Approaches for improving the safety and efficacy of adenoviral gene therapy

Cancer is a devastating disease with poor prognosis and no curative treatment, when widely metastatic. Conventional therapies, such as chemotherapy and radiotherapy, have efficacy but are not curative and systemic toxicity can be considerable. Almost all cancers are caused due to changes in the genetic material of the transformed cells. Cancer gene therapy has emerged as a new treatment option, and past decades brought new insights in developing new therapeutic drugs for curing cancer. Oncolytic viruses constitute a novel therapeutic approach given their capacity to replicate in and kill specifically tumor cells as well as reaching tumor distant metastasis. Adenoviral gene therapy has been suggested to cause liver toxicity. This study shows that new developed adenoviruses, in particular Ad5/19p-HIT, can be redirected towards kidney while adenovirus uptake by liver is minimal. Moreover, low liver transduction resulted in a favorable tumor to liver ratio of virus load. Further, we established a new immunocompetent animal model Syrian hamsters. Wild type adenovirus 5 was found to replicate in Hap-T1 hamster tumors and normal tissues. There are no antiviral drugs available to inhibit adenovirus replication. In our study, chlorpromazine and cidofovir efficiently abrogated virus replication in vitro and showed significant reduction in vivo in tumors and liver. Once safety concerns were addressed together with the new given antiviral treatment options, we further improved oncolytic adenoviruses for better tumor penetration, local amplification and host system modulation. Further, we created Ad5/3-9HIF-Δ24-VEGFR-1-Ig, oncolytic adenovirus for improved infectivity and antiangiogenic effect for treatment of renal cancer. This virus exhibited increased anti-tumor effect and specific replication in kidney cancer cells. The key player for good efficacy of oncolytic virotherapy is the host immune response. Thus, we engineered a triple targeted adenovirus Ad5/3-hTERT-E1A-hCD40L, which would lead to tumor elimination due to tumor-specific oncolysis and apoptosis together with an anti-tumor immune response prompted by the immunomodulatory molecule. In conclusion, the results presented in this thesis constitute advances in our understanding of oncolytic virotherapy by successful tumor targeting, antiviral treatment options as a safety switch in case of replication associated side-effects, and modulation of the host immune system towards tumor elimination.  Pitkälle levinneen syövän ennuste on usein huono, sillä parantavia hoitomuotoja ei ole tarjolla. Perinteiset hoitomuodot, kuten solunsalpaajat ja sädehoito, ovat harvoin parantavia ja haittavaikutukset ovat usein huomattavia. Syopä syntyy soluissa tapahtuvien geneettisten muutosten seurauksena, jolloin solujen normaalit kasvunsäätelyominaisuudet muuttuvat. Viime vuosikymmenten aikana on kehitetty uusia hoitoja syöpään ja syovän geeniterapia on yksi lupaavimmista kokeellisista hoitomuodoista. Onkolyyttiset virukset monistuvat syöpäsoluissa jonka seurauksena syöpäsolu kuolee vapauttaen uusia infektiivisiä viruspartikkeleita, jotka voivat verenkierron välityksellä kulkeutua muualla elimistössä oleviin etäpesäkkeisiin. Adenovirus geeniterapia saattaa aiheuttaa toksisuutta erityisesti maksassa. Tässä työssä on osoitettu, että geneettisesti muokatut adenovirukset, erityisesti Ad5/19p-HIT, voidaan kohdistaa munuaisiin samalla vähentäen viruksen kerääntymistä maksaan. Vähäisempi kerääntyminen maksaan johtaa myös viruksen suhteellisesti parempaan kulkeutumiseen kasvainkudokseen. Tässä työssä on kehitetty myös uusi immunokompetentti Syyrianhamsteri eläinmalli. Muokkaamattoman serotyyppi 5 adenoviruksen osoitettiin monistuvan hamsterin Hap-T1 soluilla aiheutetuissa kasvaimissa sekä normaalikudoksessa. Adenoviruksen monistumisen rajoittamiseen ei ole olemassa antiviraalilääkkeitä. Tässä työssä on osoitettu että klooripromatsiini ja sidofoviiri estävät tehokkaasti adenoviruksen monistumisen soluviljelmissä ja vähentävät monistumista myös eläinten kasvaimissa ja maksassa. Työn tarkoituksena oli myös kehittää adenoviruksia jotka tehokkaammin lisääntyisivät kasvainkudoksessa ja joilla voitaisiin samalla muokata immuunijärjestelmää paremman kasvainta tuhoavan vaikutuksen aikaan saamiseksi. Immuunijärjestelmällä on osoitettu olevan ratkaisevan tärkeä merkitys onkolyyttisen virusterapian tehokkuuden määräytymisessä. Sen vuoksi työssä on kehitetty adenovirus Ad5/3-hTERT-E1A-hCD40L joka pystyy tuhoamaan kohdennetusti syöpäsolukkoa kolmella eri mekanismilla. Viruksen onkolyyttinen vaikutus tappaa syöpäsoluja mutta sen lisäksi viruksessa oleva siirtogeeni, ihmisen CD40 ligandi (hCD40L) aiheuttaa infektoitujen syöpäsolujen apoptoosin ja herättää immuunivasteen kasvainsoluja kohtaan. Työssä kehitettiin myös adenovirus Ad5/3-9HIF-Δ24-VEGFR-1-Ig joka onkolyyttisen vaikutuksen lisäksi estää tehokkaasti kasvainkudoksen verisuonten kehittymistä. Yhteenvetona, tämä työ on tuonut lisätietoa onkolyyttisen virusterapian paremmasta kohdentamisesta kasvainkudokseen, antiviraalihoitojen toimivuudesta mahdollisten haittavaikutusten rajoittamiseksi, ja mahdollisuuksista muokata immuunijärjestelmää paremman kasvainta tuhoavan vaikutuksen aikaansaamiseksi

Inducible Caspase-9 Selectively Modulates the Toxicities of CD19-Specific Chimeric Antigen Receptor-Modified T Cells

Immunotherapy with T cells expressing the chimeric antigen receptor (CAR) specific for the CD19 antigen (CD19.CAR-Ts) is a very effective treatment in B cell lymphoid malignancies. However, B cell aplasia and cytokine release syndrome (CRS) secondary to the infusion of CD19.CAR-Ts remain significant drawbacks. The inclusion of safety switches into the vector encoding the CAR is seen as the safest method to terminate the effects of CD19.CAR-Ts in case of severe toxicities or after achieving long-term sustained remissions. By contrast, the complete elimination of CD19.CAR-Ts when CRS occurs may jeopardize clinical responses as CRS and antitumor activity seem to concur. We have demonstrated, in a humanized mouse model, that the inducible caspase-9 () safety switch can eliminate CD19.CAR-Ts in a dose-dependent manner, allowing either a selective containment of CD19.CAR-T expansion in case of CRS or complete deletion on demand granting normal B cell reconstitution

Pancreatogenic type 3C diabetes

Background. The relationship between chronic pancreatitis and diabetes is well established. This form of diabetes is secondary to exocrine pancreatic disorder and is known as diabetes mellitus type 3c (T3cDM). Materials and Methods. In this retrospective study we included 261 patients, 59 patients being diagnosed with chronic pancreatitis and secondary diabetes mellitus, and admitted in the Fundeni Clinical Institute, 2nd Department of Gastroenterology or N.C. Paulescu Institute/ Carol Davila University of Medicine and Pharmacy. Results and Discussions. Patients were 22.2% women and 77.8% men, with an average age of 56.8 years and 53.4 years respectively. 63% came from urban areas. The mean duration of chronic pancreatitis was six years. Non-diabetic patients were compared with patients who were previously diagnosed with T3cDM and who had been analyzed for body mass index (BMI). Imaging investigations were also performed to confirm pseudotumors or pancreatic tumours. Patients already considered non-diabetic had basal blood glucose values and were mostly overweight and obese. In this context, insulin resistance cannot be excluded for this group of patients. Conclusions. T3cDM is a new pathological entity that needs to be explored more deeply, and that should benefit from both a diagnostic stratification and treatment

LARS-like symptoms in the general population may suggest the significance of postoperative functional problems and emotional implications of rectal surgery

Background & Aim. Sphincter-saving rectal surgery is prone to cause changes in bowel function associated with Low Anterior Resection Syndrome (LARS). Our aim was to assess LARS-like symptoms within a population of 50-80-year old in order to understand the functional disturbances and emotional impact of LARS. Materials and methods: We used a questionnaire to evaluate LARS with the following categories of symptoms: flatulence control, anal incontinence, frequency, clustering and urgency of the stools, and the psycho-emotional impact created by the presence of these symptoms. We calculated the severity of LARS on 343 responders. Results. The average age of the responders (57.4% females) was 60 years. Overall, 48.1% of those questioned had no LARS-associated symptoms, while the rest presented either minor (39.9%) or major (12%) LARS-like symptomatology according to the assessment scale. Women have a higher relative risk (1.32) of having minor or major LARS. The frequency of stools did not correlate with the overall LARS score. The psycho-emotional impact was mostly influenced by the presence of incontinence (p=0.001) and urgency (p=0.05). Discussions. The study highlights the need to integrate the initial status of patients into the overall quantification of the effects of surgery on the quality of life. Age does not influence the prevalence of LARS, but symptoms seem more prevalent in women. The psycho-emotional impact is relevant to the general population, so explanations given during the informed consent and accurate description of potential consequences of surgical intervention increase compliance to ensure better post-operative control of the symptomatology. Conclusions. Deriving a normative LARS-like score may alter the interpretation and discussion of LARS scores for future rectal cancer patients, and it also provides a better understanding of the emotional impact of such symptoms on certain population subsets or cultural groups

Acute respiratory distress syndrome particularities in oncological patients with AH1N1 influenza. Case series report

Grigore T. Popa University of Medicine and Pharmacy, Iași, România, Regional Institute of Oncology, Iași, România, The 5th International Congress of the Society of Anesthesiology and Reanimatology of the Republic of Moldova, 16th Edition of the International Course of Guidelines and Protocols in Anesthesia, Intensive Care and Emergency Medicine, 28th Meeting of the European Society for Computing and Technology in Anesthesia and Intensive Care, September 27-29, 2018, Chisinau, the Republic of MoldovaIntroduction: In oncological patients influenza may result in severe forms associated with high mortality (11-33% in solid cancers, 23% in bone marrow transplant), one of them being acute respiratory distress syndrome (ARDS). Objective: Evaluation of diagnostic, management and outcome particularities in ARDS AH1N1 influenza oncological patients. Material and methods: The study enrolled all ARDS AH1N1 influenza patients, diagnosed and managed in ICU IRO Iași in January-February 2018 during a hospital-acquired influenza outbreak. Analyzed parameters were patient related (cancer type and treatment), influenza related (virus type and identification tests), ARDS related (severity, management, outcome). Results: Out of 40 symptomatic or influenza contact asymptomatic patients, 21 tested positive for AH1N1 influenza virus, 9 out of them having ARDS managed in ICU. 5 pts presented severe ARDS in the context of post-therapeutic severe immune compromise: 4 pts post-chemotherapy medullary aplasia for hemato-oncological disease and 1 pt post-radio/chemo/surgical therapy for gynecological cancer. All received ventilatory support: 3 pts invasive ventilatory support in prone position (worst PaO2/FiO2 29-46 mmHg, nonsurvivors), 2 pts non-invasive ventilatory support (worst PaO2/FiO2 54-94 mmHg, survivors). After 25th Jan 2018 (first case) epidemiological alert and management were instituted, resulting in outbreak control on 23rd Feb 2018 (last case). Conclusions: In immuno-compromised oncological patients AH1N1 influenza may rapidly generate a hospital-acquired outbreak and severe ARDS forms associated with high mortality. Early diagnostic and management are the most effective strategies in reducing associated mortality and controlling influenza outbreak

Interplay of Epigenetics with Gynecological Cancer

Recent data on the cell deregulation that occurs during the progression to cancer underlines the cooperation between genetic and epigenetic alterations leading to a malignant phenotype. Unlike genetic alterations, the epigenetic changes do not affect the DNA sequence of the genes, but determine the regulation of gene expression acting upon the genome. Moreover, unlike genetic changes, epigenetic ones are reversible, making them therapeutic targets in various conditions in general and in cancer disease in particular. The term epigenetics includes a series of covalent modifications that regulate the methylation pattern of DNA and posttranslational modifications of histones. Gene expression can also be regulated at the posttranscriptional level by microRNAs (miRNAs), a family of small noncoding RNAs that inhibit the translation of mRNA to protein. miRNAs can act as ‘oncomiRs’, as tumor suppressors, or both. In this chapter, we will (1) summarize the current literature on the key processes responsible for epigenetic regulation: DNA methylation, histone modifications and posttranscriptional gene regulation by miRNAs; (2) evaluate aberrant epigenetic modifications as essential players in cancer progression; (3) establish the roles of microenvironment-mediated epigenetic perturbations in the development of gynecological neoplasia; (4) evaluate epigenetic factors involved in drug resistance

Pancreatogenic type 3C diabetes

Background. The relationship between chronic pancreatitis and diabetes is well established. This form of diabetes is secondary to exocrine pancreatic disorder and is known as diabetes mellitus type 3c (T3cDM). Materials and Methods. In this retrospective study we included 261 patients, 59 patients being diagnosed with chronic pancreatitis and secondary diabetes mellitus, and admitted in the Fundeni Clinical Institute, 2nd Department of Gastroenterology or N.C. Paulescu Institute/ Carol Davila University of Medicine and Pharmacy. Results and Discussions. Patients were 22.2% women and 77.8% men, with an average age of 56.8 years and 53.4 years respectively. 63% came from urban areas. The mean duration of chronic pancreatitis was six years. Non-diabetic patients were compared with patients who were previously diagnosed with T3cDM and who had been analyzed for body mass index (BMI). Imaging investigations were also performed to confirm pseudotumors or pancreatic tumours. Patients already considered non-diabetic had basal blood glucose values and were mostly overweight and obese. In this context, insulin resistance cannot be excluded for this group of patients. Conclusions. T3cDM is a new pathological entity that needs to be explored more deeply, and that should benefit from both a diagnostic stratification and treatment

Defects in Innate Immunity Render Breast Cancer Initiating Cells Permissive to Oncolytic Adenovirus

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SARS-CoV-2 Variant Surveillance in Genomic Medicine Era

In the genomic medicine era, the emergence of SARS-CoV-2 was immediately followed by viral genome sequencing and world-wide sequences sharing. Almost in real-time, based on these sequences, resources were developed and applied around the world, such as molecular diagnostic tests, informed public health decisions, and vaccines. Molecular SARS-CoV-2 variant surveillance was a normal approach in this context yet, considering that the viral genome modification occurs commonly in viral replication process, the challenge is to identify the modifications that significantly affect virulence, transmissibility, reduced effectiveness of vaccines and therapeutics or failure of diagnostic tests. However, assessing the importance of the emergence of new mutations and linking them to epidemiological trend, is still a laborious process and faster phenotypic evaluation approaches, in conjunction with genomic data, are required in order to release timely and efficient control measures

Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

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