Non-viral in vivo cytidine base editing in hepatocytes using focused ultrasound targeted microbubbles.

CRISPR-Cas9-based genome editing technologies, such as base editing, have the potential for clinical translation, but delivering nucleic acids into target cells in vivo is a major obstacle. Viral vectors are widely used but come with safety concerns, while current non-viral methods are limited by low transfection efficiency. Here we describe a new method to deliver CRISPR-Cas9 base editing vectors to the mouse liver using focused ultrasound targeted microbubble destruction (FUTMD). We demonstrate, using the example of cytosine base editing of the Pde3b gene, that FUTMD-mediated delivery of cytosine base editing vectors can introduce stop codons (up to ∼2.5% on-target editing) in mouse liver cells in vivo. However, base editing specificity is less than one might hope with these DNA constructs. Our findings suggest that FUTMD-based gene editing tools can be rapidly and transiently deployed to specific organs and sites, providing a powerful platform for the development of non-viral genome editing therapies. Non-viral delivery also reveals greater off-target base exchange in vivo than in vitro.

A Comparison of Focused and Unfocused Ultrasound for Microbubble-Mediated Gene Delivery.

We compared focused and unfocused ultrasound-targeted microbubble destruction (UTMD) for delivery of reporter plasmids to the liver and heart in mice. Optimal hepatic expression was seen with double-depth targeting at 5 and 13 mm in vivo, incorporating a low pulse repetition frequency and short pulse duration. Reporter expression was similar, but the transfection patterns were distinct, with intense foci of transfection using focused UTMD (F-UTMD). We then compared both approaches for cardiac delivery and found 10-fold stronger levels of reporter expression for F-UTMD and observed small areas of intense luciferase expression in the left ventricle. Non-linear contrast imaging of the liver before and after insonation also showed a substantially greater change in signal intensity for F-UTMD, suggesting distinct cavitation mechanisms for both approaches. Overall, similar levels of hepatic transgene expression were observed, but cardiac-directed F-UTMD was substantially more effective. Focused ultrasound presents a new frontier in UTMD-directed gene therapy.

Protein kinase C binding protein 1 inhibits hypoxia-inducible factor-1 in the heart.

AIMS: Hypoxia-inducible factor-1 alpha (HIF-1α) is a key transcription factor responsible for the induction of genes that facilitate adaptation to hypoxia. To study HIF-1 signalling in the heart, we developed a mouse model in which an oxygen-stable form of HIF-1α can be inducibly expressed in cardiac myocytes, under the regulation of tetracycline. METHODS AND RESULTS: Remarkably, expression of the transgene in mice generated two distinct phenotypes. One was the expected expression of HIF-regulated transcripts and associated changes in cardiac angiogenesis and contractility. The other was an unresponsive phenotype with much less expression of typical HIF-response genes and substantial expression of a zinc-finger protein, Protein Kinase C Binding Protein 1 (PRKCBP1). We have demonstrated that this second phenotype is due to an insertion of a fragment of DNA upstream of the PRKCBP1 gene that contains two additional canonical HIF binding sites and leads to substantial HIF binding, assessed by chromatin immunoprecipitation, and transcriptional activation. This insertion is found only in the FVB strain of mice that contributed the αMHC-tet binding protein transgene to these biallelic mice. In HEK293 cells transfected with oxygen-stable HIF-1α and PRKCBP1, we demonstrated inhibition of HIF-1 activity by a luciferase reporter assay. Using mouse primary cells and cell lines, we show that transfection with oxygen-stable HIF-1α and PRKCBP1 reduced expression of direct HIF-1 gene targets and that knockdown of PRKCBP1 removes that negative inhibition. Consistent with previous reports suggesting that PRKCBP1 modulates the chromatin landscape, we found that HL-1 cells transfected with oxygen-stable HIF-1α and PRKCBP1 have reduced global 5-methyl cytosine compared to HIF-1 alone. CONCLUSION: We show genetic, transcriptional, biochemical, and physiological evidence that PRKCBP1 inhibits HIF activity. Identification of a new oxygen-dependent and previously unsuspected regulator of HIF may provide a target for new therapeutic approaches to ischaemic heart disease.

Ultrasound directs a transposase system for durable hepatic gene delivery in mice.

Our aim was to evaluate the delivery of transposase-based vectors by ultrasound targeted microbubble destruction (UTMD) in mice. DNA vectors were attached to cationic lipid microbubbles (1-3 µm in diameter), injected intravenously and delivered to the liver by destruction of the carrier bubbles with ultrasound in burst mode at 1.0 MHz, 20-µs pulse duration, 10-Hz pulse repetition frequency and ∼1.3-MPa acoustic peak negative pressure. We evaluated the expression and genomic integration of conventional (pcDNA3) and piggyBac transposase-based (pmGENIE) reporter vectors. In vivo, we observed UTMD-mediated liver-specific expression of pmGENIE for an average of 24 d, compared with 4 d with pcDNA3. Reporter expression was located predominately near blood vessels initially, whereas expression after 3 d was more evenly distributed through the parenchyma of the liver. We confirmed random genomic integration for pmGENIE in vitro; however, integration events for pmGENIE in vivo were targeted to specific areas of chromosome 14. Our results suggest that a combination of UTMD and non-viral DNA transposase vectors can mediate weeks of hepatic-specific gene transfer in vivo, and analyses performed by non-restrictive linear amplification-mediated (nrLAM) polymerase chain reaction, cloning and sequencing identify an unexpected tropism for integration within a specific sequence on chromosome 14 in mice. UTMD delivery of transgenes may be useful for the treatment of hepatic gene deficiency disorders.

Cardiac angiogenesis directed by stable Hypoxia Inducible Factor-1.

BACKGROUND: The heterodimeric, oxygen-sensitive transcription factor Hypoxia Inducible Factor-1 (HIF-1) orchestrates angiogenesis and plays a key role in the response to ischemia and the growth of cancers. METHODS: We developed a transgenic mouse line in which expression of an oxygen-stable HIF-1α construct was controlled by a tetracycline-responsive promoter. HIF-1α expression was induced for up to 28 days in adult mouse heart, resulting in angiogenesis and progressive ventricular dysfunction. RESULTS: Gross inspection demonstrated enlarged hearts with large epicardial vessels with prominent side branches. Perfusion curves obtained by ultrasound contrast analysis demonstrated a significant increase in the myocardial red cell volume after 28 days of HIF-1α expression. Corrosion casts of cardiac vessels were made with a new low-viscosity resin that can fill the vasculature down to the level of the capillaries. Scanning electron microscopy of these casts reveal "lakes" of capillaries forming off of larger vessels after HIF expression, and support the rapid formation of mature neovascularization. Pro-angiogenic factors DLL-4, Notch-1, and PDGF-ß, were evaluated by immunohistochemistry and Western blots, and support a pattern of progressive functional neoangiogenesis. CONCLUSIONS: This study demonstrates the structural characteristics of HIF-directed angiogenesis and supports the utility of manipulation of HIF signaling to enhance perfusion and treat ischemia.

Introduction to the ultrasound targeted microbubble destruction technique.

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.