Protein structural insights into a rare PCSK9 gain-of-function variant (R496W) causing familial hypercholesterolemia in a Saudi family: whole exome sequencing and computational analysis.

Familial hypercholesterolemia (FH) is a globally underdiagnosed genetic condition associated with premature cardiovascular death. The genetic etiology data on Arab FH patients is scarce. Therefore, this study aimed to identify the genetic basis of FH in a Saudi family using whole exome sequencing (WES) and multidimensional bioinformatic analysis. Our WES findings revealed a rare heterozygous gain-of-function variant (R496W) in the exon 9 of the PCSK9 gene as a causal factor for FH in this family. This variant was absent in healthy relatives of the proband and 200 healthy normolipidemic controls from Saudi Arabia. Furthermore, this variant has not been previously reported in various regional and global population genomic variant databases. Interestingly, this variant is classified as "likely pathogenic" (PP5) based on the variant interpretation guidelines of the American College of Medical Genetics (ACMG). Computational functional characterization suggested that this variant could destabilize the native PCSK9 protein and alter its secondary and tertiary structural features. In addition, this variant was predicted to negatively influence its ligand-binding ability with LDLR and Alirocumab antibody molecules. This rare PCSK9 (R496W) variant is likely to expand our understanding of the genetic basis of FH in Saudi Arabia. This study also provides computational structural insights into the genotype-protein phenotype relationship of PCSK9 pathogenic variants and contributes to the development of personalized medicine for FH patients in the future.

GENETIC VARIANTS AND SERUM PROFILES OF CYTOKINES IN COVID-19 SEVERITY.

ABSTRACT: Background: Patients with severe coronavirus disease 2019 (COVID-19) are at an increased risk of acute respiratory distress syndrome and mortality. This is due to the increased levels of pro-inflammatory cytokines that amplify downstream pathways that are controlled by immune regulators. Objective: This study aimed to investigate the association between cytokine genetic variants, cytokine serum levels/profiles, and disease severity in critically and noncritically ill COVID-19 patients. Methods: This cross-sectional study recruited 646 participants who tested positive for severe acute respiratory syndrome coronavirus 2 from six collection sites across the United Arab Emirates. Medical files were accessed to retrieve clinical data. Blood samples were collected from all participants. Patients were divided into two clinical groups, noncritical (n = 453) and critical (n = 193), according to World Health Organization classification guidelines for COVID-19 patients. Cytokine analyses were conducted on serum of a subset of the cohort, specifically on 426 participants (noncritical, 264; critical, 162). Candidate gene analyses of 33 cytokine-related genes (2,836 variants) were extracted from a genome-wide association study to identify genetic variants with pleiotropic effects on a specific cytokine and the severity of COVID-19 disease. Results: Age, body mass index (BMI), and pre-existing medical conditions were found to be significant risk factors that contribute to COVID-19 disease severity. After correcting for age, sex, and BMI, IP-10 ( P < 0.001), IFN ( P = 0.001), IL-6 ( P < 0.001), and CXCL-16 ( P < 0.001) serum levels were significantly higher among critical COVID-19 cases, when compared with noncritically ill patients. To investigate if the genetic variants involved in the serum cytokine levels are associated with COVID-19 severity, we studied several genes. Single nucleotide polymorphisms in IL6 (rs1554606; odd ratio (OR) G = 0.67 [0.66, 0.68]; P = 0.017), IFNG (rs2069718; OR G = 0.63 [0.62, 0.64]; P = 0.001), MIP (rs799187; OR A = 1.69 [1.66, 1.72]; P = 0.034), and CXCL16 (rs8071286; OR A = 1.42 [1.41, 1.44]; P = 0.018) were found to be associated with critically ill patients. Polymorphisms in the CXCL10 , CCL2 , IL1 , CCL7 , and TNF genes were not associated with the COVID-19 critical phenotype. The genotypes of IL-6 (gene, IL6 [7p15.3]) and CXCL-16 (gene, CXCL16 [17p13.2]) were significantly associated with the serum levels of the respective cytokine in critical cases of COVID-19. Conclusion: Data obtained from measuring cytokine levels and genetic variant analyses suggest that IL-6 and CXCL-16 could potentially be used as potential biomarkers for monitoring disease progression of COVID-19 patients. The findings in this study suggest that specific cytokine gene variants correlate with serum levels of the specific cytokine. These genetic variants could be of assistance in the early identification of high-risk patients on admission to the clinic to improve the management of COVID-19 patients and other infectious diseases.

Analysis of SARS-CoV-2 viral loads in stool samples and nasopharyngeal swabs from COVID-19 patients in the United Arab Emirates.

Coronavirus disease 2019 (COVID-19) was first identified in respiratory samples and was found to commonly cause cough and pneumonia. However, non-respiratory symptoms including gastrointestinal disorders are also present and a big proportion of patients test positive for the virus in stools for a prolonged period. In this cross-sectional study, we investigated viral load trends in stools and nasopharyngeal swabs and their correlation with multiple demographic and clinical factors. The study included 211 laboratory-confirmed cases suffering from a mild form of the disease and completing their isolation period at a non-hospital center in the United Arab Emirates. Demographic and clinical information was collected by standardized questionnaire and from the medical records of the patient. Of the 211 participants, 25% tested negative in both sample types at the time of this study and 53% of the remaining patients had detectable viral RNA in their stools. A positive fecal viral test was associated with male gender, diarrhea as a symptom, and hospitalization during infection. A positive correlation was also observed between a delayed onset of symptoms and a positive stool test. Viral load in stools positively correlated with, being overweight, exercising, taking antibiotics in the last 3 months and blood type O. The viral load in nasopharyngeal swabs, on the other hand, was higher for blood type A, and rhesus positive (Rh factor). Regression analysis showed no correlation between the viral loads measured in stool and nasopharyngeal samples in any given patient. The results of this work highlight the factors associated with a higher viral count in each sample. It also shows the importance of stool sample analysis for the follow-up and diagnosis of recovering COVID-19 patients.

Gut Microbiota Interplay With COVID-19 Reveals Links to Host Lipid Metabolism Among Middle Eastern Populations.

The interplay between the compositional changes in the gastrointestinal microbiome, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility and severity, and host functions is complex and yet to be fully understood. This study performed 16S rRNA gene-based microbial profiling of 143 subjects. We observed structural and compositional alterations in the gut microbiota of the SARS-CoV-2-infected group in comparison to non-infected controls. The gut microbiota composition of the SARS-CoV-2-infected individuals showed an increase in anti-inflammatory bacteria such as Faecalibacterium (p-value = 1.72 × 10-6) and Bacteroides (p-value = 5.67 × 10-8). We also revealed a higher relative abundance of the highly beneficial butyrate producers such as Anaerostipes (p-value = 1.75 × 10-230), Lachnospiraceae (p-value = 7.14 × 10-65), and Blautia (p-value = 9.22 × 10-18) in the SARS-CoV-2-infected group in comparison to the control group. Moreover, phylogenetic investigation of communities by reconstructing unobserved state (PICRUSt) functional prediction analysis of the 16S rRNA gene abundance data showed substantial differences in the enrichment of metabolic pathways such as lipid, amino acid, carbohydrate, and xenobiotic metabolism, in comparison between both groups. We discovered an enrichment of linoleic acid, ether lipid, glycerolipid, and glycerophospholipid metabolism in the SARS-CoV-2-infected group, suggesting a link to SARS-CoV-2 entry and replication in host cells. We estimate the major contributing genera to the four pathways to be Parabacteroides, Streptococcus, Dorea, and Blautia, respectively. The identified differences provide a new insight to enrich our understanding of SARS-CoV-2-related changes in gut microbiota, their metabolic capabilities, and potential screening biomarkers linked to COVID-19 disease severity.

Promotion of incisional wound repair by human mesenchymal stem cell transplantation.

The purpose of this study was to determine the effect of transplanted human mesenchymal stem cells (hMSCs) on wound healing. In this model, full-thickness cutaneous wounds were created by incision in the skin of adult New Zealand white rabbits and treated by transplanted hMSCs into the wounds. Wound healing was evaluated by histological analysis and tensiometry over time. A total of 15 New Zealand white rabbits with 10 wounds per animal were examined in this study. Animals were treated with hMSCs and euthanised at 3, 7, 14, 21 and 80 days after manipulation. The hMSCs were labelled with a fluorescent dye (CM-DiI), suspended in phosphate-buffered saline and used to treat full-thickness incisional wounds in rabbit skin. Tensiometry and histology were used to characterise the wound-healing rate of the incisional wounds. These results showed that transplanted hMSCs significantly inhibited scar formation and increased the tensile strength of the wounds. Importantly, MSCs from genetically unrelated donors did not appear to induce an immunologic response. In conclusion, human mesenchymal stem cell therapy is a viable approach to significantly affect the course of normal cutaneous wound healing and significantly increase the tensile strength.

Optimization of capsid-incorporated antigens for a novel adenovirus vaccine approach.

Despite the many potential advantages of Ad vectors for vaccine application, the full utility of current Ad vaccines may be limited by the host anti-vector immune response. Direct incorporation of antigens into the adenovirus capsid offers a new and exciting approach for vaccination strategies; this strategy exploits the inherent antigenicity of the Ad vector. Critical to exploiting Ad in this new context is the placement of antigenic epitopes within the major Ad capsid protein, hexon. In our current study we illustrate that we have the capability to place a range of antigenic epitopes within Ad5 capsid protein hexon hypervariable regions (HVRs) 2 or 5, thus producing viable Ad virions. Our data define the maximal incorporation size at HVR2 or HVR5 as it relates to identical antigenic epitopes. In addition, this data suggests that Ad5 HVR5 is more permissive to a range of insertions. Most importantly, repeated administration of our hexon-modified viruses resulted in a secondary anti-antigen response, whereas minimal secondary effect was present after administration of Ad5 control. Our study describes antigen placement and optimization within the context of the capsid incorporation approach of Ad vaccine employment, thereby broadening this new methodology.

Improved anti-tumor therapy based upon infectivity-enhanced adenoviral delivery of RNA interference in ovarian carcinoma cell lines.

BACKGROUND: Hec1 (Highly Expressed in Cancer gene 1) has recently been shown to play an important role in the proper segregation of chromosomes during mitosis. Recently, an adenovirus delivery system carrying RNA interference (RNAi) of Hec1 has been reported in a cervical adenocarcinoma model. Adenoviral delivery systems, however, have the main limitation of poor viral infectivity due to lack of the native receptor, Coxsackie-Adenovirus Receptor (CAR), on the surface of tumor cells. We hypothesize that the viral infectivity of the adenovirus vector would be enhanced via a CAR-independent pathway by altering the targeting tropism, thus increasing the knockdown effect of Hec1 expression in ovarian carcinoma cells. METHODS: Two adenoviruses (Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3), along with a negative control (Ad-siRNA-GAPDH.F5/3), were created using homologous recombination. HEY and SKOV3.ip1 cell lines were used to perform experiments. The following assays were then used to determine RNAi knockdown efficiency: (1) quantitative PCR (QPCR), (2) Western blot, (3) MTS assay, (4) Annexin V-FITC FACS, (5) crystal violet staining. In all experiments, a negative control served as a baseline measure. RESULTS: QPCR demonstrated a 2-log viral infectivity enhancement with Ad-siRNA-Hec1.F5/3 over Ad-siRNA-Hec1. QPCR at 72 h revealed mRNA knockdown induced by Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cells, respectively (71%/60%, and 32%/78% mRNA knockdown compared to negative control). Western blot revealed translational inhibition induced by both Hec1 Ads with the least knockdown seen with Ad-siRNA-GAPDH.F5/3. FACS analysis revealed increased annexin V positivity in RNAi-infected cells, suggesting a higher rate of apoptosis. MTS assay indicated increased cell death 8 days post-infection with Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cell lines, respectively (75% vs. 35% and 43% vs. 12% viable cells). Crystal violet staining revealed increased cell death with Ad-siRNA-Hec1.F5/3 in all tested cell lines. CONCLUSIONS: RNAi against Hec1 results in gene expression knockdown and apoptosis in vitro. The infectivity-enhanced adenovirus as delivery mechanism shows potential application in future gene therapy models of RNAi in ovarian cancer.

Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds.

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-beta(1) and TGF-beta(2) precursor proteins, but an increase in expression of TGF-beta(3) precursor protein and TGF-beta type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

Targeting lung cancer using an infectivity enhanced CXCR4-CRAd.

Conventional treatments are not adequate for the majority of lung cancer patients. Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of neoplastic diseases, including non-small cell lung cancer. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect a new population of surrounding target cells, replicate again and eradicate the infected tumor cells while leaving normal cells unaffected. However, to date, there have been two main limitations to successful clinical application of these CRAd agents; i.e. poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.RGD, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter and the viral infectivity is enhanced by a capsid modification, RGD4C. This agent CRAd-CXCR4.RGD, as expected, improved both of the viral infectivity and tumor specificity as evaluated in an established lung tumor cell line and in primary tumor tissue from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as compared to three other promoters regularly used for targeting tumors. In addition, this agent has the potential of targeting multiple other tumor cell types. From these data, the CRAd-CXCR4.RGD appears to be a promising novel CRAd agent for lung cancer targeting with low host toxicity.

Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses.

Natural and genetically modified oncolytic viruses have been systematically tested as anticancer therapeutics. Among this group, conditionally replicative adenoviruses have been developed for a broad range of tumors with a rapid transition to clinical settings. Unfortunately, clinical trials have shown limited antitumor efficacy partly due to insufficient viral delivery to tumor sites. We investigated the possibility of using mesenchymal progenitor cells (MPC) as virus carriers based on the documented tumor-homing abilities of this cell population. We confirmed preferential tumor homing of MPCs in an animal model of ovarian carcinoma and evaluated the capacity of MPCs to be loaded with oncolytic adenoviruses. We showed that MPCs were efficiently infected with an adenovirus genetically modified for coxsackie and adenovirus receptor-independent infection (Ad5/3), which replicated in the cell carriers. MPCs loaded with Ad5/3 caused total cell killing when cocultured with a cancer cell line. In an animal model of ovarian cancer, MPC-based delivery of the Ad5/3 increased the survival of tumor-bearing mice compared with direct viral injection. Further, tumor imaging confirmed a decrease in tumor burden in animals treated with oncolytic virus delivered by MPC carriers compared with the direct injection of the adenovirus. These data show that MPCs can serve as intermediate carriers for replicative adenoviruses and suggest that the natural homing properties of specific cell types can be used for targeted delivery of these virions.

Oncolytic adenoviruses - selective retargeting to tumor cells.

Virotherapy is an approach for the treatment of cancer, in which the replicating virus itself is the anticancer agent. Virotherapy exploits the lytic property of virus replication to kill tumor cells. As this approach relies on viral replication, the virus can self-amplify and spread in the tumor from an initial infection of only a few cells. The success of this approach is fundamentally based on the ability to deliver the replication-competent viral genome to target cells with a requisite level of efficiency. With virotherapy, while a number of transcriptional retargeting strategies have been utilized to restrict viral replication to tumor cells, this review will focus primarily on transductional retargeting strategies, whereby oncolytic viruses can be designed to selectively infect tumor cells. Using the adenoviral vector paradigm, there are three broad strategies useful for viral retargeting. One strategy uses heterologous retargeting ligands that are bispecific in that they bind both to the viral vector as well as to a cell surface target. A second strategy uses genetically modified viral vectors in which a cellular retargeting ligand is incorporated. A third strategy involves the construction of chimeric recombinant vectors, in which a capsid protein from one virus is exchanged for that of another. These transductional retargeting strategies have the potential for reducing deleterious side effects, and increasing the therapeutic index of virotherapeutic agents.

Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system.

INTRODUCTION: In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Adenoviral vector mediated strategies for breast cancer gene therapy and virotherapy are a promising novel therapeutic platform for the treatment of breast cancer. However, the promiscuous tropism of adenoviruses (Ads) is a major concern. Employing tissue specific promoters (TSPs) to restrict transgene expression or viral replication is an effective way to increase specificity towards tumor tissues and to reduce adverse effects in non-target tissues such as the liver. In this regard, candidate breast cancer TSPs include promoters of the genes for the epithelial glycoprotein 2 (EGP-2), cyclooxygenase-2 (Cox-2), alpha-chemokine SDF-1 receptor (stromal-cell-derived factor, CXCR4), secretory leukoprotease inhibitor (SLPI) and survivin. METHODS: We employed E1-deleted Ads that express the reporter gene luciferase under the control of the promoters of interest. We evaluated this class of vectors in various established breast cancer cell lines, primary breast cancer cells and finally in the most stringent preclinical available substrate system, constituted by precision cut tissue slices of human breast cancer and liver. RESULTS: Overall, the CXCR4 promoter exhibited the highest luciferase activity in breast cancer cell lines, primary breast cancer cells and breast cancer tissue slices. Importantly, the CXCR4 promoter displayed a very low activity in human primary fibroblasts and human liver tissue slices. Interestingly, gene expression profiles correlated with the promoter activities both in breast cancer cell lines and primary breast cancer cells. CONCLUSION: These data suggest that the CXCR4 promoter has an ideal 'breast cancer-on/liver-off' profile, and could, therefore, be a powerful tool in Ad vector based gene therapy or virotherapy of the carcinoma of the breast.

Gene transfer to carcinoma of the breast with fiber-modified adenoviral vectors in a tissue slice model system.

Successful adenoviral (Ad) vector-mediated strategies for breast cancer gene therapy and virotherapy have heretofore been hindered by low transduction efficiency. This has recently been understood to result from a relative paucity of expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on primary tumor cells. To further investigate this issue, we evaluated the expression of CAR on breast cancer cell lines as well as primary breast cancer cells. With the exception of one patient sample, CAR expression was notably higher in the tumor cells from patients compared to CAR expression in the tumor cell lines. Furthermore, we explored CAR-independent targeting strategies to breast cancer tissue by exploring a panel of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs for their utility in breast cancer gene therapy and virotherapy. These targeting motifs included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7), and were tested using the breast cancer tissue slice model, which is the most stringent substrate system available. Of all the tested tropism modified Ad vectors, Ad5/3 exhibited the highest transductional efficiency in breast cancer. These preclinical results suggest that Ad5/3 is the most useful modification to achieve higher clinical efficacy of breast cancer gene therapy and virotherapy.

Targeted gene therapy for ovarian cancer.

Despite advances in therapy, advanced ovarian cancer maintains a dismal overall survival of 15-30%. Thus, the need for novel therapeutic modalities exists. Gene therapy represents one such approach and the purpose of this review is to present a logical rationale for the investigation of gene therapy for the treatment of ovarian cancer. The different strategies of gene therapy (molecular chemotherapy (prodrugs), mutation compensation, immunotherapy approaches, altered drug sensitivity, and virotherapy) for cancer treatment are discussed separately with attention to investigations with clinical applicability. Furthermore, the different viral vectors utilized for improvements in targeted therapy are presented. The advancements, discovery, and shortcomings are reviewed which lend itself to future directions. These future directions involve coxsackie-adenovirus receptor (CAR) independent pathways to improve infectivity and specificity to ovarian tumor cells, the potential of utilizing gene therapy as an imaging modality in detecting cancer, and incorporating the recently described technique of RNA interference. Due to the advancements in detection and targeting of ovarian cancer, coupled with the containment to the intraperitoneal cavity, gene therapy remains a promising treatment modality for ovarian cancer.

Intraoperative ultrasound: improved resection rates in breast-conserving surgery.

AIM: The objective of this study was the investigation of a possible improvement of tumor resection rate, i.e. R0 vs. R1 resection when intraoperative ultrasound evaluation of tissue margins is used during breast-conserving surgery (BCS). PATIENTS AND METHODS: A total of 250 cases were evaluated retrospectively. The impact of ultrasound analysis onto clean margin rates was evaluated. A subgroup analysis assessed histology, stage, and neoadjuvant therapy with respect to R0 resection rate and ultrasound evaluation. RESULTS: Of 250 BCS cases 84, (33.6%) underwent intraoperative ultrasound and 166 (66.4%) did not. Clean primary surgical margins (R0) were demonstrated for 218 (87.2%) patients after histological analysis. R0 resection was achieved in 81 (96.4%) patients in the ultrasound group compared to 137 (82.5%) in the control group. The difference between the two groups is significant. CONCLUSION: This study revealed a significant increase in R0 resection rates when intraoperative ultrasound was used to evaluate surgical margins.

Establishment of a mammary carcinoma cell line from Syrian hamsters treated with N-methyl-N-nitrosourea.

Clearly new breast cancer models are necessary in developing novel therapies. To address this challenge, we examined mammary tumor formation in the Syrian hamster using the chemical carcinogen N-methyl-N-nitrosourea (MNU). A single 50mg/kg intraperitoneal dose of MNU resulted in a 60% incidence of premalignant mammary lesions, and a 20% incidence of mammary adenocarcinomas. Two cell lines, HMAM4A and HMAM4B, were derived from one of the primary mammary tumors induced by MNU. The morphology of the primary tumor was similar to a high-grade poorly differentiated adenocarcinoma in human breast cancer. The primary tumor stained positively for both HER-2/neu and pancytokeratin, and negatively for both cytokeratin 5/6 and p63. When the HMAM4B cell line was implanted subcutaneously into syngeneic female hamsters, tumors grew at a take rate of 50%. A tumor derived from HMAM4B cells implanted into a syngeneic hamster was further propagated in vitro as a stable cell line HMAM5. The HMAM5 cells grew in female syngeneic hamsters with a 70% take rate of tumor formation. These cells proliferate in vitro, form colonies in soft agar, and are aneuploid with a modal chromosomal number of 74 (the normal chromosome number for Syrian hamster is 44). To determine responsiveness to the estrogen receptor (ER), a cell proliferation assay was examined using increasing concentrations of tamoxifen. Both HMAM5 and human MCF-7 (ER positive) cells showed a similar decrease at 24h. However, MDA-MB-231 (ER negative) cells were relatively insensitive to any decrease in proliferation from tamoxifen treatment. These results suggest that the HMAM5 cell line was likely derived from a luminal B subtype of mammary tumor. These results also represent characterization of the first mammary tumor cell line available from the Syrian hamster. The HMAM5 cell line is likely to be useful as an immunocompetent model for human breast cancer in developing novel therapies.

Enhanced Gene Delivery to Human Primary Endothelial Cells Using Tropism-Modified Adenovirus Vectors.

Endothelial cells have been noted to have relatively low expression of the native receptor for adenovirus serotype 5 (Ad5), coxsackie and adenovirus receptor (CAR), and are thus refractory to Ad5 infection. In this study, we hypothesize that increases in the infectivity of Ad5 in primary human pulmonary artery (HPAEC), coronary artery (HCAEC) and umbilical vein endothelial cells (HUVEC) can be achieved through genetic capsid modification of Ad5 to bypass CAR-dependent infection. The modifications tested in this study include incorporation of an integrin-binding RGD peptide motif (Ad5.RGD), a poly-lysine motif (Ad5.pK7), a combination of both of these peptide domains (Ad5.RGD.pK7), an adenovirus serotype 3 knob domain (Ad5/3Luc1) and canine adenovirus serotype 1 or 2 knob domains (Ad5Luc1-CK1 and Ad5Luc1-CK2). In HPAEC and HCAEC, the greatest infectivity enhancements were achieved using Ad5/3Luc1 (26-fold and 30-fold respectively). HUVEC was most readily infected by Ad5Luc1-CK1 (213-fold). These results demonstrate that gains in Ad5 infectivity in endothelial cells can be accomplished with genetic capsid modifications.

Cancer-specific targeting of a conditionally replicative adenovirus using mRNA translational control.

BACKGROUND: In view of the limited success of available treatment modalities for a wide array of cancer, alternative and complementary therapeutic strategies need to be developed. Virotherapy employing conditionally replicative adenoviruses (CRAds) represents a promising targeted intervention relevant to a wide array of neoplastic diseases. Critical to the realization of an acceptable therapeutic index using virotherapy in clinical trials is the achievement of oncolytic replication in tumor cells, while avoiding non-specific replication in normal tissues. In this report, we exploited cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of CRAd virotherapy agents. Heretofore, the achievement of replicative specificity of CRAd agents has been accomplished either by viral genome deletions or incorporation of tumor selective promoters. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. We show herein, the utility of a novel approach that combines both transcriptional and translational regulation strategies for the key goal of replicative specificity. METHODS: We describe the construction of a CRAd with cancer specific gene transcriptional control using the CXCR4 gene promoter (TSP) and cancer specific mRNA translational control using a 5'-untranslated region (5'-UTR) element from the FGF-2 (Fibroblast Growth Factor-2) mRNA. RESULTS: Both in vitro and in vivo studies demonstrated that our CRAd agent retains anti-tumor potency. Importantly, assessment of replicative specificity using stringent tumor and non-tumor tissue slice systems demonstrated significant improvement in tumor selectivity. CONCLUSIONS: Our study addresses a conceptually new paradigm: dual targeting of transgene expression to cancer cells using both transcriptional and mRNA translational control. Our novel approach addresses the key issue of replicative specificity and can potentially be generalized to a wide array of tumor types, whereby tumor selective patterns of gene expression and mRNA translational control can be exploited.

Treatment of ovarian cancer with a novel dual targeted conditionally replicative adenovirus (CRAd).

OBJECTIVES: Current virotherapy strategies for ovarian cancer have been hampered by limitations in target cell infectivity and nonspecific tissue replication. In an effort to circumvent these limitations, we evaluated various CRAds modified to incorporate novel capsid targeting motifs (RGD and chimeric Ad5/3) with a novel tissue-specific promoter (CXCR4). METHODS: Two novel CRAds (Ad5-CXCR4-F5/3 and Ad5-CXCR4-RGD) were constructed via homologous recombination and verified by PCR and DNA sequencing. The infectivity and viral replication rates of these two CRAds were analyzed via quantitative real-time PCR (QRT-PCR) in cell line experiments using three ovarian cancer cell lines (SKOV3.ip1, Hey, and OV4) and compared to that achieved with a clinical grade CRAd (delta24-RGD) to be evaluated in a Phase I trial. Cytocidal effects were determined by crystal violet staining in these same cell lines infected with different concentrations of viral particles per cell (0, 0.1, 1, 10, 100, and 500). Additionally, viral replication was evaluated by QRT-PCR in primary ovarian cancer tissue slices from multiple patients with ovarian cancer as well as in primary human normal liver tissue slices in order to establish CRAd selectivity. All experiments incorporated appropriate controls and repeated in triplicate. RESULTS: Compared to RGD-capsid CRAds (delta24-RGD and CXCR4-RGD), the F5/3-capsid CRAd (CXCR4-F5/3) demonstrated significant improvements in infection rates (p=0.025, 0.006, and 0.006) in all ovarian cancer cell lines tested (SKOV3.ip1, Hey, and OV4, respectively). In addition to improved transduction of virus into the cells, the TSP CXCR4-based CRAds demonstrated improved viral replication. Specifically, CXCR4-F5/3 further enhanced viral replication 89-fold (p=0.009, 0.010, 0.003) in the same cancer cell lines. Furthermore, CXCR4-F5/3 showed a 4-log improvement in oncolytic potential over delta24-RGD. In the ex vivo primary ovarian tissue slices, CXCR4-F5/3 showed a 58-fold improvement in viral replication (p=0.005) compared to the clinical grade delta24-RGD. Both CXCR4-F5/3 and CXCR4-RGD demonstrated significant reduction of viral replication in normal liver slices (p=0.001). CONCLUSIONS: These data suggest that a dual targeted approach is feasible for the combined enhancement of infectivity and replication in ovarian cancer with a specificity that was attenuated in normal liver tissues. In fact, CXCR4-F5/3 outperformed our best CRAd agent to date nearly 60-fold in our most stringent ex vivo model of primary ovarian cancer tissue slices and suggests that this novel agent could be useful for the treatment of ovarian cancer.

Combining high selectivity of replication via CXCR4 promoter with fiber chimerism for effective adenoviral oncolysis in breast cancer.

Conditionally replicative adenoviruses (CRAds) represent novel therapeutic agents that have been recently applied in the context of breast cancer therapy. However, deficiencies in the ability of the adenovirus to infect target tumor cells and to specifically replicate within the tumor target represent key deficiencies preventing the realization of the full potential of this therapeutic approach. Minimal expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackie and adenovirus receptor) on breast cancer cells represents a major limitation for Ad5-based virotherapy. Genetic fiber chimerism is a method to alter the tropism of Ad5-based CRAds to achieve CAR-independent infectivity of tumor cells. Here, we describe the use of a CRAd with cancer specific transcriptional control of the essential Ad5 E1A gene using the human CXCR4 gene promoter. We further modified the fiber protein of this agent by switching the knob domain with that of the adenovirus serotype 3. The oncolytic activity of this 5/3 fiber-modified CRAd was studied in breast cancer cell lines, primary breast cancer and human liver tissue slices from patients, and in a xenograft breast cancer mouse model. This infectivity enhanced CRAd agent showed improved replication and killing in breast cancer cells in vitro and in vivo with a remarkable specificity profile that was strongly attenuated in nonbreast cancer cells, as well as in normal human breast and liver tissues. In conclusion, utilization of a CRAd that combined infectivity enhancement strategies and transcriptional targeting improved the CRAd-based antineoplastic effects for breast cancer therapy.