Intravenous Injections in Neonatal Mice

Lampe, Sara E. Gombash; Kaspar, Brian K.; Foust, Kevin D.

doi:10.3791/52037

Cited by 48 publications

(31 citation statements)

References 22 publications

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“…The newborn (P1) of Ai14 mice were used for IV AAV8 or AAV9 injection following a previous report 40 . The AAV mixtures (without Cas9, AAV-Ai14-HITI (5 × 10 10 or 2 × 10 11 GC); with Cas9, AAV-Cas9 (5 × 10 10 or 2 × 10 11 GC) and AAV-Ai14-HITI (5 × 10 10 or 2 × 10 11 GC); Scramble control, AAV-Cas9 (5 × 10 10 GC) and AAV-Ai14-Scramble (5 × 10 10 GC); HDR, AAV-Cas9 (5 × 10 10 GC) and AAV-Ai14-HDR (5 × 10 10 GC)) were injected via temporal vein of the P1 mouse.…”

Section: Methodsmentioning

confidence: 99%

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

Suzuki

Tsunekawa²,

Hernández-Benítez

et al. 2016

Nature

984

971

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Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient1, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders2. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3,4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

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Section: Methodsmentioning

confidence: 99%

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

Suzuki

Tsunekawa²,

Hernández-Benítez

et al. 2016

Nature

984

971

View full text Add to dashboard Cite

show abstract

“…Newborn (P0.5) mice were used for IV injection as described (Gombash Lampe et al, 2014). The AAV mixtures (AAV9-dgRNA (1 × 10 11 GC); AAV9-tLuc reporter (1 × 10 10 GC)) were injected via temporal vein of the P0.5 mouse.…”

Section: Methods Detailsmentioning

confidence: 99%

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation

Liao

Hatanaka

Araoka

et al. 2017

Cell

366

264

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SUMMARY Current genome-editing systems generally rely on the creation of DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. The CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat several mouse models of human diseases. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to observable phenotypic changes, and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases.

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“…Intravaenous injections of scAAV9-CB-GFP or scAAV8-CB-GFP in neonatal (P1) and juvenile (P21) mice were completed as previously described (Foust et al, 2009 ; Gombash Lampe et al, in press )…”

Section: Methodsmentioning

confidence: 99%

Intravenous AAV9 efficiently transduces myenteric neurons in neonate and juvenile mice

Gombash

Cowley

Fitzgerald

et al. 2014

Front. Mol. Neurosci.

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Gene therapies for neurological diseases with autonomic or gastrointestinal involvement may require global gene expression. Gastrointestinal complications are often associated with Parkinson's disease and autism. Lewy bodies, a pathological hallmark of Parkinson's brains, are routinely identified in the neurons of the enteric nervous system (ENS) following colon biopsies from patients. The ENS is the intrinsic nervous system of the gut, and is responsible for coordinating the secretory and motor functions of the gastrointestinal tract. ENS dysfunction can cause severe patient discomfort, malnourishment, or even death as in intestinal pseudo-obstruction (Ogilvie syndrome). Importantly, ENS transduction following systemic vector administration has not been thoroughly evaluated. Here we show that systemic injection of AAV9 into neonate or juvenile mice results in transduction of 25–57% of ENS myenteric neurons. Transgene expression was prominent in choline acetyltransferase positive cells, but not within vasoactive intestinal peptide or neuronal nitric oxide synthase cells, suggesting a bias for cells involved in excitatory signaling. AAV9 transduction in enteric glia is very low compared to CNS astrocytes. Enteric glial transduction was enhanced by using a glial specific promoter. Furthermore, we show that AAV8 results in comparable transduction in neonatal mice to AAV9 though AAV1, 5, and 6 are less efficient. These data demonstrate that systemic AAV9 has high affinity for peripheral neural tissue and is useful for future therapeutic development and basic studies of the ENS.

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Intravenous Injections in Neonatal Mice

Cited by 48 publications

References 22 publications

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation

Intravenous AAV9 efficiently transduces myenteric neurons in neonate and juvenile mice

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