Viral Vectors in Gene Therapy and Clinical Applications

Developments in gene therapy, coupled with advances in genome sequencing and a greater understanding of DNA sequences, have given rise to an exciting area of research. The use of viral vectors in gene therapy has become a very promising and fast-emerging technology over the past few decades. Despite previous setbacks, the approval of viral vector therapies worldwide, with many in late-stage clinical trials has led to a significant increase in research in this area of gene therapy. Retroviral, adenoviral, adeno-associated viral, and lentiviral vectors are all key vectors currently being researched and used in clinical trials. There are many challenges with the use of viral vectors that are yet to be overcome including cost of production, the immune response, and the ability to precisely regulate the expression of the transgene. However, with increased numbers of clinical trials showing efficacy, safety, and growing financial investment, the future use of viral vectors in gene therapy is increasingly promising

Manipulating cellular microRNAs and analyzing high-dimensional gene expression data using machine learning workflows

MicroRNAs (miRNAs) are elements of the gene regulatory network and manipulating their abundance is essential toward elucidating their role in patho-physiological conditions. We present a detailed workflow that identifies important miRNAs using a machine learning algorithm. We then provide optimized techniques to validate the identified miRNAs through over-expression/loss-of-function studies. Overall, these protocols apply to any field in biology where high-dimensional data are produced. For complete details on the use and execution of this protocol, please refer to Wong et al. (2021a)

Germline and somatic cancer-associated mutations in the ATP-binding motifs of PTEN influence its subcellular localization and tumor suppressive function

Germline and somatic PTEN mutations are found in Cowden syndrome (CS) and multiple sporadic malignancies, respectively. PTEN function appears to be modulated by subcellular compartmentalization, and mislocalization may affect function. We have shown that cellular ATP levels affect nuclear PTEN levels. Here, we examined the ATP-binding capabilities of PTEN and functional consequences, relevant to cancer-associated mutations. PTEN mutation analysis of CS patients and sporadic colorectal carcinomas and comparative aminoacid analysis were utilized to identify mutations in ATP-binding motifs. The ability of wild-type (WT) or mutant PTEN to bind ATP was assessed by ATP–agarose-binding assays. Subcellular fractionation, western blotting, confocal microscopy and growth assays were used to determine relative nuclear-cytoplasmic localization and function. Somatic colorectal carcinoma-derived PTEN missense mutations were associated with nuclear mislocalization. These mutations altered cellular proliferation, apoptosis and anchorage-dependent growth. Examination of PTEN's amino acid sequence revealed these mutations resided in previously undescribed ATP-binding motifs (c.60–73; c.122–136). In contrast to WT PTEN, both cancer-associated somatic and germline-derived PTEN missense mutations, which lie within the ATP-binding motifs, result in mutant PTEN that does not bind ATP efficiently. We also show that CS patients with germline ATP-binding motif-mutations had nuclear PTEN mislocalization. Of four unrelated patients with functional germline ATP-binding domain mutations, all three female patients had breast cancers. Germline and somatic mutations within PTEN's ATP-binding domain play important pathogenic roles in both heritable and sporadic carcinogenesis by PTEN nuclear mislocalization resulting in altered signaling and growth. Manipulation of ATP may represent novel therapies in tumors with such PTEN alterations

Transcriptomic Analysis of Insulin-Secreting Murine Hepatocytes Transduced with an Integrating Adeno-Associated Viral Vector

Type 1 Diabetes (T1D) is a chronic metabolic disorder for which current treatments are unable to prevent the onset of complications. Previously, we used an adeno-associated viral vector (AAV8) to deliver furin-cleavable human insulin (INS-FUR) to the livers of diabetic non-obese diabetic (NOD) mice to reverse T1D. The use of the traditional AAV8-INS-FUR vector could not bring about normoglycemia. However, this vector, coupled with a transposon system in the AAV8/piggyBac-INS-FUR vector, was able to do so. This study aimed to investigate the transcriptomic profiles of the livers of diabetic, AAV8-INS-FUR-transduced, and AAV8/piggyBac-INS-FUR-transduced NOD mice and compare these to the normal liver to identify genetic differences resulting from delivery of the AAV8/piggyBac-INS-FUR vector which produced normoglycemia. Differential gene expression was determined by RNA-Seq analysis and differentially expressed genes from each treatment were mapped onto cellular pathways to determine the treatments’ cell signaling and downstream effects. We observed distinct differences between the piggyBac-transduced and diabetic models, particularly in terms of metabolic function and the upregulation of key pancreatic markers in the liver of piggyBac-transduced animals. The success of the AAV8/piggyBac-INS-FUR vector in achieving normoglycemia through stable transduction was evident. However, further engineering is necessary to achieve complete pancreatic transdifferentiation of liver cells

Pancreatic Transdifferentiation Using β-Cell Transcription Factors for Type 1 Diabetes Treatment

Type 1 diabetes is a chronic illness in which the native beta (β)-cell population responsible for insulin release has been the subject of autoimmune destruction. This condition requires patients to frequently measure their blood glucose concentration and administer multiple daily exogenous insulin injections accordingly. Current treatments fail to effectively treat the disease without significant side effects, and this has led to the exploration of different approaches for its treatment. Gene therapy and the use of viral vectors has been explored extensively and has been successful in treating a range of diseases. The use of viral vectors to deliver β-cell transcription factors has been researched in the context of type 1 diabetes to induce the pancreatic transdifferentiation of cells to replace the β-cell population destroyed in patients. Studies have used various combinations of pancreatic and β-cell transcription factors in order to induce pancreatic transdifferentiation and have achieved varying levels of success. This review will outline why pancreatic transcription factors have been utilised and how their application can allow the development of insulin-producing cells from non β-cells and potentially act as a cure for type 1 diabetes

Electroencephalographic prediction of global and domain specific cognitive performance of clinically active Australian nurses

Objective: To investigate the relationship between EEG activity and the global and domain specific cognitive performance of healthy nurses, and determine the predictive capabilities of these relationships. Approach: Sixty-four nurses were recruited for the present study, and data from 61 were utilised in the present analysis. Global and domain specific cognitive performance of each participant was assessed psychometrically using the Mini-mental state exam and the Cognistat, and a 32-lead monopolar EEG was recorded during a resting baseline phase and an active phase in which participants completed the Stroop test. Main results: Global cognitive performance was successfully predicted (81%–85% of variance) by a combination of fast wave activity variables in the alpha, beta and theta frequency bands. Interestingly, predicting domain specific performance had varying degrees of success (42%–99% of the variance predicted) and relied on combinations of both slow and fast wave activity, with delta and gamma activity predicting attention performance; delta, theta, and gamma activity predicting memory performance; and delta and beta variables predicting judgement performance. Significance: Global and domain specific cognitive performance of Australian nurses may be predicted with varying degrees of success by a unique combination of EEG variables. These proposed models image transitory cognitive declines and as such may prove useful in the prediction of early cognitive impairment, and may enable better diagnosis, and management of cognitive impairment

PTEN/PTENP1: ‘Regulating the regulator of RTK-dependent PI3K/Akt signalling’, new targets for cancer therapy

Abstract Regulation of the PI-3 kinase (PI3K)/Akt signalling pathway is essential for maintaining the integrity of fundamental cellular processes, cell growth, survival, death and metabolism, and dysregulation of this pathway is implicated in the development and progression of cancers. Receptor tyrosine kinases (RTKs) are major upstream regulators of PI3K/Akt signalling. The phosphatase and tensin homologue (PTEN), a well characterised tumour suppressor, is a prime antagonist of PI3K and therefore a negative regulator of this pathway. Loss or inactivation of PTEN, which occurs in many tumour types, leads to overactivation of RTK/PI3K/Akt signalling driving tumourigenesis. Cellular PTEN levels are tightly regulated by a number of transcriptional, post-transcriptional and post-translational regulatory mechanisms. Of particular interest, transcription of the PTEN pseudogene, PTENP1, produces sense and antisense transcripts that exhibit post-transcriptional and transcriptional modulation of PTEN expression respectively. These additional levels of regulatory complexity governing PTEN expression add to the overall intricacies of the regulation of RTK/PI-3 K/Akt signalling. This review will discuss the regulation of oncogenic PI3K signalling by PTEN (the regulator) with a focus on the modulatory effects of the sense and antisense transcripts of PTENP1 on PTEN expression, and will further explore the potential for new therapeutic opportunities in cancer treatment

Expression Profile of Sphingosine Kinase 1 Isoforms in Human Cancer Tissues and Cells: Importance and Clinical Relevance of the Neglected 1b-Isoform

Background.Overexpression of sphingosine kinase 1 (SphK1) is casually associated with many types of cancer, and inhibitors of SphK1 sensitize tumors to chemotherapy. SphK1 is expressed as two major isoforms, SphK1a and SphK1b. To date, no information has been reported on the SphK1 isoform expression profile and its clinical relevance. Objective. The objective is to examine the expression profile of the SphK1a and SPhK1b isoforms in human cancer and noncancer tissues and cell lines and explore their clinical relevance. Methods. We used PCR to qualitatively examine the expression profile of these two isoforms in breast, liver, and prostate cancer tissues plus paired adjacent tissues and in 11 cancer and normal cell lines (breast, cervical, bone, prostate, colon, brain, mesothelioma tumor and benign, and human kidney cells). Results. We found that SphK1a was ubiquitously expressed in all cancer cells and tissues tested; in contrast, SphK1b was only expressed in selective cell types in breast, prostate, and lung cancer. Conclusions. Our data suggest that SphK1a is important for generic SphK1/S1P functions, and SphK1b mediates specialized and/or unique pathways in a specific type of tissue and could be a biomarker for cancer. This discovery is important for future SphK1-related cancer research and may have clinical implications in drug development associated with SphK1-directed cancer treatment

Differential regulation of Annexin A1, A2, A5 and A6 expression at the transcriptional level in MCF-7 breast cancer cells.

<p>p14ARF expression was induced by the addition of 5mM IPTG for 15h. Quantitation of ANXA1, ANXA2, ANXA5 and ANXA6 expression was analyzed at 15h post p14ARF induction using the Taqman fast master mix and pre-optimized primer and probe sets. Data were normalized to levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data have been expressed as fold change in expression post p14ARF induction by IPTG at the 15h time point relative to control (2^−ΔΔCt). Experiments were performed in duplicate in which each set of experiments contained technical triplicates. Statistical differences between groups were determined using a two tailed, paired t-test. *p < 0.02, **p < 0.003 respectively.</p

Differential regulation of Annexin A1, A2, A5 by p14ARF-p53 occurs in MCF-7 but not in U2OS osteosarcoma cells.

<p>p14ARF expression was induced in MCF-7p14ARF and U2OSp14ARF cells by the addition of 5mM IPTG at? 15h 24h and 72h, PBS was added to control cells. Expression and localization of p14ARF expression in (A) U2OS cells, (B) MCF-7 pre- and post IPTG induction using immunofluorescence microscopy. (C) Top panel shows a representative western blot analysis of annexin A1 and A2 protein expression in MCF-7 and U2OS cells at 24h and 72h time points after p14ARF induction. β-actin was used as a loading control. The MCF-7p14ARF and U2OSp14ARF protein samples shown are from the same western blot. Bottom panel, P21, a p53 transcriptional target, was used to confirm p53 activation in the cell lysates. β-actin was used as a loading control. (D) Gene expression of p14ARF was induced by the addition of 5mM IPTG for 15h in U2OSp14ARF cells. Vehicle control contained PBS in place of IPTG. Transcriptional regulation of ANXA1, ANXA2, ANXA5 and ANXA6 was analyzed at 15h post p14ARF induction using RT-qPCR. Data were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data have been expressed as fold change in expression post p14ARF induction relative to the control (2^−ΔΔCt). Experiments were performed in duplicate in which each set of experiments contained technical triplicates. Statistical differences between groups were determined using a two tailed, paired t-test.</p