Synthesis of peptide-siRNA conjugates via internal sulfonylphosphoramidate modifications and evaluation of their in vitro activity.

Conjugates of therapeutic oligonucleotides (ONs) including peptide conjugates, provide a potential solution to the major challenge of specific tissue delivery faced by this class of drugs. Conjugations are often positioned terminal at the ONs, although internal placement of other chemical modifications are known to be of critical importance. The introduction of internal conjugation handles in chemically modified ONs require highly specialized and expensive nucleoside phosphoramidites. Here, we present a method for synthesizing a library of peptide-siRNA conjugates by conjugation at internal phosphorous positions via sulfonylphosphoramidate modifications incorporated into the sense strand. The sulfonylphosphoramidate modification offers benefits as it can be directly incorporated into chemically modified ONs by simply changing the oxidation step during synthesis, and furthermore holds the potential to create multifunctionalized therapeutic ONs. We have developed a workflow using a novel pH-controlled amine-to-amine linker that yields peptide-siRNA conjugates linked via amide bonds, and we have synthesized conjugates between GLP1 peptides and a HPRT1 siRNA as a model system. The in vitro activity of the conjugates was tested by GLP1R activity and knockdown of the HPRT1 gene. We found that conjugation near the 3'-end is more favorable than certain central internal positions and different internal conjugation strategies were compared.

Restoration of KMT2C/MLL3 in human colorectal cancer cells reinforces genome-wide H3K4me1 profiles and influences cell growth and gene expression.

BACKGROUND: The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects of KMT2C expression in CRC cells, we restored one allele to wild type KMT2C in the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant. RESULTS: Gene editing resulted in increased KMT2C expression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects of KMT2C expression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation. CONCLUSIONS: Here, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.

Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering.

Many branches of biology depend on stable and predictable recombinant gene expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined integration sites. However, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated expression cassette. Lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integration site and the expression cassette components. Here we show in a systematic manner how multiple components contribute to final net expression of recombinant genes in a characterized integration site. We develop a CRISPR/Cas9-based toolbox for construction of mammalian cell lines with targeted integration of a landing pad, containing a recombinant gene under defined 5' proximal regulatory elements. Generated site-specific recombinant cell lines can be used in a streamlined recombinase-mediated cassette exchange for fast screening of different expression cassettes. Using the developed toolbox, we show that different 5' proximal regulatory elements generate distinct and robust recombinant gene expression patterns in defined integration sites of CHO cells with a wide range of transcriptional outputs. This approach facilitates the generation of user-defined and product-specific gene expression patterns for programmable mammalian cell engineering.

Using Titer and Titer Normalized to Confluence Are Complementary Strategies for Obtaining Chinese Hamster Ovary Cell Lines with High Volumetric Productivity of Etanercept.

The selection of clonally derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number are being used to assess suspension-adapted clones when grown statically in microtiter plates. However, no reported study so far has performed a direct head-to-head comparison of these two early reporters for predicting clone performance. Therefore, a screening platform for high-throughput analysis of titer and confluence of etanercept-producing clones is developed. Then an unbiased comparison of clone ranking based on either titer or titer normalized to confluence (TTC) is performed. Using two different suspension cultivation vessels, the authors demonstrate that titer- or TTC-based ranking gives rise to the selection of clones with similar volumetric productivity in batch cultures. Therefore, using both titer- and TTC-based ranking is proposed, allowing for selection of distinct clones with both high integral of viable cell density (IVCD) and high specific productivity features, respectively. This contributes to selection of a versatile panel of clones that can be further characterized and from which the final producer clone can be selected that best fits the production requirements.

Corrigendum: Insufficient antibody validation challenges oestrogen receptor beta research.

This corrects the article DOI: 10.1038/ncomms15840.

Insufficient antibody validation challenges oestrogen receptor beta research.

The discovery of oestrogen receptor ß (ERß/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ERα (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ERß antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ERß in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ERß protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions.

Chinese hamster ovary (CHO) cells are the preferred cell factory for the production of therapeutic glycoproteins. Although efforts primarily within bioprocess optimization have led to increased product titers of recombinant proteins (r-proteins) expressed in CHO cells, post-transcriptional bottlenecks in the biosynthetic pathway of r-proteins remain to be solved. To this end, the ectopic expression of transgenes (effector genes) offers great engineering potential. However, studies on effector genes have in some cases led to inconsistent results. Whereas this can in part be attributed to product specificity, other experimental and cellular factors are likely important contributors to these conflicting results. Here, these factors are reviewed and discussed with the objective of guiding future studies on effector genes.

Stratification of Hepatocellular Carcinoma Patients Based on Acetate Utilization.

Hepatocellular carcinoma (HCC) is a deadly form of liver cancer that is increasingly prevalent. We analyzed global gene expression profiling of 361 HCC tumors and 49 adjacent noncancerous liver samples by means of combinatorial network-based analysis. We investigated the correlation between transcriptome and proteome of HCC and reconstructed a functional genome-scale metabolic model (GEM) for HCC. We identified fundamental metabolic processes required for cell proliferation using the network centric view provided by the GEM. Our analysis revealed tight regulation of fatty acid biosynthesis (FAB) and highly significant deregulation of fatty acid oxidation in HCC. We predicted mitochondrial acetate as an emerging substrate for FAB through upregulation of mitochondrial acetyl-CoA synthetase (ACSS1) in HCC. We analyzed heterogeneous expression of ACSS1 and ACSS2 between HCC patients stratified by high and low ACSS1 and ACSS2 expression and revealed that ACSS1 is associated with tumor growth and malignancy under hypoxic conditions in human HCC.

Interleukin-1α gene variants influence bone mineral density and the risk of osteoporotic hip fractures in elderly Slovenian people.

BACKGROUND: Osteoporosis is a skeletal disorder, characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to increased risk of fracture. Recently, the role of age-related pro-inflammatory cytokines, such as interleukin (IL)-1α, in stimulating bone resorption has been suggested. As osteoporosis has a strong genetic background, the aim of our study was to evaluate the association of two IL-1α gene single nucleotide polymorphisms (SNPs) rs2071375 (+12534G>A) and rs17651 (+4845G>T) with osteoporotic phenotypes as well as to find the association with IL-1α gene expression in human bone tissue. METHODS: Genotyping was performed in 671 Slovenian participants, 125 elderly men, 490 post- and 56 premenopausal women. Bone mineral density (BMD) at the lumbar spine, femoral neck and total hip were measured. Biochemical markers of bone turnover were measured in women. RESULTS: Significant association of GG/TA haplotype with higher femoral neck and total hip BMD in elderly men and women was shown (p=0.009 and 0.030, respectively). In men, the association of the GG/GG haplotype with higher femoral neck BMD was of limited statistical significance (p=0.050). In women, significant association of studied genetic variants with serum C-terminal crosslinking telopeptides of type I collagen and bone alkaline phosphatase were found (p=0.033 and 0.029, respectively). No influence on IL-1α expression was found. Finally, significantly lower odds ratio for hip fracture associated with the presence of TA haplotype was found (p=0.026). CONCLUSIONS: Our results of the association of IL-1α gene single nucleotide polymorphisms (SNPs) rs2071375 (+12534G>A) and rs17651 (+4845G>T) with osteoporotic features indicate its role in pathogenesis of osteoporosis. However, these findings need further functional and clinical confirmation.