Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity.

The capsid protein purity of adeno-associated virus (AAV) is considered a critical quality attribute of AAV-based gene therapy products. However, the analytical methods currently available to monitor the viral capsid proteins, which are present in extremely low concentrations, have limited sensitivity and robustness, thus limiting their general applicability. As a result, there is an urgent need to develop robust separation methods with highly sensitive detection. In this article, we describe the first denaturation and fluorescence labeling procedure for AAV capsid proteins using the pyrylium dye Chromeo™ P503, enabling the establishment of the first capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method combined with laser-induced fluorescence (LIF) detection for AAV. Upon optimization using a quality-by-design approach, the newly developed method features a simple and robust one-step sample preparation workflow resulting in consistently labeled and denatured viral protein samples, which can subsequently be separated and quantified by CE-LIF. The method has been validated to be accurate and precise with a linear range of 50-150% of the nominal concentration of 2.0 × 1011 vector genomes per mL (vg/mL). The detection limit and quantitation limit were established to be 8.0 × 107 vg/mL (∼0.8 ng/mL) and 4.2 × 108 vg/mL (∼4 ng/mL), respectively, representing the highest sensitivity achieved for AAV capsid protein quantitation reported to date and a linear dynamic range of 8.0 × 107-3.0 × 1011 vg/mL. A comparison of the CE-SDS LIF method with existing methods, such as CE-SDS ultraviolet and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SYPRO Ruby stain, indicated that the new method has superior resolution and a significant increase in signal intensity. Capsid protein purity analysis of multiple AAV serotypes, including AAV5, scAAVrh10, AAV2, and AAV6, has been demonstrated for the first time using the same method, indicating the newly developed AAV labeling procedure and CE-LIF analysis could serve as a Quality Control-friendly platform and best-in-class analytical method for the control of AAV capsid protein purity.

RNA Splicing Factor Mutations That Cause Retinitis Pigmentosa Result in Circadian Dysregulation.

Circadian clocks regulate multiple physiological processes in the eye, but their requirement for retinal health remains unclear. We previously showed that Drosophila homologs of spliceosome proteins implicated in human retinitis pigmentosa (RP), the most common genetically inherited cause of blindness, have a role in the brain circadian clock. In this study, we report circadian phenotypes in murine models of RP. We found that mice carrying a homozygous H2309P mutation in Pre-mRNA splicing factor 8 (Prpf8) display a lengthened period of the circadian wheel-running activity rhythm. We show also that the daily cycling of circadian gene expression is dampened in the retina of Prpf8-H2309P mice. Surprisingly, molecular rhythms are intact in the eye cup, which includes the retinal pigment epithelium (RPE), even though the RPE is thought to be the primary tissue affected in this form of RP. Downregulation of Prp31, another RNA splicing factor implicated in RP, leads to period lengthening in a human cell culture model. The period of circadian bioluminescence in primary fibroblasts of human RP patients is not significantly altered. Together, these studies link a prominent retinal disorder to circadian deficits, which could contribute to disease pathology.

Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA.

Ocular gene therapy with recombinant adeno-associated virus (AAV) has shown vector-mediated gene augmentation to be safe and efficacious in the retina in one set of diseases (retinitis pigmentosa and Leber congenital amaurosis (LCA) caused by RPE65 deficiency), with excellent safety profiles to date and potential for efficacy in several additional diseases. However, size constraints imposed by the packaging capacity of the AAV genome restrict application to diseases with coding sequence lengths that are less than 5,000 nt. The most prevalent retinal diseases with monogenic inheritance are caused by mutations in genes that exceed this capacity. Here, we designed a spliceosome mediated pre-mRNA trans-splicing strategy to rescue expression of CEP290, which is associated with Leber congenital amaurosis type 10 (LCA10) and several syndromic diseases including Joubert syndrome. We used this reagent to demonstrate editing of CEP290 in cell lines in vitro and in vivo in a mini-gene mouse model. This study is the first to show broad editing of CEP290 transcripts and in vivo proof of concept for editing of CEP290 transcripts in photoreceptors and paves the way for future studies evaluating therapeutic effects.

PDLIM7 and CDH18 regulate the turnover of MDM2 during CDK4/6 inhibitor therapy-induced senescence.

CDK4/6 inhibitors are being used to treat a variety of human malignancies. In well-differentiated and dedifferentiated liposarcoma their clinical promise is associated with their ability to downregulate the MDM2 protein. The downregulation of MDM2 following treatment with CDK4/6 inhibitors also induces many cultured tumor cell lines derived from different types of malignancies to progress from quiescence into senescence. Here we used cultured human cell lines and defined a role for PDLIM7 and CDH18, regulating MDM2 protein in CDK4/6 inhibitor-treated cells. Materials from our previous phase II trials with palbociclib were then used to demonstrate that expression of CDH18 protein was associated with response, measured as both progression-free survival and overall survival. This supports the hypothesis that the biologic transition from quiescence to senescence has clinical relevance for this class of drugs.

The tumor suppressor SirT2 regulates cell cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation.

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 deposition through the deacetylation of H4K16Ac (acetylation of H4K16) and determines the levels of H4K20me2/3 throughout the cell cycle. SirT2 binds and deacetylates PR-Set7 at K90, modulating its chromatin localization. Consistently, SirT2 depletion significantly reduces PR-Set7 chromatin levels, alters the size and number of PR-Set7 foci, and decreases the overall mitotic deposition of H4K20me1. Upon stress, the interaction between SirT2 and PR-Set7 increases along with the H4K20me1 levels, suggesting a novel mitotic checkpoint mechanism. SirT2 loss in mice induces significant defects associated with defective H4K20me1-3 levels. Accordingly, SirT2-deficient animals exhibit genomic instability and chromosomal aberrations and are prone to tumorigenesis. Our studies suggest that the dynamic cross-talk between the environment and the genome during mitosis determines the fate of the subsequent cell cycle.