Porcine Teschovirus-1 Research Articles

1030. A Rhesus Macaque Transplant Model for Drug Selection of Hematopoietic Stem Cells Transduced with MGMT Vectors

Gene transfer of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) into hematopoietic stem cells (HSCs) results in efficient in vivo selection of murine HSCs after treatment with temozolomide (TMZ) and O6-benzylguanine (BG). In addition, MGMT gene transfer into HSCs can protect hematopoiesis from the toxic side effects of chemotherapeutic agents in the treatment of malignant diseases. We are studying whether this drug resistance system will be efficacious in non-human primates using a rhesus macaque autologous transplantation model. In our first two transplant cases, we used an MSCV-P140K-IRES-GFP vector to transduce bone marrow cells that had been harvested after treatment with SCF and G-CSF, and then purified on a Miltenyi CliniMACS column using the anti-CD34 MoAb clone 563. Both animals engrafted after total body irradiation. The first animal showed relatively high levels of GFP+ B cells (40%) and lower levels of GFP+ granulocytes (3%) in the peripheral blood prior to treatment with 2 courses of TMZ and BG, 88 and 98 days after transplant. After the second course of drug selection, there was an increase in GFP+ granulocytes from 3% to a peak of 18%. These levels gradually declined back to baseline over the next 75 days. Subsequent treatment courses did not result in further selection. The second transplant case had less than 1% GFP+ granulocytes in the peripheral blood, probably due to an insufficient dose of total body irradiation, and no evidence of in vivo selection following drug treatment. In order to achieve higher levels of marking prior to selection, and to test whether inclusion of HOXB4 would improve the efficiency of selection, we developed a series of SIV vectors which co-expressed various combinations of P140K-MGMT, HOXB4, and GFP using the self-cleavage peptide 2A from porcine teschovirus-1 (P2A). A tricistronic SIV vector was constructed that incorporated all three genes by using different 2A sequences from the porcine teschovirus-1 and the asigna virus. Western blot analysis showed complete cleavage of each protein with no fusion proteins detected. We then generated amphotropic pseudotyped vectors with average titers ranging from 2 |[acute]| 105 to 1.2 |[acute]| 106 per milliliters on HeLa cells. Transduction of rhesus CD34+ cells with these SIV vectors resulted in around 40% GFP+ cells immediately after transduction for the MGMT-P2A-GFP vector and the MGMT-P2A-HOXB4-T2A-GFP vector, and about 20% marking for the HOXB4-P2A-GFP vector. In the presence of 800 mM TMZ, about 25% of CFU-C were drug resistant in vitro using the SIV vectors containing the MGMT expression cassette. For both vectors expressing HOXB4, expansion of GFP+ CD34+ cells was noted over 12-day in vitro cultures, with GFP+ cells increasing from 20% to 40% in most experiments. Our efforts are now focused on further evaluation of these SIV vectors in vivo, in transplanted rhesus macaques.

Unique characteristics of a picornavirus internal ribosome entry site from the porcine teschovirus-1 talfan.

The teschoviruses constitute a recently defined picornavirus genus. Most of the genome sequence of the porcine teschovirus-1 (PTV) Talfan and several other strains is known. We now demonstrate that initiation of protein synthesis occurs at nucleotide (nt) 412 on the PTV Talfan RNA and that nt 1 to 405 contains an internal ribosome entry site (IRES) that functions efficiently in vitro and within mammalian cells. In comparison with other picornavirus IRES elements, the PTV IRES is relatively short and lacks a significant polypyrimidine tract near the 3' end. Expression of an enterovirus 2A protease, which induces cleavage of eIF4G within the translation initiation complex eIF4F, has little effect on the PTV IRES activity within BHK cells. The PTV IRES has a unique set of properties and represents a new class of picornavirus IRES element.