Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells

Abstract

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2 degreesCFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2 degreesCFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2 degreesCFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2 degreesCFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0-10% of the vector containing 2 degreesCFU-S from 8 recipients and expression in >50% of the vector-containing 2 degreesCFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo.

Figure 1

Overview of the murine serial bone marrow transplant model.

Figure 2

Diagram of the retroviral vectors used in the serial transplants. The relevant differences are highlighted. MPSV, LTR from the myeloproliferative sarcoma virus; NCR, negative control region; dl PBS, PBS from the dl587rev virus; Thy, 214-bp hypomethylation signal from the murine Thy-1 gene promoter/enhancer.

Figure 3

Northern blot analysis of vector-derived RNA from individual 2°CFU-S. Total cellular RNA was isolated and electrophoresed on a 1.2% agarose gel and was transferred to a nylon membrane. The membrane was hybridized with a ³²P-labeled neo gene probe, was stripped, and was reprobed with a labeled β-actin cDNA to confirm the presence and integrity of the RNA.

Figure 4

Southern blots to determine the extent of proviral methylation. (A) The marrow used to repopulate the animals represented was derived from primary recipient LN A. Total genomic DNA was isolated from 2°CFU-S containing the LN vector and was analyzed for methylation at the SmaI site by Southern blot analysis as described in Materials and Methods. Densitometry was performed to quantitate the intensities of the SmaI-resistant fragments (upper bands) and SmaI-sensitive fragments (lower bands). The proportion of the total density measured from both bands that was contributed by the SmaI-resistant fragment represents the extent of proviral DNA methylation. (B) The marrow used to repopulate the animals represented was transduced with the LN vector and was derived from primary recipient LNC. Total genomic DNA was extracted and analyzed as above.

Figure 5

Proviral methylation data grouped by vector and expression profile. For each construct, the data are segregated into two columns: nonexpressing (−) on the left and expressing (+) on the right. Within each column, the extent of methylation values measured by densitometry are plotted against the y axis (0–100%).