Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability.

Despite recent advances in nucleic acid delivery systems with the success of LNP vehicles, adeno-associated virus (AAV) remains the leading platform for targeted gene delivery due to its low immunogenicity to humans, high transduction efficiency, and range of serotypes with varying tropisms. Depending on the therapeutic goals and serotype used, different production conditions may be more amenable, generating an ever-growing need for rapid yet robust analytical techniques to support the high-quality manufacturing of AAV. A critical bottleneck exists for assessing full capsids where rapid, high-throughput techniques capable of analyzing a range of serotypes are needed. Here, we present a rapid, high-throughput analytical technique, microfluidic electrophoresis, for the assessment of full capsids compatible with AAV1, AAV2, AAV6, AAV8, and AAV9 without the need for assay modifications or optimizations, and AAV5 with some constraints. The method presented in this study uses a mathematical formulation we developed previously with a reference standard to combine the independently obtained capsid protein and single-stranded DNA (ssDNA) profiles to estimate the percentage of full capsids in a sample of unknown concentration. We assessed the ability to use a single serotype (AAV8) as the reference standard regardless of the serotype of the sample being analyzed so long as the melting temperature (Tm) of the capsids is within 12 °C from the Tm of AAV8. Using this method, we are able to characterize samples ±6.1% with an average analytical turnaround time of <5 min/sample, using only 10 µL/sample at a concentration of 2.5 × 1012 VG/mL.

Validation of prostate cancer risk variants rs10993994 and rs7098889 by CRISPR/Cas9 mediated genome editing.

GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the ß-microseminoprotein (MSMB) promoter region, mediates MSMB prostate secretion levels, and is linked to mRNA expression changes in both MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in positive linkage disequilibrium with rs10993994 and associated with MSMB expression independent of rs10993994. Here, we generate a series of clones with single alleles removed by double guide RNA (gRNA) mediated CRISPR/Cas9 deletions, through which we demonstrate that each of these SNPs independently and greatly alters MSMB expression in an allele-specific manner. We further show that these SNPs have no substantial effect on the expression of NCOA4. These data demonstrate that a single SNP can have a large effect on gene expression and illustrate the importance of functional validation studies to deconvolute observed correlations. The method we have developed is generally applicable to test any SNP for which a relevant heterozygous cell line is available. AUTHOR SUMMARY: In pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in the human genome as genetic markers to perform large scale genome wide association studies (GWAS) and have so far identified more than a hundred prostate cancer risk variants. Such variants provide an unbiased and systematic new venue to study the disease mechanism, and the next big challenge is to translate these genetic associations to the causal role of altered gene function in oncogenesis. The majority of these variants are waiting to be studied and lots of them may act in oncogenesis through gene expression regulation. To prove the concept, we took rs10993994 and its linked rs7098889 as an example and engineered single cell clones by allelic-specific CRISPR/Cas9 deletion to separate the effect of each allele. We observed that a single nucleotide difference would lead to surprisingly high level of MSMB gene expression change in a gene specific and cell-type specific manner. Our study strongly supports the notion that differential level of gene expression caused by risk variants and their associated genetic locus play a major role in oncogenesis and also highlights the importance of studying the function of MSMB encoded ß-MSP in prostate cancer pathogenesis.

Androgen Deprivation Therapy Potentiates the Efficacy of Vascular Targeted Photodynamic Therapy of Prostate Cancer Xenografts.

Purpose: WST11 vascular targeted photodynamic therapy (VTP) is a local ablation approach relying upon rapid, free radical-mediated destruction of tumor vasculature. A phase III trial showed that VTP significantly reduced disease progression when compared with active surveillance in patients with low-risk prostate cancer. The aim of this study was to identify a druggable pathway that could be combined with VTP to improve its efficacy and applicability to higher risk prostate cancer tumors.Experimental Design: Transcriptome analysis of VTP-treated tumors (LNCaP-AR xenografts) was used to identify a candidate pathway for combination therapy. The efficacy of the combination therapy was assessed in mice bearing LNCaP-AR or VCaP tumors.Results: Gene set enrichment analysis identifies the enrichment of androgen-responsive gene sets within hours after VTP treatment, suggesting that the androgen receptor (AR) may be a viable target in combination with VTP. We tested this hypothesis in mice bearing LNCaP-AR xenograft tumors by using androgen deprivation therapy (ADT), degarelix, in combination with VTP. Compared with either ADT or VTP alone, a single dose of degarelix in concert with VTP significantly inhibited tumor growth. A sharp decline in serum prostate-specific antigen (PSA) confirmed AR inhibition in this group. Tumors treated by VTP and degarelix displayed intense terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining 7 days after treatment, supporting an increased apoptotic frequency underlying the effect on tumor inhibition.Conclusions: Improvement of local tumor control following androgen deprivation combined with VTP provides the rationale and preliminary protocol parameters for clinical trials in patients presented with locally advanced prostate cancer. Clin Cancer Res; 24(10); 2408-16. ©2018 AACR.

Suppression of polyamine catabolism by activated Ki-ras in human colon cancer cells.

An activated Ki-ras was expressed in the human colon adenocarcinoma cell line Caco-2 to study the effects of Ki-ras oncogene on polyamine metabolism during gastrointestinal tumorigenesis. Multiple clones selected for expression of the mutant Ki-ras transgene displayed a suppression of transcription of a key catabolic enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT). Gene expression analysis, with cDNA microarrays, showed that Ki-ras transfected clones had decreased levels of expression, compared to mock transfected cells, of peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor family and an important regulator of cell proliferation and differentiation. The activated Ki-ras suppressed SSAT expression by a mechanism involving the PPARgamma response element (PPRE) located at +48 bp relative to the transcription start site of the SSAT gene. Transient expression of the PPARgamma protein in Ki-ras expressing Caco-2 clones, or treatment with the PPARgamma ligand ciglitazone, led to an increase in the SSAT promoter activity. A MEK1/2 inhibitor PD98059 induced transcription of both PPARgamma and SSAT genes in the activated Ki-ras clones, suggesting that the mitogen-activated protein kinases (MAPKs) were involved in the regulation of SSAT expression by PPARgamma. We concluded that mutated Ki-ras suppressed SSAT via a transcriptional mechanism involving the PPARgamma signaling pathway.