Gene therapy is a promising treatment option for cancer. However, its utility may be limited due to expression in off-target cells. Cancer-specific promoters such as telomerase reverse transcriptase ...(TERT), survivin, and chemokine receptor 4 (CXCR4) have enhanced activity in a variety of human and murine cancers, however, little has been published regarding these promoters in dogs. Given the utility of canine cancer models, the activity of these promoters along with adenoviral E2F enhanced E1a promoter (EEE) was evaluated in a variety of canine tumors, both from the endogenous gene and from exogenously administered constructs. Endogenous expression levels were measured for cTERT, cSurvivin, and cCXCR4 and were low for all three, with some non-malignant and some tumor cell lines and tissues expressing the gene. Expression levels from exogenously supplied promoters were measured by both the number of cells expressing the construct and the intensity of expression in individual cells. Exogenously supplied promoters were active in more cells in all tumor lines than in normal cells, with the EEE promoter being most active, followed by cTERT. The intensity of expression varied more with cell type than with specific promoters. Ultimately, no single promoter was identified that would result in reliable expression, regardless of the tumor type. Thus, these findings imply that identification of a pan-cancer promoter may be difficult. In addition, this data raises the concern that endogenous expression analysis may not accurately predict exogenous promoter activity.
Extracting sufficient quantity and quality RNA from bone is essential for downstream application, such as transcriptomic sequencing, to evaluate gene expression. Isolation of RNA from bone presents a ...unique challenge owing to the hypocellular, brittle and mineralized matrix, which makes homogenizing the tissue difficult and provides little RNA to work with. Removal of contaminating tissue, such as bone marrow and connective tissue, is essential for isolating RNA that is unique to osteoblasts, osteoclasts and osteocytes. This study established a method to effectively isolate RNA from normal canine bone cells using the phalanges, without contamination from other tissue types, for downstream transcriptomic analysis.
A combination of physical manipulation to remove exterior tissue, washing and centrifugation to remove cells and fat within the diaphysis, homogenization using a mortar and pestle on dry ice prior to bead dissociation, followed by acid guanidinium thiocyanate-phenol-chloroform extraction and column purification yielded sufficient quantity and quality RNA.
Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface ...receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.
Despite the high safety profile demonstrated in clinical trials, the immunogenicity of adeno-associated virus (AAV)-mediated gene therapy remains a major hurdle. Specifically, T-cell-mediated immune ...responses to AAV vectors are related to loss of efficacy and potential liver toxicities. As post-translational modifications in T cell epitopes have the potential to affect immune reactions, the cellular immune responses to peptides derived from spontaneously deamidated AAV were investigated. Here, we report that highly deamidated sites in AAV9 contain CD4 T cell epitopes with a Th1 cytokine pattern in multiple human donors with diverse human leukocyte antigen (HLA) backgrounds. Furthermore, some peripheral blood mononuclear cell (PBMC) samples demonstrated differential T cell activation to deamidated or non-deamidated epitopes. Also, in vitro and in silico HLA binding assays showed differential binding to the deamidated or non-deamidated peptides in some HLA alleles. This study provides critical attributes to vector-immune-mediated responses, as AAV deamidation can impact the immunogenicity, safety, and efficacy of AAV-mediated gene therapy in some patients.
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Deamidation of AAV vectors occurs after long-term storage. Here, we show that deamidation of AAV-derived peptides can increase T cell immunogenicity in some individuals and decrease it in others. This subject-to-subject variability is associated with differential binding to some HLA molecules.
Adenoviruses are the most widely used vectors in cancer gene therapy. Adenoviruses vectors are well characterized and are easily manipulated. Adenovirus serotype 5 (Ad5) is the most commonly used ...human serotype. Ad5 internalization into host cells is a combined effect of binding of Ad5 fiber knob with the coxsackie virus and adenovirus receptor (CAR) and binding of RGD motifs in viral penton to cell surface integrins (αvβ3, αvβ5). Ad5's wide range of host-cell transduction and lack of integration into the host genome have made it an excellent choice for cancer therapeutics. However, Ad5 has limited ability to transduce cells of hematopoietic origin. It has been previously reported that low or no expression of CAR is a potential obstacle to Ad5 infection in hematopoietic origin cells. In addition, we have previously reported that low levels of cell surface integrins (αvβ3, αvβ5) may inhibit Ad5 infection in canine lymphoma cell lines. In the current report we have examined the ability of an Ad5 vector to infect human (HEK293) and canine non-cancerous (NCF and PBMC), canine non-hematopoietic origin cancer (CMT28, CML7, and CML10), and canine hematopoietic origin cancer (DH82, 17-71, OSW, MPT-1, and BR) cells. In addition, we have quantified CAR, αvβ3 and αvβ5 integrin transcript expression in these cells by using quantitative reverse transcriptase PCR (q-RT-PCR). Low levels of integrins were present in MPT1, 17-71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. CAR mRNA levels were comparatively higher in MPT1, 17-71, OSW, and PBMC cells. This report confirms and expands the finding that low or absent expression of cell surface integrins may be the primary reason for the inability of Ad5-based vectors to transduce cells of lymphocytic origin and some myeloid cells but this is not true for all hematopoietic origin cells. For efficient use of Ad5-based therapeutic vectors in cancers of lymphocytic origin, it is important to address the defects in cell surface integrins.
Oncolytic virotherapy is a promising therapeutic approach designed to selectively eradicate cancer cells but to spare normal cells. Oncolytic adenoviruses (Ad) are an outstanding and common vehicle ...for efficient cancer gene therapy. Selection of appropriate intermediate animal models is a basic requirement for successful cancer virotherapy. Some unique characteristics such as inter-individual and intratumoral heterogeneity, similar genomic sequence and instability to humans, and similar environmental exposures validate the dog as a suitable intermediate animal model of cancer and other complex human diseases. Although adenoviruses are the most commonly used viral vector for gene therapy, several concerns, such as normal cell/organ toxicity, lack of suitable cell surface receptors to allow viral entry to the desired cancer cell, and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of these vectors. Hence, new approaches to improve the transductional and transcriptional targeting efficacy of Ad are required for adapting the infectivity of adenovirus in disseminated canine tumors such as multicentric lymphoma, which can serve as a model for human non-Hodgkin’s lymphoma (NHL). Our goal is to explore mechanisms to target canine tumors at both the level of transduction (and infection) and transcription. Targeted modification of several viral components, such as the viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for conducting the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is time consuming and expensive. It is solely dependent on the presence of unique restriction enzyme sites that may or may not be present in the target site. Clustered regularly interspaced short palindromic repeat (CRISPR) along with an RNA-guided nuclease called Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in various cells and organisms. Thus, we utilized in vitro CRISPR/Cas9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification in the viral genome. We have conducted CRISPR/Cas9-mediated insertional mutagenesis and successfully inserted the RFP (red fluorescent protein) reporter construct into the CAV2 genome. We have also utilized the CRISPR/Cas9 system to conduct wild type gene replacement with our desired ligand in the CAV-2 genome to construct tumor-targeted vectors. Results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. For transcriptional targeting, several previously identified tumor up-regulated promoters, CXCR4, Survivin and TERT along with an E2F modified Canine Adenovirus 2 E1A promoter (EEE), were tested for their expression in various canine tumor cells/tissues by employing RT-qPCR and GFP reporter gene expression levels to measure endogenous and exogenous promoter activity, respectively. Endogenous expression levels measured for cTERT, cSurvivin, and cCXCR4 were low for all three, with some non-malignant and some tumor cell lines and tissues expressing the gene. Expression levels from exogenously supplied promoters were measured by both the number of cells expressing the construct and the intensity of expression in individual cells. Exogenously supplied promoters were active in more cells in all tumor lines than in normal cells, with the EEE promoter being most active, followed by cTERT. The intensity of expression varied more with cell type than with specific promoters. Ultimately, no single promoter was identified that would result in reliable expression, regardless of the tumor type. Thus, these findings imply that the three investigated promoters are unsuitable for use as lymphoma-specific promoters. In addition, this data raises the concern that endogenous expression analysis may not accurately predict exogenous promoter activity. We hope that these approaches and findings will help us to construct oncolytic CAV-2 suitable to eliminate current obstacles for oncolytic virotherapy in multicentric canine tumors such as lymphoma. In addition, these approaches may be translated to human patients and will allow adenoviruses to be successfully utilized as gene therapy vectors for a wide range of autoimmune and other diseases beyond hematopoietic malignancies.
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