Genetic and process engineering for the production of protein therapeutics for the treatment of CNS disorders

Neurological disorders constitute the major cause of disability adjusted-life years (DALYs). Alzheimer’s disease and other dementias are included in the group of the most prevalent disorders in this field (Feigin et al., 2017). Nevertheless, the urgency of the treatment of these pathologies is not met by efficacious drug development, as indicated by a recent statistical analysis measuring the likelihood of approval of new drugs by disease area (Hay et al., 2014). In neurology only 8.4% of the candidate drugs have been approved in the 2006 – 2015 decade and the drugs categorized as “Large Molecules” were characterized by 13.2% rate of success (Hay et al., 2014). This statistical analysis would suggest that only those molecules proving robust Proof of Mechanism (PoM) and Proof of Principle (PoP) during their early development deserve the risk for further development. On the other hand, proteins used in protein replacement therapies (PRTs) constitute an exception in this scenario as the iter for their approval is generally more straightforward (Gorzelany and De Souza, 2013). In this case the main hurdles involving the drug development directly coincide with the production, the purification and the stable formulation of the final product rather than the assessment of its efficacy or toxicity (Saccardo, Corchero and Ferrer-Miralles, 2016). In the present document we describe the approaches followed in the implementation of biotechnological processes for the production of two different proteins with potential applications in the treatment of central nervous system (CNS) disorders. In the first chapter we present the establishment of a simple and efficient pipeline for the E. coli recombinant expression and purification of the bacterial toxin named CNF1. This 114 kDa protein is involved in a series of infectious diseases (Ho et al., 2018), but it has also been demonstrated to be promising in the treatment of severe neurological pathologies like Alzheimer’s disease, Parkinson disease, Rett syndrome and epilepsy (Maroccia et al., 2018). Nevertheless, during the development of the project we decided not to pursue any further attempt in the clinical development of CNF1 as a drug because of the lack of robust, clear and completely demonstrated PoM and PoP. However, the proposed procedure for the purification and final formulation of the product outperforms the others reported in the literature in terms of yield, purity and stability and it can be easily employed in the future for further structural and functional analyses in toxicological and immunological perspectives. The reproducibility of the entire pipeline has been demonstrated repeating the production and purification protocols dozens of times at intervals of several months. Moreover, the stability of the final product was routinely ascertained using SDS-PAGE, size-exclusion chromatography, DLS and activity assays. In the second chapter of this thesis we describe the employment of Pseudoalteromonas haloplanktis TAC125 in the production of a human protein to be used in a PRT. Although the quality of the product achievable in this host seemed better than the one previously obtained with other expression systems, the overall yields remained low. Slight improvements in this sense were achieved by the genetic engineering of the coding sequence of the target protein and by the implementation of alternative expression plasmids. Nevertheless, further studies demonstrated that the whole expression platform – the host and the plasmids – are affected by imperfections and bottlenecks whose correction is pivotal for a satisfying recombinant production. This kind of issues is typical of unconventional and less explored recombinant bacteria, but there are several examples in the literature about how they can be systematically overcome. Hence, a series of measures to be taken for the improvement of this microbial factory are proposed

Bacterial Production of CDKL5 Catalytic Domain: Insights in Aggregation, Internal Translation and Phosphorylation Patterns

: Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in human brain development and functioning. Mutations in CDKL5, especially in its catalytic domain, cause a severe developmental condition named CDKL5 deficiency disorder. Nevertheless, molecular studies investigating the structural consequences of such mutations are still missing. The CDKL5 catalytic domain harbors different sites of post-translational modification, such as phosphorylations, but their role in catalytic activity, protein folding, and stability has not been entirely investigated. With this work, we describe the expression pattern of the CDKL5 catalytic domain in Escherichia coli demonstrating that it predominantly aggregates. However, the use of solubility tags, the lowering of the expression temperature, the manual codon optimization to overcome an internal translational start, and the incubation of the protein with K+ and MgATP allow the collection of a soluble catalytically active kinase. Interestingly, the resulting protein exhibits hypophosphorylation compared to its eukaryotic counterpart, proving that bacteria are a useful tool to achieve almost unmodified CDKL5. Posing questions about the CDKL5 autoactivation mechanism and the determinants for its stability, this research provides a valuable platform for comparative biophysical studies between bacterial and eukaryotic-expressed proteins, contributing to our understanding of neurodevelopmental disorders associated with CDKL5 dysfunction

Getting value from the waste: recombinant production of a sweet protein by Lactococcus lactis grown on cheese whey

Background Recent biotechnological advancements have allowed for the adoption of Lactococcus lactis, a typical component of starter cultures used in food industry, as the host for the production of food-grade recombinant targets. Among several advantages, L. lactis has the important feature of growing on lactose, the main carbohydrate in milk and a majoritarian component of dairy wastes, such as cheese whey. Results We have used recombinant L. lactis NZ9000 carrying the nisin inducible pNZ8148 vector to produce MNEI, a small sweet protein derived from monellin, with potential for food industry applications as a high intensity sweetener. We have been able to sustain this production using a medium based on the cheese whey from the production of ricotta cheese, with minimal pre-treatment of the waste. As a proof of concept, we have also tested these conditions for the production of MMP-9, a protein that had been previously successfully obtained from L. lactis cultures in standard growth conditions. Conclusions Other than presenting a new system for the recombinant production of MNEI, more compliant with its potential applications in food industry, our results introduce a strategy to valorize dairy effluents through the synthesis of high added value recombinant proteins. Interestingly, the possibility of using this whey-derived medium relied greatly on the choice of the appropriate codon usage for the target gene. In fact, when a gene optimized for L. lactis was used, the production of MNEI proceeded with good yields. On the other hand, when an E. coli optimized gene was employed, protein synthesis was greatly reduced, to the point of being completely abated in the cheese whey-based medium. The production of MMP-9 was comparable to what observed in the reference conditions.info:eu-repo/semantics/publishedVersio

Clinical Features, Cardiovascular Risk Profile, and Therapeutic Trajectories of Patients with Type 2 Diabetes Candidate for Oral Semaglutide Therapy in the Italian Specialist Care

Introduction: This study aimed to address therapeutic inertia in the management of type 2 diabetes (T2D) by investigating the potential of early treatment with oral semaglutide. Methods: A cross-sectional survey was conducted between October 2021 and April 2022 among specialists treating individuals with T2D. A scientific committee designed a data collection form covering demographics, cardiovascular risk, glucose control metrics, ongoing therapies, and physician judgments on treatment appropriateness. Participants completed anonymous patient questionnaires reflecting routine clinical encounters. The preferred therapeutic regimen for each patient was also identified. Results: The analysis was conducted on 4449 patients initiating oral semaglutide. The population had a relatively short disease duration (42%  60% of patients, and more often than sitagliptin or empagliflozin. Conclusion: The study supports the potential of early implementation of oral semaglutide as a strategy to overcome therapeutic inertia and enhance T2D management

Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression

Brain tumors in children under 3 years of age: recent experience (1987-1997) in 39 patients

Brain tumors in children under 3 years of age differ in clinical presentation and pathological behavior from those in older patients. In this study, we reviewed data from 39 children (24 males and 15 females) under 3 years of age who were treated for intracranial brain tumors since the introduction of magnetic resonance imaging. The purpose was to assess correlations between clinicoradiological and treatment-related factors, and their impact on prognosis. The following factors were analyzed: sex, age, duration of symptoms, intracranial hypertension on admission, tumor location, surgical removal and histology. Associations between these factors and correlations with prognosis were determined using bivariate analyses (chi(2) test) and Kaplan-Meier survival curves. Collins' concept of a period of risk recurrence was tested. Mean follow-up was 41 months (range 0-136). In March 1998, 20 children were still alive (51.2%) with a mean survival time of 65 months (range 2-136). The incidence of supratentorial tumors was significantly higher in children less than 1 year old (p = 0.027). Lateral tumors were 9/10 (90%) supratentorial versus only 7/26 (27%) midline tumors (p = 0.001). Outcome (dead or alive) was significantly better (p = 0.037) for low-grade astrocytomas (9/12 = 75% survival) when compared to ependymomas (2/6 = 33%) and primitive neuroectodermal tumors (3/12 = 25%). Total tumor removal was achieved in 20 cases and was associated with a better outcome (65 vs. 33% survival; p = 0.049). Survival analysis confirmed a worse prognosis for children with ependymomas and primitive neuroectodermal tumors (p = 0.011) and revealed a worse survival for children with intracranial hypertension on admission (p = 0.047). Total tumor removal was associated with a longer survival, although not significantly (p = 0. 077). Finally, we found no exceptions to Collins' law