Influence of Autologous In Vitro Activation of Ovaries by Stem Cells and Growth Factors on Endocrine and Reproductive Function of Patients with Ovarian Insufficiency-A Clinical Trial Study.

BACKGROUND: Premature ovarian failure (POF) can be found in 1% of women at the age of 35-40, mostly due to unknown causes. PI3K-Akt signaling is associated with both ovarian function and growth of primordial follicles. In this study, we examined the effects of autologous in vitro ovarian activation with stem cells and autologous growth factors on reproductive and endocrine function in patients with ovarian impairment. MATERIALS AND METHODS: The longitudinal prospective observational study included 50 patients (between 30 and 50 years) with a diagnosis of POF and infertility. This multicenter study was performed at Jevremova Special Hospital in Belgrade, Saint James Hospital (Malta), and Remedica Skoplje Hospital, between 2015 and 2018. All patients went through numerous laboratory testings, including hormonal status. The autologous bone marrow mesenchymal stem cells (BMSCs) and growth factors were used in combination for activation of ovarian tissue before its re-transplantation. The software package SPSS 20.0 was used for statistical analysis of the results. RESULTS: Differences in follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (PG) hormone concentrations before and after 3, 6, and 12 months post-transplantation were tested in correlation with the volume of transplanted ovarian tissue. A significant correlation (P=0.029) was found between the change in E2 level after 3 months and the volume of re-transplanted tissues. Also after re-transplantation, 64% of the patients had follicles resulting in aspiration of oocytes in 25% of positive women with follicles. CONCLUSION: The SEGOVA method could potentially solve many human reproductive problems in the future due to the large number of patients diagnosed with POF, as well asthe possibility of delaying menopause, thus improving the quality of life and general health (Registration number: NCT04009473).

Combinatorial targeting of 2 different steps in adenoviral DNA replication by herpes simplex virus thymidine kinase and artificial microRNA expression for the inhibition of virus multiplication in the presence of ganciclovir.

BACKGROUND: Human adenoviruses are a frequent threat to immunocompromised patients, and disseminated disease is associated with severe morbidity and mortality. Current drugs are not capable of preventing all fatalities, thus indicating the need for alternative treatment strategies. Adenoviruses can be rendered susceptible to antiherpetic prodrugs such as ganciclovir (GCV), upon expression of the herpes simplex virus thymidine kinase (HSV-TK) gene in adenovirus-infected cells. Furthermore, adenoviruses are amenable to post-transcriptional gene silencing via small interfering RNAs (siRNAs) or artificial micro RNAs (amiRNAs). RESULTS: In this study, we combined these 2 approaches by constructing a combinatorial gene expression cassette that comprises the HSV-TK gene and multiple copies of an amiRNA directed against the mRNA encoding the adenoviral preterminal protein (pTP). HSV-TK gene expression was controlled by the adenoviral E4 promoter, which is activated in the presence of the adenoviral E1 gene products (i.e., when adenovirus is present in the cell). When inserted into a replication-deficient (E1-, E3-deleted) adenoviral vector, this cassette effectively inhibited the replication of wild-type adenovirus in vitro. The reduction rate mediated by the combinatorial approach was higher compared to that achieved by either of the 2 approaches alone, and these obvious additive effects became most pronounced when the GCV concentration was low. CONCLUSIONS: The concept presented here has the potential to aid in the inhibition of wild-type adenovirus replication. Furthermore, the combinatorial expression cassette may constitute a safeguard to potentially control unintended replication of adenoviral vectors and to prevent immune responses provoked by them.

An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs.

Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude.

Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery.

Human adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugs have a limited effect on adenoviruses, and existing treatment modalities often fail to prevent fatal outcome. Silencing of viral genes by short interfering RNAs (siRNAs) holds a great promise in the treatment of viral infections. The aim of the present study was to identify adenoviral candidate targets for RNA interference-mediated inhibition of adenoviral replication. We investigated the impact of silencing of a set of early, middle, and late viral genes on the replication of adenovirus 5 in vitro. Adenovirus replication was inhibited by siRNAs directed against the adenoviral E1A, DNA polymerase, preterminal protein (pTP), IVa2, hexon, and protease genes. Silencing of early and middle genes was more effective in inhibiting adenovirus multiplication than was silencing of late genes. A siRNA directed against the viral DNA polymerase mRNA decreased viral genome copy numbers and infectious virus progeny by several orders of magnitude. Since silencing of any of the early genes directly or indirectly affected viral DNA synthesis, our data suggest that reducing viral genome copy numbers is a more promising strategy for the treatment of adenoviral infections than is reducing the numbers of proteins necessary for capsid generation. Thus, adenoviral DNA replication was identified as a key target for RNAi-mediated inhibition of adenovirus multiplication. In addition, the E1A transcripts emerged as a second important target, because its knockdown markedly improved the viability of cells at late stages of infection.

Targeted expression of herpes simplex virus thymidine kinase in adenovirus-infected cells reduces virus titers upon treatment with ganciclovir in vitro.

BACKGROUND: Adenoviruses are a frequent cause of life-threatening infections in immunocompromised patients. Available therapeutics still cannot completely prevent fatal outcomes. By contrast, herpes viruses are well treatable with prodrugs such as ganciclovir (GCV), which are selectively activated in virus-infected cells by virus-encoded thymidine kinases. This effective group of prodrugs is not applicable to adenoviruses and other DNA viruses because they lack those kinases. METHODS: To render adenoviruses amenable to GCV treatment, we generated an adenoviral vector-based delivery system for targeted expression of herpes simplex virus thymidine kinase (HSV-TK) in wild-type adenovirus 5 (wt Ad5)-infected cells. HSV-TK expression was largely restricted to wt virus-infected cells by transcription of the gene from the Ad5 E4 promoter. Its activity is dependent on the adenoviral E1A gene product which is not produced by the vector but is only provided in cells infected with wt adenovirus. The anti-adenoviral effect of HSV-TK expression and concomitant treatment with GCV was assessed in vitro in four different cell lines or primary cells. RESULTS: E4 promoter-mediated HSV-TK background expression was sufficiently low to prevent cytotoxicity in the presence of low-levels GCV in cells not infected with wt Ad5. However, expression was several-fold increased in wt Ad5-infected cells and treatment with low levels of GCV efficiently inhibited wt Ad5 DNA replication. Genome copy numbers and output of infectious particles were reduced by up to > 99.99% and cell viability was greatly increased. CONCLUSIONS: We extended the concept of enzyme/prodrug therapy to adenovirus infections by selectively sensitizing adenovirus-infected cells to treatment with GCV.