TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice

Toro-Montoya, Ana Isabel; Grunebaum, Eyal

doi:10.1172/jci25052

Cited by 67 publications

(51 citation statements)

References 56 publications

(78 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The most compelling evidence was the sustained near-normal levels of dystrophin expression in skeletal muscles and enhanced dystrophin expression in cardiac muscle, with improved muscle pathology and function after 3 months of treatment. This is consistent with previous reports showing lack of immunogenicity with similar peptides in animal models (26,27). The use of unnatural amino acids in the peptide sequence of the PPMO most likely contributes to the lack of immunogenicity.…”

Section: Discussionsupporting

confidence: 92%

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Moulton

Iversen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

219

257

View full text Add to dashboard Cite

Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cellpenetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.M utations in the dystrophin gene underlie two forms of muscular dystrophy: Duchenne and Becker muscular dystrophy (DMD and BMD). DMD is caused mainly by nonsense and frame-shift mutations with little or no production of functional dystrophin protein, leading to disease onset in early childhood with lethal consequences. BMD is caused by mutations that typically create shortened but in-frame transcripts with production of partially functional dystrophin, leading to variable and often overt symptoms (1-3). Most DMD mutations occur within the rod domain, which spans more than half the length of the protein, but seems to have limited functional importance (4, 5). Antisense therapy uses specific oligomers to remove the mutated or additional exon(s) that disrupt the reading frame, thus restoring the expression of shortened forms of dystrophin protein retaining critical functions (6-11).We previously demonstrated that i.m. delivery of a specific 2Ј-O-methyl phosphorothioate antisense oligonucleotide (2ЈOMeAON) was able to skip targeted dystrophin exon 23 in mdx mouse, a model of DMD (9). This mouse carries a nonsense point mutation within exon 23 and lacks dystrophin expression (except in a few rare revertant fibers) in all muscles, including the heart (12, 13). Skipping the mutated exon 23 restored both the reading frame and dystrophin expression, with functional improvement of the treated muscles (14) [supporting information (SI) Fig. S1a]. Recently we showed that a phosphorodiamidate morpholino oligomer (PMO), E23ϩ7-18 targeting the junction of exon 23 and intron 23 of mouse dystrophin (referred to as PMOE23 hereafter), was able to induce up to functional levels of dystrophin expression in some skeletal muscles by regular i.v. injections in mdx mice (15). However, dystrophin expression induced by both 2ЈOMeAON and PMO required high doses and was highly variable between muscles and myofibers in terms of observed efficacy. Of greater conc...

show abstract

Section: Discussionsupporting

confidence: 92%

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Moulton

Iversen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

219

257

View full text Add to dashboard Cite

show abstract

“…Several protein transduction domains have been tested, but derivatives of the transactivator of transcription (TAT) domain of HIV have proven to be the most effective. The delivery of a variety of proteins into all tissues in mice, including passage across the blood-brain barrier, and the safety of the TAT domain have been demonstrated in detail (10,11).…”

mentioning

confidence: 99%

Effective Blockage of Both the Extrinsic and Intrinsic Pathways of Apoptosis in Mice by TAT-crmA

Krautwald

Ziegler²,

Rölver³

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Evidence accumulates that in clinically relevant cell death, both the intrinsic and extrinsic apoptotic pathway synergistically contribute to organ failure. In search for an inhibitor of apoptosis that provides effective blockage of these pathways, we analyzed viral proteins that evolved to protect the infected host cells. In particular, the cowpox virus protein crmA has been demonstrated to be capable of blocking key caspases of both pro-apoptotic pathways. To deliver crmA into eukaryotic cells, we fused the TAT protein transduction domain of HIV to the N terminus of crmA. In vitro, the TAT-crmA fusion protein was efficiently translocated into target cells and inhibited apoptosis mediated through caspase-8, caspase-9, and caspase-3 after stimulation with ␣-Fas, etoposide, doxorubicin, or staurosporine. The extrinsic apoptotic pathway was investigated following ␣-Fas stimulation. In vivo 90% of TAT-crmA-treated animals survived an otherwise lethal dose of ␣-Fas and showed protection from Fas-induced organ failure. To examine the intrinsic apoptotic pathway, we investigated the survival of mice treated with an otherwise lethal dose of doxorubicin. Whereas all control mice died within 31 days, 40% of mice that concomitantly received intraperitoneal injections of TAT-crmA survived. To test the ability to comprehensively block both the intrinsic and extrinsic apoptotic pathway in a clinically relevant setting, we employed a murine cardiac ischemia-reperfusion model. TAT-crmA reduced infarction size by 40% and preserved left ventricular function. In summary, these results provide a proof of principle for the inhibition of apoptosis with TAT-crmA, which might provide a new treatment option for ischemia-reperfusion injuries.Apoptotic processes are centrally involved during organogenesis in complex organisms but may also cause organ failure in response to a variety of harmful stimuli. Certain infectious diseases, intoxications, or fulminant immune responses may trigger an overwhelming apoptotic response (1). Moreover, minimizing apoptotic organ failure due to ischemia-reperfusion injuries that occur during stroke or myocardial infarction remains a major challenge in clinical settings. Each year, ϳ1.5 million myocardial infarction cases are recorded in the United States. Myocardial infarction is the leading cause of death in both Europe and the United States. Therefore, transient blockage of apoptosis is of clinical importance to protect cardiac function and to save lives.Apoptosis is controlled by the extrinsic death receptor pathway and the intrinsic mitochondrial pathway. Notably, both pathways converge at caspase-3, leading to activation of other proteases. Caspases have more than 400 different substrates that interfere with transcription, translation, DNA cleavage, cytoskeleton assembly, and membrane trafficking. To prevent this "death by a thousand cuts," it is important to block caspases in both the intrinsic and extrinsic pathways as well as caspases involved in the execution process of apoptosis (2). The extrins...

show abstract

“…The authors detected the same maximal levels of specific antibodies against the human PNP, either given as TAT-PNP or as nonfused PNP. These findings suggest that the immune response observed in TAT-PNP-treated mice was not against TAT itself, but rather against the xenogeneic human PNP protein (33). Thus, TAT itself did not increase the immunogenicity of the human protein.…”

Section: Discussionmentioning

confidence: 83%

“…We believe that the rapid intracellular transduction of TAT fusion proteins would protect it from exposure to the immune system. This issue was addressed in an important in vivo study, where a human purine nucleoside phosphorylase (PNP) was given to PNP knockout mice (PNP -/-) in the form of TAT-PNP (33). The immuno-response was measured by the presence of specific IgG antibodies after 2-3 wks of treatment with TAT-PNP compared with treatment with the nonfused PNP.…”

Section: Discussionmentioning

confidence: 99%

Replacement of the C6ORF66 Assembly Factor (NDUFAF4) Restores Complex I Activity in Patient Cells

Marcus¹,

Lichtenstein²,

Saada

et al. 2013

Mol Med

View full text Add to dashboard Cite

Disorders of the oxidative phosphorylation (OXPHOS) system frequently result in a severe multisystem disease with the consequence of early childhood death. Among these disorders, isolated complex I deficiency is the most frequently diagnosed, accounting for one-third of all cases of respiratory chain deficiency. We chose to focus on complex I deficiency, caused by mutation in the assembly factor chromosome 6, open reading frame 66 (C6ORF66; NADH dehydrogenase [ubiquinone] complex I assembly factor 4 [NDUFAF4]) protein. We used the approach of cell-and organelle-directed protein/enzyme replacement therapy, with the transactivator of transcription (TAT) peptide as the moiety delivery system. This step will enable us to deliver the wild-type assembly factor C6ORF66 into patient cells and their mitochondria, leading to the proper assembly and function of complex I and, as a result, to a functional OXPHOS system. We designed and constructed the TAT-ORF fusion protein by gene fusion techniques, expressed the protein in an Escherichia coli expression system and highly purified it. Our results indicate that TAT-ORF enters patients' cells and their mitochondria rapidly and efficiently. TAT-ORF is biologically active and led to an increase in complex I activity. TAT-ORF also increased the number of patient cells and improved the activity of their mitochondria. Moreover, we observed an increase in ATP production, a decrease in the content of mitochondria and a decrease in the level of reactive oxygen species. Our results suggest that this approach of protein replacement therapy for the treatment of mitochondrial disorders is a promising one.

show abstract

TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice

Cited by 67 publications

References 56 publications

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Effective Blockage of Both the Extrinsic and Intrinsic Pathways of Apoptosis in Mice by TAT-crmA

Replacement of the C6ORF66 Assembly Factor (NDUFAF4) Restores Complex I Activity in Patient Cells

Contact Info

Product

Resources

About