Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets.

BACKGROUND: The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels. RESULTS: In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells. CONCLUSION: The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.

Dynamic changes in immune cell populations by AXL kinase targeting diminish liver inflammation and fibrosis in experimental MASH.

Background and aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Methods: Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. Results: In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB- NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Conclusion: Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

AXL targeting by a specific small molecule or monoclonal antibody inhibits renal cell carcinoma progression in an orthotopic mice model.

AXL tyrosine kinase activation enhances cancer cell survival, migration, invasiveness, and promotes drug resistance. AXL overexpression is typically detected in a high percentage of renal cell carcinomas (RCCs) and is strongly associated with poor prognosis. Therefore, AXL inhibition represents an attractive treatment option in these cancers. In this preclinical study, we investigated the antitumor role of a highly selective small molecule AXL inhibitor bemcentinib (BGB324, BerGenBio), and a newly developed humanized anti-AXL monoclonal function blocking antibody tilvestamab, (BGB149, BerGenBio), in vitro and an orthotopic RCC mice model. The 786-0-Luc human RCC cells showed high AXL expression. Both bemcentinib and tilvestamab significantly inhibited AXL activation induced by Gas6 stimulation in vitro. Furthermore, tilvestamab inhibited the downstream AKT phosphorylation in these cells. The 786-0-Luc human RCC cells generated tumors with high Ki67 and vimentin expression upon orthotopic implantation in athymic BALB/c nude mice. Most importantly, both bemcentinib and tilvestamab inhibited the progression of tumors induced by the orthotopically implanted 786-0 RCC cells. Remarkably, their in vivo antitumor effectiveness was not significantly enhanced by concomitant administration of a multi-target tyrosine kinase inhibitor. Bemcentinib and tilvestamab qualify as compounds of potentially high clinical interest in AXL overexpressing RCC.

Enhanced gene expression from retroviral vectors.

BACKGROUND: Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter. RESULTS: By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods. We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the retroviral genome by T7 RNA polymerase. When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression - up to 3.5 times greater than conventional retroviral LTR-driven expression. CONCLUSION: Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with in vitro transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the in vitro transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.

Expanding the spectrum of genetic elements transferable by retroviral vectors.

Retroviral vectors are powerful tools to study gene function. However, conventional methods require a cellular transcription step to generate the genomic RNA for viral production. This limits the scope of genetic elements that may be transferred by these vectors, excluding many key gene regulatory signals, including RNA editing motifs, alternative splicing, and various promoter/enhancer constellations, as well as cytotoxic genes. To address this problem, we devised a simple approach where in vitro-synthesized vector genomic RNA is transfected into the cytoplasm of a packaging cell, allowing immediate viral particle assembly. We demonstrate that high-titer retroviruses that efficiently transduce mammalian cell lines and primary cells are readily generated. Importantly, we show that an intron-containing expression cassette can be transferred by this method, leading to increased expression levels in the target cell. Further, we demonstrate that the cap structure is not required for retroviral packaging, thus avoiding translation of vector-encoded genes in the packaging cell. This allows the retroviral transfer of cytotoxic genes or proteins that otherwise inhibit viral production.

S100A14 inhibits proliferation of oral carcinoma derived cells through G1-arrest.

Altered expression of S100A14 has been reported in various human cancers including oral squamous cell carcinomas (OSCCs). Its biological functions in carcinogenesis, however, are largely unknown. This study aimed to investigate the functional role of S100A14 in tumor cell proliferation and its possible functional association with p53. S100A14 protein was found to be gradually down-regulated during the transition from normal to dysplastic and carcinoma cells in an in vitro human OSCC progression model. When over-expressed by employing retroviral expression vector, S100A14 inhibited proliferation of CaLH3 and OSCC1, OSCC cell-lines harboring wild type (wt) p53, by inducing G1-arrest. This G1-arrest correlated with up-regulation of p21 both in the CaLH3 and OSCC1 cell-lines. shRNA mediated silencing of p53 led to partial suppression of p21 in S100A14 over-expressing CaLH3 cells, indicating that p21 up-regulation was, at least, partly dependent on p53. We further demonstrated that nuclear accumulation of p53 occurred with over-expression of S100A14 in CaLH3 cells. Our data suggest a novel role of S100A14 in OSCC cell proliferation by inducing G1-arrest and also indicate a functional link between S100A14 and the tumor suppressor protein p53.

Drug target discovery using retroviruses.

Contemporary drug target discovery relies on a continuum of genetic and chemical-based screening technologies. These approaches conflate pharmaceutical and genetic principles, providing a conceptual platform that links dominant genetics with drug action. Thus, phenotypic genetic screens using vector-expressed dominant genetic effectors - trans-acting molecules that modulate gene function, such as peptides or RNA interference triggers - can reveal genes whose inhibition engenders a therapeutic effect. The correlation of this genetic inhibition with a specific protein activity defines a drug target candidate. Retroviruses provide a unique opportunity to stably deliver a variety of dominant genetic effectors to mammalian cells in a flexible predetermined fashion and are a favoured system for phenotypic screening. Here, the authors review recent innovations and approaches to therapeutic target discovery using retroviral vectors.