AAV vector-mediated in vivo reprogramming into pluripotency

Senís, Elena; Mosteiro, Lluc; Wilkening, Stefan; Wiedtke, Ellen; Nowrouzi, Ali; Afzal, Saira; Fronza, Raffaele; Landerer, Henrik; Abad, María; Niopek, Dominik; Schmidt, Manfred; Serrano, Manuel; Grimm, Dirk

doi:10.1038/s41467-018-05059-x

Cited by 52 publications

(35 citation statements)

References 80 publications

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“…[12] However, for the safe clinical use of this vector it will be important to confirm that the high prevalence of random AAV integration previously reported for AAV delivery of OSKM factors is not also observed with this approach. [21] Moreover, the limited capacity of AAV vectors prevents delivery of the reprogramming factors and the tetracycline transactivator protein within a single polycistronic vector. [12] Indeed, adenoviral vectors offer a significant advantage given their limited integration, increased transgene capacity and transient expression without the need for additional pharmacological control.…”

Section: Discussionmentioning

confidence: 99%

“…Investigations that have utilized exogenous gene delivery for in vivo reprogramming have been limited by either the efficiency of the delivery vehicle [4,8] or the integrating nature of vectors leading to sustained reprogramming and teratoma formation. [20,21] It is therefore essential that alternative gene delivery vectors are explored for potential in vivo applications of transient and partial reprogramming. Despite the known efficiency and transiency of adenoviral mediated gene delivery, the use of this vector for in vivo reprogramming toward pluripotency has not been previously reported.…”

Section: Introductionmentioning

confidence: 99%

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Adenoviral Mediated Delivery of OSKM Factors Induces Partial Reprogramming of Mouse Cardiac Cells In Vivo

Kisby

Lázaro

Fisch

et al. 2020

Advanced Therapeutics

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The induction of in vivo reprogramming toward pluripotency has been demonstrated in several tissues utilizing either transgenic inducible mice or gene delivery approaches. However, the effects of exogenous reprogramming factor expression in the mammalian heart have not been previously reported. The present study aims to investigate the response of cardiac cells to ectopic Oct3/4, Sox2, Klf4, and cMyc (OSKM) expression in vivo using a non-integrating adenoviral vector. Direct intramyocardial injection of this vector achieves effective and transient OSKM overexpression in the healthy heart and after myocardial infarction. The expression of these factors induces transient upregulation of a number of endogenous pluripotency (endo-Oct3/4, Gdf3) and reprogramming related (Cdh1, Fut4) genes, confirming the induction of cell reprogramming. Despite the initiation of reprogramming, markers of fully de-differentiated cells including Nanog remain silenced, consistent with a partially reprogrammed state. Furthermore, no indications of tumorigenesis or teratoma formation are observed. Overall, these data suggest that adenoviral mediated OSKM delivery can be utilized to induce partial in vivo reprogramming. However, the absence of any clear regenerative effects after myocardial infarction indicates that further optimization of vector mediated reprogramming strategies is essential to overcome barriers to therapeutic efficacy.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Adenoviral Mediated Delivery of OSKM Factors Induces Partial Reprogramming of Mouse Cardiac Cells In Vivo

Kisby

Lázaro

Fisch

et al. 2020

Advanced Therapeutics

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“…AAV vectors were titrated using quantitative real-time PCR (qRT-PCR) as described in 75 . For the CAG_Cre::GFP construct, the primers and probe GFP_fwd, GFP_rev and GFP_probe were used, while Cre_fwd, Cre_rev and Cre_probe were used for the CMV_Cre construct (Table S1).…”

Section: Methodsmentioning

confidence: 99%

In vivohigh-throughput screening of novel adeno-associated viral capsids targeting adult neural stem cells in the subventricular zone

Dehler

Cerrizuela

et al. 2021

Preprint

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The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 177 intraventricularly injected barcoded AAV variants profiled by RNA sequencing. Quantification of barcoded AAV mRNAs identified two synthetic capsids, AAV9_A2 and AAV1_P5, both of which transduce active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of promoter and dose of injected viral genomes enabled labeling of 30-60% of the NSC compartment, which was validated by FACS analyses and single cell RNA sequencing. Importantly, transduced NSC readily produced neurons. The present study identifies AAV variants with a high regional tropism towards the v-SVZ with high efficiency in targeting adult NSCs, thereby paving the way for preclinical testing of regenerative gene therapy.

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“…Surprisingly, continuous induction of OSK for over a year had no discernable negative effect on the mice ( Fig.1b and Extended Data Fig. 2b) 20 , ostensibly because we avoided high-level expression in the intestine (Extended Data Fig.2c-e), thus avoiding the dysplasia and weight loss seen in other studies 15 .…”

mentioning

confidence: 88%

“…Next, we tested if a similar restoration was possible in mice. To deliver and control OSK expression in vivo, we engineered a tightly regulated adeno-associated viral (AAV) vector under the control of tetracycline response element (TRE) promoter ( Fig.1a) 19,20 . The TRE promoter can be activated either by rtTA in the presence of DOX (the "Tet-ON" system) or by tTA in the absence of DOX ("Tet-OFF").…”

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confidence: 99%

Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming

Krishnan

Brommer

et al. 2019

Preprint

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2Ageing is a degenerative process leading to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise, which disrupts youthful gene expression patterns that are required for cells to function optimally and recover from damage 1-3 .Changes to DNA methylation patterns over time form the basis of an 'ageing clock' 4,5 , but whether old individuals retain information to reset the clock and, if so, whether this would improve tissue function is not known. Of all the tissues in the body, the central nervous system (CNS) is one of the first to lose regenerative capacity 6,7 . Using the eye as a model tissue, we show that expression of Oct4, Sox2, and Klf4 genes (OSK) in mice resets youthful gene expression patterns and the DNA methylation age of retinal ganglion cells, promotes axon regeneration after optic nerve crush injury, and restores vision in a mouse model of glaucoma and in normal old mice. This process, which we call recovery of information via epigenetic reprogramming or REVIVER, requires the DNA demethylases Tet1 and Tet2, indicating that DNA methylation patterns don't just indicate age, they participate in ageing. Thus, old tissues retain a faithful record of youthful epigenetic information that can be accessed for functional age reversal.The metaphor of the epigenetic landscape, first invoked by Waddington to explain embryonic development 8,9 , is increasingly seen as relevant to the other end of life 9. Evidence from yeast and mammals supports an Information Theory of Ageing, in which the loss of epigenetic information disrupts youthful gene expression patterns 1-3 , leading to cellular dysfunction and senescence. 10In mammals, progressive DNA methylation changes serve as an epigenetic clock, but whether they are merely an effect or a driver of ageing is not known 4,5 . In cell culture, the ectopic expression of the four Yamanaka transcription factors, namely Oct4, Sox2, Klf4, and c-Myc (OSKM) 11 , can reprogram somatic cells to become pluripotent stem cells, a process that erases most DNA methylation marks and leads to the loss of cellular identity 4,12 . In vivo, ectopic, transgene-mediated expression of these four genes alleviates progeroid symptoms in a mouse model of Hutchison-Guilford Syndrome, indicating that OSKM might counteract normal ageing 13 . Continual expression of all four factors, however, induces teratomas 14 or causes death within days 13 , ostensibly due to tissue dysplasia 15 .Ageing is generally considered a unidirectional process akin an increase in entropy, butliving systems are open, not closed, and in some cases can fully reset biological age, examples 3 being "immortal" cnidarians and the cloning of animals by nuclear transfer 16 . Having previously found evidence for epigenetic noise as an underlying cause of ageing 2,3 , we wondered whether mammalian cells might retain a faithful copy of epigenetic information from earlier in life, analogous to Shannon's "observer" system in Information Theory, essentially a back-up copy of the original si...

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AAV vector-mediated in vivo reprogramming into pluripotency

Cited by 52 publications

References 80 publications

Adenoviral Mediated Delivery of OSKM Factors Induces Partial Reprogramming of Mouse Cardiac Cells In Vivo

Adenoviral Mediated Delivery of OSKM Factors Induces Partial Reprogramming of Mouse Cardiac Cells In Vivo

In vivohigh-throughput screening of novel adeno-associated viral capsids targeting adult neural stem cells in the subventricular zone

Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming

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