Generation of CRISPR engineered prostate cancer cell line models to study androgen receptor signalling in advanced prostate cancer

PhD ThesisProstate cancer resistance to AR targeted therapies due to the emergence of AR point mutations and AR splice variants that cannot be targeted by the currently available agents comprise a major clinical challenge. There is paucity of models that accurately reflect the mechanisms of AR regulation in advanced disease. This highlights the high demand of generating novel disease relevant models. A CRISPR pipeline was developed to generate cell line models which harbour specific point mutations in the LBD of AR as well as stop codons in AR exon 5 which resulted in AR-FL knock-out so that the remaining endogenous AR-Vs could be studied discriminately of interfering AR-FL. Using a streptavidin-tagged Cas9 in conjugation with a biotinylated donor template resulted in high donor template knock-in efficiencies and yielded (i) an ARW741L CWR22Rv1 cell line derivative and (ii) an AR-FL knock-out cell line derivative called CWR22Rv1-AR-EK (Exon Knock-out). CWR22Rv1-AR-EK cells retained all endogenous AR-Vs following AR gene editing. AR-Vs acted unhindered following AR-FL deletion to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrated that AR-Vs drive expression of a cohort of cell cycle and DNA damage response genes and depletion of AR-Vs sensitised cells to ionising radiation. To date, elimination of AR-Vs by pharmacological inhibition remains challenging. However, disruption of AR pre-mRNA splicing is nowadays a highly attractive option. A CRISPR-based approach, called CRIME (Cas9-directed Rapid Immunoprecipitation Mass Spectrometry of Endogenous proteins) was developed to isolate and identify the AR-V7-specific spliceosome, in association with a nuclease deficient Cas9 at AR cryptic exon 3. Mass spec-derived hits were screened for their ability to alter AR-V7 mRNA levels in CWR22Rv1 cells. SRSF3 was identified as a potential AR-V7 splicer which promotes cryptic exon 3 skipping in normal prostate. In CRPC, SRSF3 is significantly downregulated and hence cryptic exon 3 inclusion in the mature AR-V7 mRNA transcript is permitted leading to generation of AR-V7

Ex vivo gene modification therapy for genetic skin diseases—recent advances in gene modification technologies and delivery

Genetic skin diseases, also known as genodermatoses, are inherited disorders affecting skin and constitute a large and heterogeneous group of diseases. While genodermatoses are rare with the prevalence rate of less than 1 in 50,000 - 200,000, they frequently occur at birth or early in life and are generally chronic, severe, and could be life-threatening. The quality of life of patients and their families are severely compromised by the negative psychosocial impact of disease, physical manifestations, and the lack or loss of autonomy. Currently, there are no curative treatments for these conditions. Ex-vivo gene modification therapy that involves modification or correction of mutant genes in patients' cells in-vitro and then transplanted back to patients to restore functional gene expression has being developed for genodermatoses. In this review, the ex-vivo gene modification therapy strategies for genodermatoses are reviewed, focusing on current advances in gene modification and correction in patients' cells and delivery of genetically modified cells to patients with discussions on gene therapy trials which have been performed in this area

The disruption of protein-protein interactions as a therapeutic strategy for prostate cancer

This is an accepted manuscript of an article published by Elsevier in Pharmacological Research on 16/08/2020, available online: https://doi.org/10.1016/j.phrs.2020.105145 The accepted version of the publication may differ from the final published version.Prostate cancer (PCa) is one of the most common male-specific cancers worldwide, with high morbidity and mortality rates associated with advanced disease stages. The current treatment options of PCa are prostatectomy, hormonal therapy, chemotherapy or radiotherapy, the selection of which is usually dependent upon the stage of the disease. The development of PCa to a castration-resistant phenotype (CRPC) is associated with a more severe prognosis requiring the development of a new and effective therapy. Protein-protein interactions (PPIs) have been recognised as an emerging drug modality and targeting PPIs is a promising therapeutic approach for several diseases, including cancer. The efficacy of several compounds in which target PPIs and consequently impair disease progression were validated in phase I/II clinical trials for different types of cancer. In PCa, various small molecules and peptides proved successful in inhibiting important PPIs, mainly associated with the androgen receptor (AR), Bcl-2 family proteins, and kinases/phosphatases, thus impairing the growth of PCa cells in vitro. Moreover, a majority of these compounds require further validation in vivo and, preferably, in clinical trials. In addition, several other PPIs associated with PCa progression have been identified and now require experimental validation as potential therapeutic loci. In conclusion, we consider the disruption of PPIs to be a promising though challenging therapeutic strategy for Pca. Agents which modulate PPIs might be employed as a monotherapy or as an adjunct to classical chemotherapeutics to overcome drug resistance and improve efficacy. The discovery of new PPIs with important roles in disease progression, and of novel optimized strategies to target them, are major challenges for the scientific and pharmacological communities.We thank the Portuguese Foundation for Science and Technology(FCT), European Union, QREN, FEDER and COMPETE for funding iBiMED (UIDB/04501/2020, POCI-01-0145-FEDER-23007628 and UID/BIM/04501/2019) and an individual scholarship from BM (SFRH/BD/146032/2019)

An update in the management of ocular surface squamous neoplasia

Introduction: Management of ocular surface squamous neoplasia (OSSN) has been a matter of debate among ocular oncologists. While surgical excision with tissue biopsy has been the gold standard, topical chemotherapy with agents such as interferon-α2b, 5-fluorouracil, and mitomycin C, as well as less invasive diagnostic modalities, are becoming increasingly popular. Areas covered: This report provides a comprehensive review of recent data and addresses the key controversies in the management of OSSN. A MEDLINE database search was performed using the keywords ‘ocular surface squamous neoplasia,’ ‘conjunctival intraepithelial neoplasia,’ ‘conjunctival squamous cell carcinoma,’ ‘corneal intraepithelial neoplasia,’ ‘CIN,’ and ‘OSSN.’ All resulting articles published in English until December 2020 were then reviewed for pertinent references. Expert opinion: Although tissue biopsy remains the gold standard for the diagnosis and management of OSSN, the advent of new and sophisticated imaging modalities is likely to change the approach to OSSN diagnosis for the majority of patients, while the use of topical chemotherapeutic and immunotherapeutic agents may become the mainstay of treatment. © 2021 Informa UK Limited, trading as Taylor & Francis Group

An update in the management of ocular surface squamous neoplasia

Management of ocular surface squamous neoplasia (OSSN) has been a matter of debate among ocular oncologists. While surgical excision with tissue biopsy has been the gold standard, topical chemotherapy with agents such as interferon-α2b, 5-fluorouracil, and mitomycin C, as well as less invasive diagnostic modalities, are becoming increasingly popular. This report provides a comprehensive review of recent data and addresses the key controversies in the management of OSSN. A MEDLINE database search was performed using the keywords 'ocular surface squamous neoplasia,' 'conjunctival intraepithelial neoplasia,' 'conjunctival squamous cell carcinoma,' 'corneal intraepithelial neoplasia,' 'CIN,' and 'OSSN.' All resulting articles published in English until December 2020 were then reviewed for pertinent references. Although tissue biopsy remains the gold standard for the diagnosis and management of OSSN, the advent of new and sophisticated imaging modalities is likely to change the approach to OSSN diagnosis for the majority of patients, while the use of topical chemotherapeutic and immunotherapeutic agents may become the mainstay of treatment

A Neural Dynamic Architecture That Autonomously Builds Mental Models

Reasoning and other mental operations are believed to rely onmental models. Arguments have been made that mental mod-els share representational substrate with perception. Here, wedemonstrate that a neural dynamic architecture that perceptu-ally grounds language may also support the building of men-tal models. Supplied with a sequence of simple premises thatspecify the colors of object pairs as well as their spatial rela-tion, the architecture builds a mental model of the describedscene. We show how the neural processes of the architec-ture evolve in response to both determinate and indeterminatepremises. For indeterminate premises, we demonstrate thatthe preferred mental models observed in human participantsemerge from the underlying neural dynamics