Long-Term Assessment of AAV-Mediated Zinc Finger Nuclease Expression in the Mouse Brain

Zahur, Muzna; Tolö, Johan; Bähr, Mathias; Kügler, Sebastian

doi:10.3389/fnmol.2017.00142

Cited by 9 publications

(7 citation statements)

References 41 publications

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“…Of note, these drug effects examined in the polyneuropathy and carrageenan model appeared to persist unchanged for at least 6-7 weeks. Long term expression has been similarly noted in other gene therapy studies 57,[93][94][95] . Importantly, these effects were unaccompanied by any detectable adverse motor effects or changes in bladder function.…”

Section: In Vivo Spinal Na V 17 Knock Downsupporting

confidence: 61%

Long-lasting Analgesia via Targetedin vivoEpigenetic Repression of Nav1.7

Moreno

Catroli

Alemán

et al. 2019

Preprint

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One sentence summary:In situ epigenome engineering approach for genomically scarless, durable, and non-addictive management of pain. ABSTRACTCurrent treatments for chronic pain rely largely on opioids despite their unwanted side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing, with the voltage-gated sodium channel, Na V 1.7 (SCN9A), being perhaps the most promising candidate for analgesic drug development.Specifically, a hereditary loss-of-function mutation in Na V 1.7 leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence similarity between Na V subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of Na V 1.7 via genome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins as a potential treatment for chronic pain.Towards this end, we first optimized the efficiency of Na V 1.7 repression in vitro in Neuro2A cells, and then by the lumbar intrathecal route delivered both genomeengineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain and BzATP-induced pain. Our results demonstrate: one, effective repression of Na V 1.7 in lumbar dorsal root ganglia; two, reduced thermal hyperalgesia in the inflammatory state; three, decreased tactile allodynia in the neuropathic state; and four, no changes in normal motor function. We anticipate this genomically scarless and non-addictive pain amelioration approach enabling Long-lasting Analgesia via Targeted in vivo Epigenetic Repression of Nav1.7, a methodology we dub pain LATER, will have significant therapeutic potential, such as for preemptive administration in 2 anticipation of a pain stimulus (pre-operatively), or during an established chronic pain state.

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Section: In Vivo Spinal Na V 17 Knock Downsupporting

confidence: 61%

Long-lasting Analgesia via Targetedin vivoEpigenetic Repression of Nav1.7

Moreno

Catroli

Alemán

et al. 2019

Preprint

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“…CT-01 hiPSC-derived neurons were transduced with the EGFP-based genetically encoded calcium indicator (GECI) GCaMP3.5 as previously reported 31 and Ca 2+ live imaging was performed at DIV 25 using a Zeiss AxioVert observer Z1 microscope equipped with an AxioCam 503m_S815 camera and imaged using Zen pro v2.3.64 software. The calcium influx was measured using hand drawn ROIs around whole cells.…”

Section: Methodsmentioning

confidence: 99%

“…Lab-made ECL was used and protein detection was performed with the help of X-ray detection system. Equal loading was verified using MemCode (Thermo Scientific; 24580) as previously described 32 .…”

Section: Methodsmentioning

confidence: 99%

Homogenous generation of dopaminergic neurons from multiple hiPSC lines by transient expression of transcription factors

et al. 2019

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A major hallmark of Parkinson's disease is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The pathophysiological mechanisms causing this relatively selective neurodegeneration are poorly understood, and thus experimental systems allowing to study dopaminergic neuron dysfunction are needed. Induced pluripotent stem cells (iPSCs) differentiated toward a dopaminergic neuronal phenotype offer a valuable source to generate human dopaminergic neurons. However, currently available protocols result in a highly variable yield of dopaminergic neurons depending on the source of hiPSCs. We have now developed a protocol based on HBA promoter-driven transient expression of transcription factors by means of adeno-associated viral (AAV) vectors, that allowed to generate very consistent numbers of dopaminergic neurons from four different human iPSC lines. We also demonstrate that AAV vectors expressing reporter genes from a neuron-specific hSyn1 promoter can serve as surrogate markers for maturation of hiPSC-derived dopaminergic neurons. Dopaminergic neurons differentiated by transcription factor expression showed aggravated neurodegeneration through α-synuclein overexpression, but were not sensitive to γ-synuclein overexpression, suggesting that these neurons are well suited to study neurodegeneration in the context of Parkinson’s disease.

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“…Importantly, the long-term expression of ZFNs in central neural system neurons induced significant gene repression activity for at least 6 months 251,252 and did not induce measurable inflammation or neurodegeneration. 253 TALENs were the second endonucleases to be evaluated in gene editing. 243,254,255 A single TALE motif recognizes one nucleotide, and an array of TALEs can associate with a longer sequence.…”

Section: Brain Gene Editingmentioning

confidence: 99%