A lentivirus-vectored feline erythropoietin gene therapy strategy in tissue culture and rodent models for the potential treatment of chronic renal disease-associated anemia.

OBJECTIVE: The aim of this study was to assess the efficacy and safety of a third-generation lentivirus-based vector encoding the feline erythropoietin (EPO) (feEPO) gene in vitro and in rodent models in vivo. This vector incorporates a genetic mechanism to facilitate the termination of the therapeutic effect in the event of supraphysiologic polycythemia, the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene." ANIMALS: CFRK cells and replication-defective lentiviral vectors encoding feEPO were used for in vitro experiments. Eight Fischer rats were enrolled in the pilot in vivo study, 24 EPO-deficient mice were used in the initial mouse study, and 15 EPO-deficient mice were enrolled in the final mouse study. METHODS: Efficacy of a third-generation lentivirus encoding feEPO was determined in vitro using western blot assays. Subsequently, in a series of rodent experiments, animals were administered the viral vector in progressively increasing inoculation doses with serial measurements of blood packed cell volume (PCV) over time. RESULTS: We documented production of feEPO protein in transduced CRFK cells with subsequent cessation of production when treated with the HSV-TK substrate ganciclovir. In vivo, we demonstrated variably persistent elevated PCV values in treated rats and mice with eventual return to baseline values over time. CLINICAL RELEVANCE: These results provide justification for a lentiviral gene therapy approach to the treatment of nonregenerative anemia associated with chronic renal disease in cats.

Biopsy of an intracardiac paraganglioma in a dog using a fluoroscopically guided endovascular technique.

A 10-year-old female spayed mixed breed dog was evaluated for diarrhea and vomiting. Diagnostic imaging demonstrated the presence of an intracardiac mass. A modified Seldinger technique was used to access the right jugular vein, and an endomyocardial biopsy forceps was introduced through a sheath to obtain several biopsies. Histopathology and immunohistochemistry demonstrated a paraganglioma. The dog underwent 1 fraction of radiotherapy and l-asparaginase chemotherapy and was discharged. The dog developed a pulmonary thromboembolism 2 days after radiotherapy and chemotherapy, and the owner elected humane euthanasia. Although long-term assessment of treatment response was unable to be performed, this novel diagnostic option could be considered for similar cases due to success in obtaining a histopathologic diagnosis, which is essential in developing a disease-specific treatment plan. This report also describes the use of radiotherapy for primary treatment of an intracardiac neoplasm, which can be a consideration in the future.

An RNA-Directed Gene Editing Strategy for Attenuating the Infectious Potential of Feline Immunodeficiency Virus-Infected Cells: A Proof of Concept.

Modern antiretroviral therapy for immunodeficiency viruses, although remarkably effective in controlling viral transcription, and overt virus-associated morbidity, has failed to absolutely eradicate retroviruses from their infected hosts as a result of proviral integration in long-lived reservoir cells. Immunodeficiency virus-infected patients are therefore consigned to lifelong antiviral therapy as a means to control viremia, viral transmission, and infection-associated morbidity. Unfortunately, lifelong antiviral therapies can be difficult for patients to continuously maintain and may be associated with therapy-specific morbidities. Patient advocates have argued for new methods to achieve retroviral eradication. As a proof-of-concept study, a lentivirus-delivered RNA-directed gene editing strategy was utilized in a series of in vitro experiments in an attempt to attenuate the feline immunodeficiency virus (FIV) proviral load, viral transcription, and production of infectious virions. We found that a feline T lymphocyte cell line (MCH5-4) treated with an FIV-specific clustered regularly interspersed short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing tool resulted in a reduction of cell-free viral RNA relative to control cells. Decreased infectious potential was demonstrated in a two-step FIV infection study-naïve MCH5-4 cells infected with cell-free FIV harvested from FIV-infected and CRISPR lentivirus-treated cells had less integrated proviral DNA than control cells. This study represents the initial steps towards the development of an effective method of proviral eradication in an immunodeficiency virus-infected host.