Generation of genetically engineered CHO cell lines to support the production of a difficult to express therapeutic protein (original) (raw)
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Nucleic Acids Research, 2008
Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.
Development of Mitochondrial Gene Replacement Therapy
Journal of Bioenergetics and Biomembranes, 2004
Many “classic” mitochondrial diseases have been described that arise from single homoplasmic mutations in mitochondrial DNA (mtDNA). These diseases typically affect nonmitotic tissues (brain, retina, muscle), present with variable phenotypes, can appear sporadically, and are untreatable. Evolving evidence implicates mtDNA abnormalities in diseases such as Alzheimer's, Parkinson's, and type II diabetes, but specific causal mutations for these conditions remain to be defined. Understanding the mtDNA genotype–phenotype relationships and developing specific treatment for mtDNA-based diseases is hampered by inability to manipulate the mitochondrial genome. We present a novel protein transduction technology (“protofection”) that allows insertion and expression of the human mitochondrial genome into mitochondria of living cells. With protofection, the mitochondrial genotype can be altered, or exogenous genes can be introduced to be expressed and either retained in mitochondria or be directed to other organelles. Protofection also delivers mtDNA in vivo, opening the way to rational development of mitochondrial gene replacement therapy of mtDNA-based diseases.
Progress and prospects: gene therapy for mitochondrial DNA disease
Gene Therapy, 2008
Defects of the mitochondrial genome cause a wide variety of clinical disorders. Except for rare cases where surgery or transplant is indicated, there is no effective treatment for patients. Genetic-based therapies are consequently being considered. On account of the difficulties associated with mitochondrial (mt) transfection, alternative approaches whereby mitochondrial genes can be engineered and introduced into the nucleus (allotopic expression) are being attempted with some success, at least in cultured cells. Defects in the activities of multi-subunit complexes of the oxidative phosphorylation apparatus have been circumvented by the targeted expression of simple single subunit enzymes from other species (xenotopic expression). Although far from the clinic, these approaches show promise. Similarly, nuclear transfection with genes encoding restriction endonucleases or sequence-specific zinc finger-binding proteins destined for mitochondria has also proved successful in targeting mtDNA-borne pathogenic mutations. This is particularly important, as mutated mtDNA is often found in cells that also contain normal copies of the genome, a situation termed heteroplasmy. Shifting the levels of heteroplasmy towards the normal mtDNA has become the goal of a variety of invasive and non-invasive methods, which are also highlighted in this review.
Re-engineering the mitochondrial genomes in mammalian cells
Anatomy & Cell Biology, 2010
Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. Th ey have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. Th e human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the signifi cant advances in discovering the mtDNA defects, however, there are currently no eff ective therapies for these clinically devastating diseases due to the lack of technology for introducing specifi c modifi cations into the mitochondrial genomes and for generating accurate mtDNA disease models. Th e ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own eff orts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.
Journal of Controlled Release, 2018
To achieve mitochondrial gene therapy, developing a mitochondrial transgene expression system that produces therapeutic proteins in mitochondria of disease cells is essential. We previously reported on the design of pCMV-mtLuc (CGG) containing a CMV promotor and a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system, and showed that the mitochondrial transfection of pCMV-mtLuc (CGG) resulted in the efficient production of the Nluc luciferase protein in human HeLa cells. This mitochondrial transfection was achieved using a MITO-Porter, a liposome-based carrier for delivering a cargo to mitochondria via membrane fusion. We report herein that mitochondrial transfection using the MITO-Porter results in mitochondrial transgene expression in G625A fibroblasts obtained from a patient with a mitochondrial disease. We investigated the effect of promoters and the basic structure of pCMV-mtLuc (CGG) on gene expression efficiency, and were able to construct a high performance mitochondrial DNA vector, pCMV-mtLuc (CGG) [hND4] that contains a human mitochondrial endogenous gene. We also constructed an RP/KALA-MITO-Porter composed of the KALA peptide (cellpenetrating peptide) with a mitochondrial RNA aptamer to enhance cellular uptake and mitochondrial targeting. Finally, the mitochondrial transfection of pCMV-mtLuc (CGG) [hND4] in G625A fibroblasts using the RP/KALA-MITO-Porter resulted in strong mitochondrial transgene expression.
Journal of Biomedical Science, 2002
The restriction endonucleaseSmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993T→G mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F0F1-ATPase. Our ultimate goal is to applySmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA)
Pharmaceutical Research
Purpose We describe a novel strategy for expression of GFP in mammalian mitochondria. Methods The key components of the strategy were an artificially created mitochondrial genome pmtGFP and a DQAsome transfection system. Results Using immunofluorescence and a combination of immunohistochemical and molecular based techniques, we show that DQAsomes are capable of delivering the pmtGFP construct to the mitochondrial compartment of the mouse macrophage cell line RAW264.7, albeit at low efficiency (1–5%), resulting in the expression of GFP mRNA and protein. Similar transfection efficiencies were also demonstrated in a range of other mammalian cell lines. Conclusions The DQAsome-transfection technique was able to deliver the exogenous DNA into the cellular mitochondria and the pmtGFP was functional. Further optimization of this strategy would provide a flexible and rapid way to generate mutant cells and useful animal models of mitochondrial disease.
Molecular Therapy - Nucleic Acids, 2020
Here, we report on validating a mitochondrial gene therapy by delivering nucleic acids to mitochondria of diseased cells by a MITO-Porter, a liposome-based carrier for mitochondrial delivery. We used cells derived from a patient with a mitochondrial disease with a G625A heteroplasmic mutation in the tRNA Phe of the mitochondrial DNA (mtDNA). It has been reported that some mitochondrial gene diseases are caused by heteroplasmic mutations, in which both mutated and wildtype (WT) genes are present, and the accumulation of pathological mutations leads to serious, intractable, multi-organ diseases. Therefore, the decrease of the mutated gene rate is considered to be a useful gene therapy strategy. To accomplish this, wild-type mitochondrial pre-tRNA Phe (pre-WT-tRNA Phe), prepared by in vitro transcription, was encapsulated in the MITO-Porter. The pre-WT-tRNA Phe encapsulated in the MITO-Porter was transfected into diseased mitochondrial cells, and the resulting mutant levels were examined by an amplification refractory mutation system (ARMS)-quantitative PCR. The mutation rate of tRNA Phe was decreased, and this therapeutic effect was sustained even on the 8th day after transfection. Furthermore, mitochondrial respiratory activity of the disease cells was increased after the transfection of therapeutic pre-WT-tRNA Phe. These results support the conclusion that the mitochondrial delivery of therapeutic nucleic acids represents a viable strategy for mitochondrial gene therapy.