MSCs loaded with oncolytic reovirus: migration and in vivo virus delivery potential for evaluating anti-cancer effect in tumor-bearing C57BL/6 mice

Background and aims Several oncolytic viruses applications have been approved in the clinic or in different phases of clinical trials. However, these methods have some rudimentary problems. Therefore, to enhance the delivery and quality of treatment, considering the advantage of cell carrier-based methods such as Mesenchymal Stem Cells (MSC) have been proposed. This study was designed to evaluate the performance and quality of cancer treatment based on MSCs loaded by oncolytic reovirus in the cancerous C57BL/6 mouse model. Also, we evaluated MSCs migration potency in vitro and in vivo following the oncolytic reovirus infection. Methods C57BL/6 mice were inoculated with TC-1 cell lines and tumors were established in the right flank. Mice were systemically treated with reovirus, MSCs-loaded with reovirus, MSCs, and PBS as a control in separated groups. Effects of infected AD-MSCs with reovirus on tumor growth and penetration in the tumor site were monitored. All groups of mice were monitored for two months in order to therapeutic and anticancer potential. After treatments, tumor size alteration and apoptosis rate, as well as cytokine release pattern was assessed. Results The results of the current study indicated that the effect of reovirus infection on AD-MSCs is not devastating the migration capacity especially in MOI 1 and 5 while intact cells remain. On the other hand, MSCs play an efficient role as a carrier to deliver oncolytic virus into the tumor site in comparison with systemic administration of reovirus alone. Apoptosis intensity relies on viral titration and passing time. Followed by systemic administration, treatment with oncolytic reovirus-infected AD-MSCs and MSCs alone had shown significant inhibition in tumor growth. Also, treatment by reovirus causes an increase in IFN-γ secretion. Conclusion The results of in vitro and in vivo study confirmed the tumor-homing properties of infected AD-MSCs and the significant antitumor activity of this platform. Hence, our results showed that the cell carrier strategy using oncolytic reovirus-loaded AD-MSCs enhanced virus delivery, infiltration, and antitumor activity can be effectively applied in most cancers.

Experimental Reptarenavirus Infection of Boa constrictor and Python regius

Boid inclusion body disease (BIBD) causes losses in captive snake populations globally. BIBD is associated with the formation of cytoplasmic inclusion bodies (IBs), which mainly comprise reptarenavirus nucleoprotein (NP). In 2017, BIBD was reproduced by cardiac injection of boas and pythons with reptarenaviruses, thus demonstrating a causative link between reptarenavirus infection and the disease. Here, we report experimental infections of Python regius (n = 16) and Boa constrictor (n = 16) with three reptarenavirus isolates. First, we used pythons (n = 8) to test two virus delivery routes: intraperitoneal injection and tracheal instillation. Viral RNAs but no IBs were detected in brains and lungs at 2 weeks postinoculation. Next, we inoculated pythons (n = 8) via the trachea. During the 4 months following infection, snakes showed transient central nervous system (CNS) signs but lacked detectable IBs at the time of euthanasia. One of the snakes developed severe CNS signs; we succeeded in reisolating the virus from the brain of this individual and could demonstrate viral antigen in neurons. In a third attempt, we tested cohousing, vaccination, and sequential infection with multiple reptarenavirus isolates on boas (n = 16). At 10 months postinoculation, all but one snake tested positive for viral RNA in lung, brain, and/or blood, but none exhibited the characteristic IBs. Three of the four vaccinated snakes seemed to sustain challenge with the same reptarenavirus; however, neither of the two snakes rechallenged with different reptarenaviruses remained uninfected. Comparison of the antibody responses in experimentally versus naturally reptarenavirus-infected animals indicated differences in the responses. IMPORTANCE In the present study, we experimentally infected pythons and boas with reptarenavirus via either intraperitoneal injection or tracheal instillation. The aims were to experimentally induce boid inclusion body disease (BIBD) and to develop an animal model for studying disease transmission and pathogenesis. Both virus delivery routes resulted in infection, and infection via the trachea could reflect the natural route of infection. In the experimentally infected snakes, we did not find evidence of inclusion body (IB) formation, characteristic of BIBD, in pythons or boas. Most of the boas (11/12) remained reptarenavirus infected after 10 months, which suggests that they developed a persistent infection that could eventually have led to BIBD. We demonstrated that vaccination using recombinant protein or an inactivated virus preparation prevented infection by a homologous virus in three of four snakes. Comparison of the antibody responses of experimentally and naturally reptarenavirus-infected snakes revealed differences that merit further studies.

Endovascular Selective Intra-Arterial Infusion of Mesenchymal Stem Cells Loaded With Delta-24 in a Canine Model

BACKGROUND Delta-24-RGD, an oncolytic adenovirus, shows promise against glioblastoma. To enhance virus delivery, we recently demonstrated that human bone marrow-derived mesenchymal stem cells loaded with Delta-24-RGD (hMSC-D24) can eradicate glioblastomas in mouse models. There are no studies examining the safety of endovascular selective intra-arterial (ESIA) infusions of MSC-D24 in large animals simulating human clinical situations. OBJECTIVE To perform canine preclinical studies testing the feasibility and safety of delivering increasing doses of hMSCs-D24 via ESIA infusions. METHODS ESIA infusions of hMSC-D24 were performed in the cerebral circulation of 10 normal canines in the target vessels (internal carotid artery [ICA]/P1) via transfemoral approach using commercially available microcatheters. Increasing concentrations of hMSC-D24 or particles (as a positive control) were injected into 1 hemisphere; saline (negative control) was infused contralaterally. Toxicity (particularly embolic stroke) was assessed on postinfusion angiography, diffusion-weighted magnetic resonance imaging, clinical exam, and necropsy. RESULTS ESIA injections were performed in the ICA (n = 7) or P1 (n = 3). In 2 animals injected with particles (positive control), strokes were detected by all assays. Of 6 canines injected with hMSC-D24 through the anterior circulation, escalating dose from 2 × 106 cells/20 mL to 1 × 108 cells/10 mL resulted in no strokes. Two animals had ischemic and hemorrhagic strokes after posterior cerebral artery catheterization. A survival experiment of 2 subjects resulted in no complications detected for 24-h before euthanization. CONCLUSION This novel study simulating ESIA infusion demonstrates that MSCs-D24 can be infused safely at least up to doses of 1 × 108 cells/10 mL (107 cells/ml) in the canine anterior circulation using commercially available microcatheters. These findings support a clinical trial of ESIA infusion of hMSCs-D24.

Experimental Reptarenavirus Infection of Boa constrictor and Python regius

ABSTRACTBoid inclusion body disease (BIBD) causes losses in captive constrictor snake populations globally. BIBD associates with formation of cytoplasmic inclusion bodies (IB) which mainly comprise reptarenavirus nucleoprotein (NP). In 2017, BIBD was reproduced by cardiac injection of boas and pythons with reptarenaviruses, thus demonstrating a causative link between reptarenavirus infection and the disease. Herein, we report experimental infections of pythons (N=16) and boas (N=16) with three reptarenavirus isolates. First, we used pythons (N=8) to test two virus delivery routes: intraperitoneal injection and tracheal instillation. Independent of the delivery route, we detected viral RNA but no IBs in tissues two weeks post inoculation. Next, we inoculated pythons (N=8) via the trachea. During the four month following the infection snakes showed transient central nervous system (CNS) signs but lacked detectable IB at the time of euthanasia. One of the snakes developed severe CNS signs and we succeeded in re-isolating the virus from the brain of this individual, and could demonstrate viral antigen in neurons. In a third attempt, we tested co-housing, vaccination, and sequential infection with multiple reptarenavirus isolates on boas (N=16). At 10 months post inoculation all except one snake tested positive for viral RNA but none exhibited the characteristic IB. Analysis of the antibody responses demonstrated lower neutralizing but higher anti-reptarenavirus NP titers in experimentally versus naturally reptarenavirus infected boas. Our findings suggest that in addition to reptarenavirus infection, other factors, e.g. the antibody response, contribute to BIBD pathogenesis.IMPORTANCEA 2017 study demonstrated cardiac reptarenavirus injection to induce boid inclusion body disease (BIBD) in pythons and boas. In the present study, we experimentally infected pythons and boas with reptarenavirus via either intraperitoneal injection or tracheal instillation. We found both virus delivery routes to result in infection; though the latter could reflect the natural route of infection. In the experimentally infected snakes, we did not find evidence of inclusion body (IB) formation, characteristic to BIBD, in pythons or in boas. Most of the snakes (11/12) studied were reptarenavirus infected after ten-month follow up, which suggests that they could eventually have developed BIBD. We further found differences between the antibody responses of experimentally and naturally reptarenavirus infected snakes, which could indicate that the pathogenesis of BIBD involves factors additional to reptarenavirus infection. As snakes are poikilotherm, also the housing conditions could have an effect.

A Mouse Model of Oropharyngeal Papillomavirus-Induced Neoplasia Using Novel Tools for Infection and Nasal Anesthesia

Human head and neck cancers that develop from the squamous cells of the oropharynx (Oropharyngeal Squamous Cell Carcinomas or OPSCC) are commonly associated with the papillomavirus infection. A papillomavirus infection-based mouse model of oropharyngeal tumorigenesis would be valuable for studying the development and treatment of these tumors. We have developed an efficient system using the mouse papillomavirus (MmuPV1) to generate dysplastic oropharyngeal lesions, including tumors, in the soft palate and the base of the tongue of two immune-deficient strains of mice. To maximize efficiency and safety during infection and endoscopy, we have designed a nose cone for isoflurane-induced anesthesia that takes advantage of a mouse’s need to breathe nasally and has a large window for oral manipulations. To reach and infect the oropharynx efficiently, we have repurposed the Greer Pick allergy testing device as a virus delivery tool. We show that the Pick can be used to infect the epithelium of the soft palate and the base of the tongue of mice directly, without prior scarification. The ability to induce and track oropharyngeal papillomavirus-induced tumors in the mouse, easily and robustly, will facilitate the study of oropharyngeal tumorigenesis and potential treatments.

Gene Transfer in Rodent Nervous Tissue Following Hindlimb Intramuscular Delivery of Recombinant Adeno-Associated Virus Serotypes AAV2/6, AAV2/8, and AAV2/9

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus–mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.