Precision delivery of RAS-inhibiting siRNA to KRAS driven cancer via peptide-based nanoparticles

Over 95% of pancreatic adenocarcinomas (PDACs), as well as a large fraction of other tumor types, such as colorectal adenocarcinoma, are driven by KRAS activation. However, no direct RAS inhibitors exist for cancer therapy. Furthermore, the delivery of therapeutic agents of any kind to PDAC in particular has been hindered by the extensive desmoplasia and resultant drug delivery challenges that accompanies these tumors. Small interfering RNA (siRNA) is a promising modality for anti-neoplastic therapy due to its precision and wide range of potential therapeutic targets. Unfortunately, siRNA therapy is limited by low serum half-life, vulnerability to intracellular digestion, and transient therapeutic effect. We assessed the ability of a peptide based, oligonucleotide condensing, endosomolytic nanoparticle (NP) system to deliver siRNA to KRAS-driven cancers. We show that this peptide-based NP is avidly taken up by cancer cell

A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications.

RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications

Decrease in gyrase A protein expression in _E. coli_ cells inhibited by antisense ribozymes

RNase P complexed with external guide sequence (EGS) represents a novel nucleic-acid-based gene interference approach to modulate gene expression. Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of protein expression of _E. coli_ gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of _E. coli_, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. Western blot was done to investigate the downregulation of gyrase A protein. The results showed a significant decrease of gyrase A suggesting the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins

Baseline anti-NS4a antibodies in combination with on-treatment quantitative HCV-RNA reliably identifies nonresponders to pegylated interferon-ribavirin combination therapy after 4 weeks of treatment

Background Early detection of nonresponders to hepatitis C therapy limits unnecessary exposure to treatment and its side-effects. A recent algorithm combining baseline anti-NS4a antibodies and on-treatment quantitative PCR identified nonresponders to a combination of interferon and ribavirin after 1 week of treatment. Aim To validate a stopping rule based on baseline anti-NS4a antibody levels and early on-treatment virological response in treatment-naive genotype 1 chronic hepatitis C patients treated with the current standard pegylated interferon and ribavirin combination therapy. Methods Eighty-nine genotype 1 patients from the Dynamically Individualized Treatment of hepatitis C Infection and Correlates of Viral/Host dynamics Study treated for 48 weeks with standard 180 mu g pegylated interferon (PEG-IFN)-alpha-2a (weekly) and ribavirin 1000-1200mg (daily) were analysed. Baseline anti-NS4a antibody enzyme-linked immunosorbent assay (NS4a AA 1687-1718) was performed on pretreatment serum. Hepatitis C virus-RNA was assessed at days 0, 1, 4, 7, 8, 15, 22, 29, weeks 6, 7, 8, 10, 12 and 6 weekly thereafter until end of treatment. Multiple regression logistic analysis was performed. Results Overall 54 of 89 (61%) patients achieved sustained virological response. A baseline anti-NS4a antibody titre less than 1/1250 correlated with absence of favourable initial viral decline according to variable response types (P=0.015). The optimal algorithm was developed using the combination of the absence of anti-NS4a Ab (= 100.000 IU/ml at week 4. This algorithm has a specificity of 43% and negative predictive value of 100% to detect nonresponse to standard PEG-IFN-alpha-2a and ribavirin therapy at fourth week of therapy (intention-to-treat analysis). Conclusion The decision to stop the therapy in genotype 1 chronic hepatitis C patients treated with PEG-IFN-alpha-2a and ribavirin can be confidently made after 4 weeks of treatment based on the absence of baseline anti-NS4a Ab and a week-4 hepatitis C virus-RNA above 100.000 IU/ml. Eur J Gastroenterol Hepatol 22:1443-1448 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

CRISPR/Cas9‐mediated genome editing: from basic research to translational medicine

The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.Portuguese Foundation for Science and Technology: UID/BIM/04773/2013 1334 Spanish Ministry of Science, Innovation and Universities RTI2018-094629-B-I00 Portuguese Foundation for Science and Technology SFRH/BPD/100434/2014 European Union (EU) 748585 LPCC-NRS/Terry Fox grantsinfo:eu-repo/semantics/publishedVersio

Sequential emergence and clinical implications of viral mutants with K70E and K65R mutation in reverse transcriptase during prolonged tenofovir monotherapy in rhesus macaques with chronic RT-SHIV infection.

BackgroundWe reported previously on the emergence and clinical implications of simian immunodeficiency virus (SIVmac251) mutants with a K65R mutation in reverse transcriptase (RT), and the role of CD8+ cell-mediated immune responses in suppressing viremia during tenofovir therapy. Because of significant sequence differences between SIV and HIV-1 RT that affect drug susceptibilities and mutational patterns, it is unclear to what extent findings with SIV can be extrapolated to HIV-1 RT. Accordingly, to model HIV-1 RT responses, 12 macaques were inoculated with RT-SHIV, a chimeric SIV containing HIV-1 RT, and started on prolonged tenofovir therapy 5 months later.ResultsThe early virologic response to tenofovir correlated with baseline viral RNA levels and expression of the MHC class I allele Mamu-A*01. For all animals, sensitive real-time PCR assays detected the transient emergence of K70E RT mutants within 4 weeks of therapy, which were then replaced by K65R mutants within 12 weeks of therapy. For most animals, the occurrence of these mutations preceded a partial rebound of plasma viremia to levels that remained on average 10-fold below baseline values. One animal eventually suppressed K65R viremia to undetectable levels for more than 4 years; sequential experiments using CD8+ cell depletion and tenofovir interruption demonstrated that both CD8+ cells and continued tenofovir therapy were required for sustained suppression of viremia.ConclusionThis is the first evidence that tenofovir therapy can select directly for K70E viral mutants in vivo. The observations on the clinical implications of the K65R RT-SHIV mutants were consistent with those of SIVmac251, and suggest that for persons infected with K65R HIV-1 both immune-mediated and drug-dependent antiviral activities play a role in controlling viremia. These findings suggest also that even in the presence of K65R virus, continuation of tenofovir treatment as part of HAART may be beneficial, particularly when assisted by antiviral immune responses