Improved Retroviral Vectors for Hematopoietic Stem Cell Protection andIn VivoSelection

Abstract

Therapeutic gene transfer into hematopoietic cells is critically dependent on the evolution of methods that allow ex vivo expansion, high-frequency transduction, and selection of gene-modified long-term repopulating cells. Progress in this area needs elaboration of defined culture and transduction conditions for long-term repopulating cells and improvement of gene transfer systems. We have optimized retroviral vector constructions based on murine leukemia viruses (MuLV) to overcome the transcriptional repression encountered with the use of conventional Moloney MuLV (MoMuLV) vectors in early hematopoietic progenitor cells (HPC). Novel retroviral vectors, termed FMEV (for Friend-MCF/MESV hybrid vectors), were cloned that mediate greatly improved gene expression in the myeloerythroid compartment. Transfer of the selectable marker multidrug resistance 1 (mdr1), FMEV, in contrast to conventional MoMuLV-related vectors currently in use for clinical protocols, mediated background-free selectability of transduced human HPC in the presence of myeloablative doses of the cytostatic agent paclitaxel in vitro. Furthermore, FMEV also greatly improved chemo-protection of hematopoietic progenitor cells in a murine model system in vivo. Finally, when a second gene was transferred along with mdr1 in an FMEV-backbone, close to 100% coexpression was observed in multidrug-resistant colonies. These observations have significant consequences for a number of ongoing and planned gene therapy trials, for example, stem cell protection to reduce the myelotoxic side effects of anticancer chemotherapy, correction of inherited disorders involving hematopoietic cells, and antagonism of HIV infection.

Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates

“…(57). This design has a number of modifications in the 5′ untranslated leader sequences to remove all AUG codons and all viral coding sequences; thus, the probability of expression of unwanted peptides and the potential for homologous recombination is essentially eliminated (57)(58)(59). The backbone for the MGMT P140K gammaretrovirus construct (provided by D.B.…”

Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation

“…Although the mode of gene delivery has been diverse, hematopoietic stem cell studies have primarily used either retroviral or adenoviral vectors. [1][2][3][4][5] Overall, there has been low stem cell transduction efficiency and some controversy as to the safety when using these vectors. For many current modes of gene transfer (with the exception of adenoviral and lentiviral systems), cell division is required.…”

True

“…For example, inclusion of the enhancer elements of the polycythemic strain of the spleen focus-forming virus (SFFVp) was shown to be superior to the classical MoMuLV enhancer configuration for transduction of hematopoietic cells. [23][24][25] Also, intron-based retroviral vectors, 26 as well as inclusion of post-transcriptional elements such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), 27,28 have been used for increased transgene expression.…”

“…232 Modification of the basic MoMuLV vectors in order to optimize transcription resulted in greatly improved retroviral vectors for use in hematopoietic cell-based gene therapy. [23][24][25] To this end, the so-called FMEV vectors combining the LTR of Friend mink (FM) cell focus-forming viruses with the 5Ј untranslated leader region of the murine embryonic stem cells virus (MESV) were developed to achieve high transgene expression in hematopoietic progenitor and stem cells. 233,234 …”