Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics.

PURPOSE: To systematically evaluate human rod opsin (hRHO) mRNA for potential target sites sensitive to posttranscriptional gene silencing (PTGS) by hammerhead ribozyme (hhRz) or RNA interference (RNAi) in human cells. To develop a comprehensive strategy to identify and optimize lead candidate agents for PTGS gene therapeutics. METHODS: In multidisciplinary RNA drug discovery, computational mRNA accessibility and in vitro experimental methods using reverse transcription-polymerase chain reaction (RT-PCR) were used to map accessibility in full-length hRHO transcripts. HhRzs targeted predicted accessible and inaccessible sites and were screened for cellular knockdown using a bicistronic reporter construct. Lead hhRz and RNAi PTGS agents were rationally optimized for target knockdown in human cells. RESULTS: Systematic screening of hRHO mRNA targeting agents resulted in lead candidate identification of a novel hhRz embedded in an RNA scaffold. Rational optimization strategies identified a minimal 725 hhRz as the most active agent. Recently identified tertiary accessory elements did not enhance activity. A 725-short-hairpin RNA (shRNA) agent exerts log-order knockdown. Silent modulation of the 725-hhRz target site in hRHO mRNA resulted in resistance to knockdown. CONCLUSIONS: Combining rational RNA drug design with cell-based screening allowed rapid identification of lead agents targeting hRHO. Optimization strategies identified the agent with highest intracellular activity. These agents have therapeutic potential in a mutation-independent strategy for adRP, or other degenerations where hRHO is a target. This approach can be broadly applied to any validated target mRNA, regardless of the disease. TRANSLATIONAL RELEVANCE: This work establishes a platform approach to develop RNA biologicals for the treatment of human disease.

A Discovery with Potential to Revitalize Hammerhead Ribozyme Therapeutics for Treatment of Inherited Retinal Degenerations.

Hammerhead ribozymes (hhRzs), RNA enzymes capable of site-specific cleavage of arbitrary target mRNAs, have faced significant hurdles in development and optimization as gene therapeutics for clinical translation. Chemical and biological barriers must be overcome to realize an effective therapeutic. A new Facilitated ribozyme has been identified with greatly enhanced kinetic properties that lead new insight on the capacity of ribozymes to target mutant genes to treat inherited retinal degenerations.

Insulin secretagogue use and circulating inflammatory C-C chemokine levels in breast cancer patients.

Monocytes' infiltration into the tumor tissue and their activation to tumor-associated macrophages is an essential step in tumor development, also playing a critical role in an eventual metastasis. Stimulation of endogenous insulin production by oral insulin secretagogue treatment has the potential to interfere with the production and release of C-C chemokines, a group of potent inflammatory cytokines acting as monocyte chemo-attractants and influencing their behavior in the tumor microenvironment. Studied plasma samples were collected under a previously reported study design involving a population of women diagnosed with breast cancer presenting with or without type 2 diabetes mellitus at the time of breast cancer diagnosis (Wintrob et al., 2017, 2016) [1,2]. The data presented here shows the relationship between pre-existing use of insulin secretagogue, the inflammatory C-C chemokine profiles at the time of breast cancer diagnosis, and subsequent cancer outcomes. A Pearson correlation analysis stratified by secretagogue use and controls was implemented to evaluate the relationship between the investigated biomarkers and respectively each of these biomarkers and the other relevant reported cytokine datasets derived from the same patient population (Wintrob et al., 2017, 2016) [1,2].

Data report on inflammatory C-C chemokines among insulin-using women with diabetes mellitus and breast cancer.

Injectable insulin use may interfere with pro-inflammatory cytokines' production and, thus, play a role in the activation of tumor-associated macrophages - a process mainly influenced by inflammatory C-C chemokines. The data presented shows the relationship between pre-existing use of injectable insulin in women diagnosed with breast cancer and type 2 diabetes mellitus, the inflammatory C-C chemokine profiles at the time of breast cancer diagnosis, and subsequent cancer outcomes. A Pearson correlation analysis stratified by insulin use and controls is also provided. We present the observed relationship between the investigated C-C chemokines and between each of these biomarkers and previously reported adipokines levels in this study population [1].

Circulating adipokines data associated with insulin secretagogue use in breast cancer patients.

Oral drugs stimulating endogenous insulin production (insulin secretagogues) may have detrimental effects on breast cancer outcomes. The data presented shows the relationship between pre-existing insulin secretagogues use, adipokine profiles at the time of breast cancer (BC) diagnosis and subsequent cancer outcomes in women diagnosed with BC and type 2 diabetes mellitus (T2DM). The Pearson correlation analysis evaluating the relationship between adipokines stratified by T2DM pharmacotherapy and controls is also provided. This information is the extension of the data presented and discussed in "Insulin use, adipokine profiles and breast cancer prognosis" (Wintrob et al., in press) [1].

Insulin use, adipokine profiles and breast cancer prognosis.

BACKGROUND: Type-2 diabetes mellitus (T2DM) and breast cancer (BC) share common cytokine signaling changes resultant from adipose tissue dysfunction. This modified adipokine signaling was shown to be directly associated with changes in the body mass index (BMI) and diet and it is expected to also be influenced by T2DM pharmacotherapy. We evaluated the relationship between pre-existing diabetes treatment, circulating adipokine levels at cancer diagnosis, and long-term outcomes. METHODS: All incident BC cases were reviewed (01/01/2003-12/31/2009, N=2194). Each of the subjects with baseline T2DM (cases) was matched with two other subjects without T2DM (controls) based on the following criteria: age, BMI, ethnicity, menopausal status and tumor stage. All cases and controls with available baseline plasma and tumor biopsies, and being surgery and BC treatment naïve, were included (N1=97, N2=194). Clinical history and vital status were documented. Adipokine levels (adiponectin, leptin, TNF-α, CRP, IL-1ß, IL-1Ra, IL-6, and C-peptide) were assessed by either ELISA or Luminex® assays. Cancer outcomes were assessed by Kaplan-Meier analysis; associations between categorical variables were assessed by Fisher's exact test, categorical and continuous variables by Kruskal-Wallis or Wilcoxon Rank-Sum test, where appropriate. Multivariate adjustments (MVP, multivariate p-value) were performed accounting for age, tumor stage, BMI, estrogen receptor (ER) status and cumulative comorbidity. All biomarker correlations were assessed by the Pearson method. Utilization of insulin and insulin secretagogues was associated with ER (-) phenotype (p=0.008, p=0.043) and poorer BC outcomes (p=0.012, p=0.033). Insulin users were found to have lower C-peptide and higher IL-6, TNF-α and CRP levels, of which elevated CRP and TNF-α were associated with poorer BC outcomes (p=0.003, MVP=0.210). Insulin remarked by higher leptin levels as compared to controls (p=0.052), but did not differ significantly from non-users. Although lower adiponectin levels were observed among non-insulin users as compared to controls (p<0.001, MVP=0.006), insulin use seemed to have restored adiponectin production. C-peptide levels were lower among insulin users as compared to non-users (p<0.001, MVP<0.001) and approached levels comparable with those of the controls. In the overall dataset, C-peptide lower than 0.75ng/ml were strongly associated with poorer survival (p=0.007, MVP=0.002). Among insulin users, C-peptide levels were inversely correlated with IL-1ß and IL-1Ra levels only after full adjustment (p=0.012, p=0.030); the correlation was unremarkable in other groups. CONCLUSION: Insulin use is associated with elevated leptin, CRP, TNFα, and lower C-peptide and also linked to poor BC outcomes. More research is needed to verify these findings; however, we are among the first to correlate pharmacotherapy use, measures of adipose tissue dysfunction and cancer outcomes.