Mitochondria Isolated from Liver Contain the Essential Factors Required for RNA/DNA Oligonucleotide- Targeted Gene Repair

Chen, Zongyu; Felsheim, Rod; Wong, Phillip; Augustin, Lance B.; Metz, Richard; Kren, Betsy T.; Steer, Clifford J.

doi:10.1006/bbrc.2001.5156

Cited by 37 publications

(21 citation statements)

References 30 publications

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“…For example, the delivery of wild-type mtDNA (circular DNA) to mitochondria in diseased cells would decrease the proportion of mutated mtDNA, thereby suppressing mitochondrial diseases [7,12]. On the other hand, oligo DNA (linear DNA), as a therapeutic agent for muted mtDNA, was investigated, in order to repair mutated mtDNA and inhibit the replication of mutated mtDNA [34,35]. Moreover, the use of a restriction enzyme (protein) would be expected to be useful and productive for the selective digestion of mutated mtDNA [36].…”

Section: Discussionmentioning

confidence: 99%

Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter

Yamada

Harashima

2012

Biomaterials

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Mitochondrial dysfunction has been implicated in a variety of human diseases. It is now well accepted that mutations and defects in the mitochondrial genome form the basis of these diseases. Therefore, mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve such an innovative a strategy, it will be necessary to deliver therapeutic agents into mitochondria in living cells. We report here on a novel an approach to accomplish this via the use of a Dual Function (DF)-MITO-Porter, aimed at the mitochondrial genome, so-called mitochondrial DNA (mtDNA). The DF-MITO-Porter, an innovative a nano carrier for mitochondrial delivery, has the ability to penetrate the endosomal and mitochondrial membranes via step-wise membrane fusion. We first constructed a DF-MITO-Porter encapsulating DNase I protein as a bioactive cargo. It was expected that mtDNA would be digested, when the DNase I was delivered to the mitochondria.We observed the intracellular trafficking of the carriers, and then measured mitochondrial activity and mtDNA-levels after the delivery of DNase I by the DF-MITO-Porter. The findings confirm that the DF-MITO-Porter effectively delivered the DNase I into the mitochondria, and provides a demonstration of its potential use in therapies that are selective for the mitochondrial genome. 3

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Section: Discussionmentioning

confidence: 99%

Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter

Yamada

Harashima

2012

Biomaterials

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“…Bacteria transformed with control dual antibioticresistant plasmids exhibited~70% reduction in colonies recovered on medium containing antibiotics other than ampicillin. 30 In the lacZ plasmid-based assay, the number of repaired plasmids (blue color on plates containing Xgal) versus the total number of plasmids recovered (ampicillin resistance) indicates the frequency of repair. The repair of the point mutation also reintroduces an EcoRI restriction endonuclease site, permitting screening by restriction fragment length polymorphism.…”

Section: Figure 2 Serum From a Gunn Rat Before And After Treatment Wimentioning

confidence: 99%

“…We established that mitochondrial extracts exhibit a dosedependent repair activity (unpublished observations) that was five-fold greater than chimeraplast-mediated repair activity. 27,30,34 The increased frequency of repair using single-stranded oligonucleotides rather than chimeraplasts was observed using either nuclear or mitochondrial extracts. Again, no change in repair frequency was observed between extracts isolated from quiescent or proliferating cells.…”

Section: Figure 2 Serum From a Gunn Rat Before And After Treatment Wimentioning

confidence: 99%

“…26 Extracts were prepared from nuclei and highly purified mitochondria isolated from rat liver, and the authenticity and purity of the extracts established by Western blot, enzymatic activity and electron microscopy. [28][29][30] Using this PCR-independent bacterial read-out system and extracts derived from rat liver, we have observed a chimeraplast-specific and mitochondrial protein extract dose-dependent growth of dual resistant colonies, ranging from 0.003 ± 0.002% to 0.05 ± 0.002%. This rate of repair in vitro did not differ significantly from that observed in parallel reactions using rat liver nuclear extract.…”

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Modification of hepatic genomic DNA using RNA/DNA oligonucleotides

Kren¹,

Chen

Felsheim

et al. 2002

Gene Ther

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Inherited metabolic diseases impose a significant worldwide burden both financially and socially. Although there have been advances in treating these disorders, alternative therapeutic approaches are being developed to correct the responsible genetic defects. Gene augmentation using viral vectors and transgene expression to replace an absent or defective protein has shown limited success in treating a variety of genetic disorders. In fact, the mechanisms by which cells and organisms protect against viral infections have presented significant barriers for long-term stable expression of the introduced transgene. In part, due to the disadvantages of using biological vectors, a variety of different approaches for nonviral gene delivery have been developed. 1 In particular, targeting of nonviral delivery systems to specific tissues by receptor/ligand interactions has been exploited for in vivo gene transfer. 2 By targeting the asialoglycoprotein receptor (ASGPR), gene delivery to hepatocytes has been significantly improved both in vitro 3 and in vivo. 4,5 Thus, we have focused on using nonviral approaches to correct the genetic defect in hepatocytes using galactosylated compounds attached directly to polycations, or incorporated in liposomes. 6 The strategy of in situ genomic repair using chimeric RNA/DNA molecules (chimeraplasts) developed from studies investigating molecular aspects of DNA repair. It became apparent that there was a significant increase in pairing efficiency between an oligonucleotide Յ50 bases and a genomic DNA target if RNA replaced DNA in a portion of the targeting oligonucleotide. 7,8 The original chimeric 68-mer design incorporated 10 2'-O-methylated RNA residues flanking each side of a 5 bp stretch of DNA, poly-T hairpin loops, a 3' GC clamp and a complementary all-DNA strand resulting in a stable, nucleaseresistant duplex molecule. The 25 bp homology segment between the chimeraplast and its genomic target is constructed such that it includes one mismatch with the DNA sequence of the gene. This engineered mismatch permits the directed alteration of the genomic target using the chimeraplast as a template for the intended correction of target sequence. It is postulated that the 'mismatched' chimeraplast complexes with the genomic DNA and creates the illusion of a genetic mutation leading to the recruitment of endogenous repair functions. 9,10 This novel approach of gene repair, based on in vitro experimental evidence, was shown by Yoon et al 11 and Cole-Strauss et al 12 to be capable of introducing targeted single base conversions in episomal and genomic DNA in cultured cells. We then reported that the chimeraplast

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“…Chen and co-workers demonstrated that, not only nucleus, but also mitochondria contain the machinery required for the repair of genomic mutations using oligodeoxynucleotides (ODN). Therefore, the delivery of therapeutic ODN to the mitochondrial matrix is promising for the treatment of such mitochondrial diseases (Chen et al, 2001). …”

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confidence: 99%

Mitochondrial drug delivery and mitochondrial disease therapy – An approach to liposome-based delivery targeted to mitochondria

et al. 2007

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Recent progress in genetics and molecular biology has provided useful information regarding the molecular mechanisms associated with the mitochondrial diseases. Genetic approaches were initiated in the late 1980s to clarify the gene responsible for various mitochondrial diseases, and information concerning genetic mutations is currently used in the diagnosis of mitochondrial diseases. Moreover, it was also revealed that mitochondria play a central role in apoptosis, or programmed cell death, which is closely related to the loss of physiological functions of tissues. Therefore, drug therapies targeted to the mitochondria would be highly desirable. In spite of the huge amount of mechanism-based studies of mitochondrial diseases, effective therapies have not yet been established mainly because of the lack of an adequate delivery system. To date, numerous investigators have attempted to establish a mitochondrial drug delivery system. However, many problems remain to be overcome before a clinical application can be achieved. To fulfill a drug delivery targeted to mitochondria, we first need to establish a method to encapsulate various drugs, proteins, peptides, and genes into a drug carrier depending on their physical characteristics. Second, we need to target it to a specific cell. Finally, multi-processes of intracellular trafficking should be sophisticatedly regulated so as to release a drug carrier from the endosome to the cytosol, and thereafter to deliver to the mitochondria. In this review, we describe the current state of the development of mitochondrial drug delivery systems, and discuss the advantage and disadvantage of each system. Our current efforts to develop an efficient method for the packaging of macromolecules and regulating intracellular trafficking are also summarized.Furthermore, novel concept of "Regulation of intramitochondrial trafficking" is proposed herein as a future challenge to the development of a mitochondrial drug delivery system. Yamada, Y., et al 3 Keywords:Mitochondrial drug delivery; Mitochondrial protein therapy; Mitochondrial gene therapy; Transferrin and GALA system; multifunctional envelope-type nano device (MEND);Regulation of intramitochondrial trafficking. Yamada, Y., et al 4

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Mitochondria Isolated from Liver Contain the Essential Factors Required for RNA/DNA Oligonucleotide- Targeted Gene Repair

Cited by 37 publications

References 30 publications

Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter

Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter

Modification of hepatic genomic DNA using RNA/DNA oligonucleotides

Mitochondrial drug delivery and mitochondrial disease therapy – An approach to liposome-based delivery targeted to mitochondria

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