted integration of a rAAV vector into the AAVS1 region. Virology 2012; 433:356-66. [PMID: 22981435 DOI: 10.1016/j.virol.2012.08.015] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2012] [Revised: 05/25/2012] [Accepted: 08/03/2012] [Indexed: 11/22/2022]
Abstract
Adeno-associated virus (AAV) has been reported to integrate in a site-specific manner into chromosome 19 (a site designated AAVS1), a phenomenon that could be exploited for ex vivo targeted gene therapy. Recent studies employing LM-PCR to determine AAV integration loci; however, have, contrary to previous results with less reliable methods, concluded that the proclivity for AAV integration at AAVS1 is minimal. We tested this conclusion employing LM-PCR protocols designed to avoid bias. Hep G2 cells were infected with rAAV2-GFP and coinfected with wt AAV2 to supply Rep in trans. Sorted cells were cloned and cultured. In 26 clones that retained fluorescence, DNA was extracted and AAV-genomic junctions amplified by two LM-PCR methods. Sequencing was performed without bacterial cloning. Of these 26 clones it was possible to assign a genomic integration site to 14, of which 9 were in the AAVS1 region. In three additional clones, rAAV integration junction were to an integrated wt AAV genome while two were to an rAAV genome. We also show that integration of the AAV-GFP genome can be achieved without cointegration of the AAV genome. Based on the pattern of integrants we propose, for potential use in ex vivo targeted gene therapy, a simplified PCR method to identify clones that have rAAV genomes integrated into AAVS1.
Collapse
|
|
14
|
Sebastiano V, Maeder ML, Angstman JF, Haddad B, Khayter C, Yeo DT, Goodwin MJ, Hawkins JS, Ramirez CL, Batista LFZ, Artandi SE, Wernig M, Joung JK. In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases. Stem Cells 2012; 29:1717-26. [PMID: 21898685 DOI: 10.1002/stem.718] [Citation(s) in RCA: 264] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.
Collapse
Affiliation(s)
- Vittorio Sebastiano
- Department of Pathology, Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford California, USA
| | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
15
|
Targeted In Situ Gene Correction of Dysfunctional APOE Alleles to Produce Atheroprotective Plasma ApoE3 Protein. Cardiol Res Pract 2012; 2012:148796. [PMID: 22645694 PMCID: PMC3356902 DOI: 10.1155/2012/148796] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2011] [Accepted: 01/30/2012] [Indexed: 02/07/2023] Open
Abstract
Cardiovascular disease is the leading worldwide cause of death. Apolipoprotein E (ApoE) is a 34-kDa circulating glycoprotein, secreted by the liver and macrophages with pleiotropic antiatherogenic functions and hence a candidate to treat hypercholesterolaemia and atherosclerosis. Here, we describe atheroprotective properties of ApoE, though also potential proatherogenic actions, and the prevalence of dysfunctional isoforms, outline conventional gene transfer strategies, and then focus on gene correction therapeutics that can repair defective APOE alleles. In particular, we discuss the possibility and potential benefit of applying in combination two technical advances to repair aberrant APOE genes: (i) an engineered endonuclease to introduce a double-strand break (DSB) in exon 4, which contains the common, but dysfunctional, ε2 and ε4 alleles; (ii) an efficient and selectable template for homologous recombination (HR) repair, namely, an adeno-associated viral (AAV) vector, which harbours wild-type APOE sequence. This technology is applicable ex vivo, for example to target haematopoietic or induced pluripotent stem cells, and also for in vivo hepatic gene targeting. It is to be hoped that such emerging technology will eventually translate to patient therapy to reduce CVD risk.
Collapse
|
|
16
|
Mali P, Cheng L. Concise review: Human cell engineering: cellular reprogramming and genome editing. Stem Cells 2012; 30:75-81. [PMID: 21905170 DOI: 10.1002/stem.735] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Cell engineering is defined here as the collective ability to both reset and edit the genome of a mammalian cell. Until recently, this had been extremely challenging to achieve as nontransformed human cells are significantly refractory to both these processes. The recent success in reprogramming somatic cells into induced pluripotent stem cells that are self-renewable in culture, coupled with our increasing ability to effect precise and predesigned genomic editing, now readily permits cellular changes at both the genetic and epigenetic levels. These dual capabilities also make possible the generation of genetically matched, disease-free stem cells from patients for regenerative medicine. The objective of this review is to summarize the key enabling developments on these two rapidly evolving research fronts in human cell engineering, highlight unresolved issues, and outline potential future research directions.
Collapse
Affiliation(s)
- Prashant Mali
- Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | | |
Collapse
|
|
17
|
Abstract
The discovery of induced pluripotent stem (iPS) cells has broadened the promises of regenerative medicine through the generation of syngeneic replacement cells or tissues via the differentiation of patient-specific iPS cells. To apply iPS cell-mediated therapy to patients with genetic disorders, however, genome-editing technologies with high efficiency and specificity are needed. Recently, several targeted genome-editing strategies mediated by zinc finger nuclease and transcription activator-like effector nuclease have been applied to human and mouse iPS cells. Furthermore, spontaneous homologous recombination can restore genotype to wild type in mouse iPS cells heterozygous for genetic mutations. Through genome editing, the clinical application of patient-specific genetic mutation-free iPS cells to genetic disorders can finally be realized.
Collapse
Affiliation(s)
- Li-Tao Cheng
- Stem Cell Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | | | | |
Collapse
|
|
18
|
Asuri P, Bartel MA, Vazin T, Jang JH, Wong TB, Schaffer DV. Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells. Mol Ther 2012; 20:329-38. [PMID: 22108859 PMCID: PMC3277219 DOI: 10.1038/mt.2011.255] [Citation(s) in RCA: 94] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2011] [Accepted: 10/27/2011] [Indexed: 12/12/2022] Open
Abstract
Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFN- mediated double-stranded DNA breaks.
Collapse
Affiliation(s)
- Prashanth Asuri
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720-1462, USA
| | | | | | | | | | | |
Collapse
|
|
19
|
Pan H, Zhang W, Zhang W, Liu GH. Find and replace: editing human genome in pluripotent stem cells. Protein Cell 2011; 2:950-6. [PMID: 22173708 DOI: 10.1007/s13238-011-1132-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2011] [Accepted: 12/04/2011] [Indexed: 12/14/2022] Open
Abstract
Genetic manipulation of human pluripotent stem cells (hPSCs) provides a powerful tool for modeling diseases and developing future medicine. Recently a number of independent genome-editing techniques were developed, including plasmid, bacterial artificial chromosome, adeno-associated virus vector, zinc finger nuclease, transcription activator-like effecter nuclease, and helper-dependent adenoviral vector. Gene editing has been successfully employed in different aspects of stem cell research such as gene correction, mutation knock-in, and establishment of reporter cell lines (Raya et al., 2009; Howden et al., 2011; Li et al., 2011; Liu et al., 2011b; Papapetrou et al., 2011; Sebastiano et al., 2011; Soldner et al., 2011; Zou et al., 2011a). These techniques combined with the utility of hPSCs will significantly influence the area of regenerative medicine.
Collapse
Affiliation(s)
- Huize Pan
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | | | | | | |
Collapse
|
|
20
|
Osiak A, Radecke F, Guhl E, Radecke S, Dannemann N, Lütge F, Glage S, Rudolph C, Cantz T, Schwarz K, Heilbronn R, Cathomen T. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases. PLoS One 2011; 6:e28911. [PMID: 22194948 PMCID: PMC3237556 DOI: 10.1371/journal.pone.0028911] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2011] [Accepted: 11/17/2011] [Indexed: 12/18/2022] Open
Abstract
Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.
Collapse
Affiliation(s)
- Anna Osiak
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
- Institute of Virology (CBF), Charité Medical School, Berlin, Germany
| | - Frank Radecke
- Institute for Transfusion Medicine, University of Ulm, Ulm, Germany
| | - Eva Guhl
- Institute of Virology (CBF), Charité Medical School, Berlin, Germany
| | - Sarah Radecke
- Institute for Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Service Baden-Württemberg – Hessen, Ulm, Germany
| | - Nadine Dannemann
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Fabienne Lütge
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Silke Glage
- Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany
| | - Cornelia Rudolph
- JRG Genetic and Epigenetic Integrity, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Tobias Cantz
- JRG Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Klaus Schwarz
- Institute for Transfusion Medicine, University of Ulm, Ulm, Germany
- Institute for Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Service Baden-Württemberg – Hessen, Ulm, Germany
| | - Regine Heilbronn
- Institute of Virology (CBF), Charité Medical School, Berlin, Germany
| | - Toni Cathomen
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
- Institute of Virology (CBF), Charité Medical School, Berlin, Germany
| |
Collapse
|
|
21
|
Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors. Mol Ther 2011; 20:424-31. [PMID: 22146343 DOI: 10.1038/mt.2011.266] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
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.
Collapse
|
|
22
|
Hirsch ML, Fagan BM, Dumitru R, Bower JJ, Yadav S, Porteus MH, Pevny LH, Samulski RJ. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells. PLoS One 2011; 6:e27520. [PMID: 22114676 PMCID: PMC3219675 DOI: 10.1371/journal.pone.0027520] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2011] [Accepted: 10/18/2011] [Indexed: 11/19/2022] Open
Abstract
Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.
Collapse
Affiliation(s)
- Matthew L. Hirsch
- Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States of America
- * E-mail: (MLH); (RJS)
| | - B. Matthew Fagan
- Human Embryonic Stem Cell Core Facility, University of North Carolina, Chapel Hill, North Carolina, United States of America
| | - Raluca Dumitru
- Department of Cell and Developmental Biology, Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, United States of America
| | - Jacquelyn J. Bower
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America
| | - Swati Yadav
- Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States of America
| | - Matthew H. Porteus
- Department of Pediatrics-Cancer Biology, Stanford University, Palo Alto, California, United States of America
| | - Larysa H. Pevny
- Department of Genetics, University of North Carolina, Chapel Hill, North Carolina, United States of America
- Department of Cell and Developmental Biology, Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, United States of America
| | - R. Jude Samulski
- Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States of America
- Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, United States of America
- * E-mail: (MLH); (RJS)
| |
Collapse
|
|
23
|
Normal collagen and bone production by gene-targeted human osteogenesis imperfecta iPSCs. Mol Ther 2011; 20:204-13. [PMID: 22031238 DOI: 10.1038/mt.2011.209] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease.
Collapse
|
|
24
|
Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. Blood 2011; 118:4599-608. [PMID: 21881051 DOI: 10.1182/blood-2011-02-335554] [Citation(s) in RCA: 235] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (β(s)/β(s)). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the β(s) locus, we achieved precise conversion of 1 mutated β(s) to the wild-type β(A) in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected β(s)/β(A) heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.
Collapse
|
|
25
|
MacMillan ML, Hughes MR, Agarwal S, Daley GQ. Cellular therapy for fanconi anemia: the past, present, and future. Biol Blood Marrow Transplant 2011; 17:S109-14. [PMID: 21195298 DOI: 10.1016/j.bbmt.2010.11.027] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Allogeneic hematopoietic cell transplantation (HCT) remains the only proven curative therapy for the hematologic manifestation of Fanconi anemia (FA). Over the past 2 decades, major advances have been made such that transplant outcomes have markedly improved. With the development of in vitro fertilization and preimplantation genetic diagnosis, HLA-matched sibling donor umbilical blood transplantation may be an option for more patients with FA. Recently, the use of pluripotent stem cells has been explored as a novel approach to model the hematopoietic developmental defects in FA, and to provide a potential source of autologous stem cells that can be genetically manipulated and used to generate corrected hematopoietic progenitors.
Collapse
Affiliation(s)
- Margaret L MacMillan
- The Division of Pediatric Hematology, Oncology, and Blood and Marrow Transplantation, University of Minnesota Blood and Marrow Transplant Program, Minneapolis, Minnesota, USA.
| | | | | | | |
Collapse
|
|
26
|
Liu GH, Suzuki K, Qu J, Sancho-Martinez I, Yi F, Li M, Kumar S, Nivet E, Kim J, Soligalla RD, Dubova I, Goebl A, Plongthongkum N, Fung HL, Zhang K, Loring JF, Laurent LC, Izpisua Belmonte JC. Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. Cell Stem Cell 2011; 8:688-94. [PMID: 21596650 DOI: 10.1016/j.stem.2011.04.019] [Citation(s) in RCA: 174] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2011] [Revised: 04/22/2011] [Accepted: 04/29/2011] [Indexed: 11/25/2022]
Abstract
Combination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases. We show that helper-dependent adenoviral vectors (HDAdVs) provide a highly efficient and safe method for correcting mutations in large genomic regions in human induced pluripotent stem cells and can also be effective in adult human mesenchymal stem cells. This type of approach could be used to generate genotype-matched cell lines for disease modeling and drug discovery and potentially also in therapeutics.
Collapse
Affiliation(s)
- Guang-Hui Liu
- Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
27
|
Abstract
Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10(-5) to 10(-2) per infected cell. These targeting frequencies are 1-4 logs higher than those obtained by conventional transfection or electroporation approaches. A wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. Optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by Southern blots. This protocol (from vector design through a single round of targeting and screening) can be completed in ∼10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.
Collapse
Affiliation(s)
- Iram F Khan
- Department of Medicine, University of Washington, Seattle, WA, USA
| | | | | |
Collapse
|
|
28
|
|
|
29
|
Jensen NM, Dalsgaard T, Jakobsen M, Nielsen RR, Sørensen CB, Bolund L, Jensen TG. An update on targeted gene repair in mammalian cells: methods and mechanisms. J Biomed Sci 2011; 18:10. [PMID: 21284895 PMCID: PMC3042377 DOI: 10.1186/1423-0127-18-10] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2010] [Accepted: 02/02/2011] [Indexed: 11/10/2022] Open
Abstract
Transfer of full-length genes including regulatory elements has been the preferred gene therapy strategy for clinical applications. However, with significant drawbacks emerging, targeted gene alteration (TGA) has recently become a promising alternative to this method. By means of TGA, endogenous DNA repair pathways of the cell are activated leading to specific genetic correction of single-base mutations in the genome. This strategy can be implemented using single-stranded oligodeoxyribonucleotides (ssODNs), small DNA fragments (SDFs), triplex-forming oligonucleotides (TFOs), adeno-associated virus vectors (AAVs) and zinc-finger nucleases (ZFNs). Despite difficulties in the use of TGA, including lack of knowledge on the repair mechanisms stimulated by the individual methods, the field holds great promise for the future. The objective of this review is to summarize and evaluate the different methods that exist within this particular area of human gene therapy research.
Collapse
Affiliation(s)
- Nanna M Jensen
- Institute of Human Genetics, The Bartholin Building, University of Aarhus, 8000 Aarhus C, Denmark
| | | | | | | | | | | | | |
Collapse
|
|
30
|
Wobus AM, Löser P. Present state and future perspectives of using pluripotent stem cells in toxicology research. Arch Toxicol 2011; 85:79-117. [PMID: 21225242 PMCID: PMC3026927 DOI: 10.1007/s00204-010-0641-6] [Citation(s) in RCA: 84] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2010] [Accepted: 12/21/2010] [Indexed: 02/08/2023]
Abstract
The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed.
Collapse
Affiliation(s)
- Anna M Wobus
- In Vitro Differentiation Group, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany.
| | | |
Collapse
|
|
31
|
Gene therapy, gene targeting and induced pluripotent stem cells: Applications in monogenic disease treatment. Biotechnol Adv 2010; 28:715-24. [DOI: 10.1016/j.biotechadv.2010.05.019] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2010] [Revised: 05/05/2010] [Accepted: 05/11/2010] [Indexed: 12/15/2022]
|
|
32
|
Gene therapy, gene targeting and induced pluripotent stem cells: applications in monogenic disease treatment. Biotechnol Adv 2010; 29:1-10. [PMID: 20656005 DOI: 10.1016/j.biotechadv.2010.07.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2010] [Revised: 05/05/2010] [Accepted: 05/11/2010] [Indexed: 01/15/2023]
Abstract
Monogenic diseases are often severe, life-threatening disorders for which lifelong palliative treatment is the only option. Over the last two decades, a number of strategies have been devised with the aim to treat these diseases with a genetic approach. Gene therapy has been under development for many years, yet suffers from the lack of an effective and safe vector for the delivery of genetic material into cells. More recently, gene targeting by homologous recombination has been proposed as a safer treatment, by specifically correcting disease-causing mutations. However, low efficiency is a major drawback. The emergence of two technologies could overcome some of these obstacles. Terminally differentiated somatic cells can be reprogrammed, using defined factors, to become induced pluripotent stem cells (iPSCs), which can undergo efficient gene mutation correction with the aid of fusion proteins known as zinc finger nucleases (ZFNs). The amalgamation of these two technologies has the potential to break through the current bottleneck in gene therapy and gene targeting.
Collapse
|
|
33
|
Nieminen M, Tuuri T, Savilahti H. Genetic recombination pathways and their application for genome modification of human embryonic stem cells. Exp Cell Res 2010; 316:2578-86. [PMID: 20542027 DOI: 10.1016/j.yexcr.2010.06.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2010] [Revised: 05/31/2010] [Accepted: 06/06/2010] [Indexed: 12/24/2022]
Abstract
Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.
Collapse
Affiliation(s)
- Mikko Nieminen
- Division of Genetics and Physiology, Department of Biology, University of Turku, Turku, Finland
| | | | | |
Collapse
|
|
34
|
Affiliation(s)
- Jizhong Zou
- Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | | | | |
Collapse
|
|
35
|
Abstract
Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.
Collapse
|
|
36
|
Lowry WE, Quan WL. Roadblocks en route to the clinical application of induced pluripotent stem cells. J Cell Sci 2010; 123:643-51. [PMID: 20164303 PMCID: PMC10851774 DOI: 10.1242/jcs.054304] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/06/2010] [Indexed: 12/16/2022] Open
Abstract
Since the first studies of human embryonic stem cells (hESCs) and, more recently, human induced pluripotent stem cells (hiPSCs), the stem-cell field has been abuzz with the promise that these pluripotent populations will one day be a powerful therapeutic tool. Although it has been proposed that hiPSCs will supersede hESCs with respect to their research and/or clinical potential because of the ease of their derivation and the ability to create immunologically matched iPSCs for each individual patient, recent evidence suggests that iPSCs in fact have several underappreciated characteristics that might mean they are less suitable for clinical application. Continuing research is revealing the similarities, differences and deficiencies of various pluripotent stem-cell populations, and suggests that many years will pass before the clinical utility of hESCs and hiPSCs is realized. There are a plethora of ethical, logistical and technical roadblocks on the route to the clinical application of pluripotent stem cells, particularly of iPSCs. In this Essay, we discuss what we believe are important issues that should be considered when attempting to bring hiPSC-based technology to the clinic.
Collapse
Affiliation(s)
- William E Lowry
- Department of Molecular, Cell and Developmental Biology, 621 Charles Young Drive South, University of California Los Angeles, Los Angeles, CA 90095, USA.
| | | |
Collapse
|