Efficient transfer and sustained high expression of the human glucocerebrosidase gene in mice and their functional macrophages following transplantation of bone marrow transduced by a retroviral vector.

Abstract

A recombinant retroviral vector (MFG-GC) was used to study the efficiency of transduction of the human gene encoding glucocerebrosidase (GC; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45), in mouse hematopoietic stem cells and expression in their progeny. Transfer of the GC gene to CFU-S (spleen cell colony-forming units) in primary and secondary recipients was virtually 100%. In mice 4-7 months after transplantation, highly efficient transfer of the human gene to bone marrow cells capable of long-term reconstitution was confirmed by detection of one or two copies per mouse genome in hematopoietic tissues and in cultures of pure macrophages. Expression of the human gene exceeded endogenous activity by several fold in primary and secondary CFU-S, tissues from long-term reconstituted mice, and explanted macrophages cultures. These studies are evidence of the feasibility of efficient transfer of the GC gene to hematopoietic stem cells and expression in their progeny for many months after reconstitution. The results of this study strengthen the rationale for gene therapy as a treatment for Gaucher disease.

High efficiency retroviral vectors that contain no viral coding sequences

“…As additional controls, wellknown retroviral vectors MFG and LXSN were also used. 5,9,11,12 In the case of MFG, the reporter gene is coexpressed with neo as a bicistronic message, similar to the case of the MIN series vectors.…”

“…Although MFG has been shown to drive stable, high levels of gene expression in vivo as well as in vitro and produce high viral titers, 9,10 this vector contains even more viral coding sequences, 420 bp for gag, 377 bp for pol and 99 bp for env, 11,12 raising the possibility of an even higher frequency of producing RCR than the LN series vector.…”

Transfection of human macrophages by lipoplexes via the combined use of transferrin and pH-sensitive peptides

“…Their broad activity that includes tissue maintenance, immune regulation, and pathogen control makes them crucial targets for gene therapy [1]. Through the expression of therapeutic transgenes, attempts have been made to modulate the function and dysfunction of macrophages for the treatment of genetic metabolic diseases, including Gaucher's disease [2][3][4][5], to promote adoptive cellular immunotherapy against neoplastic cells [6], to repair damaged tissues [7], or to inhibit HIV replication in these cells [8,9].…”

Engineering the splice acceptor for improved gene expression and viral titer in an MLV-based retroviral vector

“…1,2 Despite their frequent use as gene delivery vehicles and the promising therapeutic effects in the SCID-X1 trial case, 3 there are still areas that need to be improved upon in order for MLV-based vectors to be effective in actual clinical settings. [4][5][6][7][8][9] First, many of these vectors contain sequences that are also present in the packaging lines. 6,8,10 A recombination between the packaging genome and the vector can result in the generation of replication-competent retrovirus (RCR).…”

“…[4][5][6][7][8][9] First, many of these vectors contain sequences that are also present in the packaging lines. 6,8,10 A recombination between the packaging genome and the vector can result in the generation of replication-competent retrovirus (RCR). [11][12][13] Second, the level of gene expression in vivo driven by MLV-based vectors, for reasons not fully understood yet, is not high enough to give clear therapeutic effects in most cases.…”