Identification and Characterization of Novel Rat Polyomavirus 2 in a Colony of X-SCID Rats by P-PIT assay.

Polyomaviruses (PyVs) are known to infect a wide range of vertebrates and invertebrates and are associated with a broad spectrum of diseases, including cancers, particularly in immune-suppressed hosts. A novel polyomavirus, designated rat polyomavirus 2 (RatPyV2), was identified from a breeding colony of rats having X-linked severe combined immunodeficiency. Using a human panpolyomavirus immunohistochemistry test (P-PIT), RatPyV2 was initially detected in the parotid salivary gland of a colony member. Rolling circle amplification using DNA from harderian and parotid glands identified a novel 5.1-kb polyomavirus genome closely related to human Washington University (WU) and Karolinska Institute (KI) and vole polyomaviruses but notably divergent from Rattus norvegicus PyV1 (RnorPyV1; also designated RatPyV1). Further screening showed RatPyV2 inclusion body infection in the lung epithelium and variably in other respiratory, reproductive, and glandular tissues of 12/12 (100%) rats. IMPORTANCE Although P-PIT was developed to detect diseases associated with known human polyomaviruses, the identification of a new polyomavirus in rats suggests that it may have utility as a broad-based screen for new, as well as known polyomaviruses. Our findings suggest that RatPyV2 may be a commensal infection of laboratory rats that can lead to disseminated disease in T cell immune-deficient rats. Infection of the X-SCID rats with RatPyV2 and Pneumocystis carinii is a potential model for coinfection pathogenesis and treatment options during transplant preclinical studies.

Systemic delivery and pre-clinical evaluation of nanoparticles containing antisense oligonucleotides and siRNAs.

By virtue of their potential to selectively silence oncogenic molecules in cancer cells, antisense oligonucleotides (ASO) and small interfering RNAs (siRNAs) are powerful tools for development of tailored anti-cancer drugs. The clinical benefit of ASO/siRNA therapeutic is, however, hampered due to poor pharmacokinetics and biodistribution, and suboptimal suppression of the target in tumor tissues. Raf-1 protein serine/threonine kinase is a druggable signaling molecule in cancer therapy. Our laboratory has developed cationic liposomes for systemic delivery of raf ASO (LErafAON) and raf siRNA (LErafsiRNA) to human tumor xenografts grown in athymic mice. LErafAON is also the first ASO containing liposomal drug tested in humans. In this article, we primarily focus on a modified formulation of systemically delivered cationic liposomes containing raf antisense oligonucleotide (md-LErafAON). The cationic liposomes were prepared using dimyristoyl 1,2-diacyl-3-trimethylammonium-propane (DMTAP), phosphatidylcholine (PC), and cholesterol (CHOL). The toxicology, pharmacokinetics, biodistribution, target selectivity, and anti-tumor efficacy studies of md-LErafAON were conducted in mice. We demonstrate that md-LErafAON is the next generation of systemically delivered and well-tolerated antisense therapeutic suitable for clinical evaluation.

Pharmacokinetics, toxicity, and efficacy of ends-modified raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome.

Raf-1 protein serine threonine kinase plays an important role in cell survival and proliferation. Antisense inhibition of Raf-1 expression has been shown to enhance the cytotoxic effects of radiation and anticancer drugs. Here we have evaluated the toxicity, pharmacokinetics, and antitumor efficacy of a novel formulation of liposome-entrapped raf antisense oligodeoxyribonucleotide (LErafAON). The LErafAON preparation showed high liposome entrapment efficiency of rafAON (>85%) and stability at room temperature. In CD2F1 mice, administration of LErafAON produced no morbidity/mortality (5-35 mg/kg/dose, i.v., x12). Dose-related elevations in liver enzymes (alanine aminotransferase and aspartate aminotransferase) and histopathological changes in liver were noted in LErafAON and blank liposome groups. No morbidity/mortality and changes in clinical chemistry or histopathology were observed in New Zealand white rabbits (3.75 mg/kg/dose, i.v., x8; 6.5 mg/kg/dose, i.v., x6) or in cynomolgous monkeys (3.75 or 6.25 mg/kg/dose, i.v., x9). Transient decrease in total hemolytic complement activity (approximately 62-74%) and increases in C3a (approximately 3-fold) and Bb levels (approximately 5-12-fold) were observed in LErafAON and blank liposome groups of monkeys. A 30 mg/kg i.v. dose of LErafAON in human prostate tumor (PC-3)-bearing BALB/c athymic mice gave a terminal plasma half-life of 27 h, and intact rafAON could be detected in plasma and in normal and tumor tissues for up to at least 48 h. In monkeys, the terminal plasma half-life of 30.36 +/- 23.87 h was observed at an i.v. dose of 6.25 mg/kg. LErafAON (25 mg/kg/dose, i.v., x10) or ionizing radiation (3.8 Gy/day, x5) treatment of PC-3 tumor-bearing athymic mice led to tumor growth arrest, whereas a combination of LErafAON and ionizing radiation treatments resulted in tumor regression. LErafAON treatment caused inhibition of Raf-1 protein expression in normal and tumor tissues in these mice (>50%, versus controls). These data have formed a basis of the clinical Phase I studies of LErafAON for cancer treatment.